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W3186930904.txt	https://www.strategybusiness.ru/jour/article/download/753/620	en		THE IMPACT OF DIGITALIZATION ON THE EFFICIENCY OF THE SALES CYCLE	Strategii biznesa	2,021	cc-by	1,513	Том 9, № 7 (2021) Business Strategies стр 204 DOI: 10.17747/2311-7184-2021-7-204-206 4.0 Влияние цифровизации на эффективность коммерческого цикла продаж Конго Даниил Михайлович АНОВО «Московский международный университет» Аннотация. Научная статья посвящена исследовательскому анализу перспективных направлений совершенствования работы отдела продаж строительной компании при помощи цифровой трансформации бизнес-процессов и внедрения готовых IT-решений. Актуальность исследования обусловлена цифровой трансформацией строительного сектора российской экономики, из-за чего все большее число конкурирующих организаций внедряют новейшие технологии при управлении эффективностью своих продаж. В рамках статьи рассмотрена характеристика направлений цифровизации коммерческого цикла продаж компании. Проанализированы преимущества и эффективность, которая формируется благодаря использованию цифровых технологий и готовых IT-решений. Ключевые слова: цифровизация; цифровые технологии; продажи; коммерческий цикл продажи; готовые IT-решения; цифровой маркетинг; цифровые продажи. The impact of digitalization on the eﬃciency of the sales cycle Kongo Daniil Mikhailovich Abstract. The scientiﬁc article is devoted to the research analysis of promising directions for improving the work of the sales department of a construction company through the digital transformation of business processes and the implementation of ready-made IT solutions. The relevance of the study is due to the digital transformation of the construction sector of the Russian economy, which is why an increasing number of competing organizations are introducing the latest technologies in managing the eﬃciency of their sales. Within the framework of the article, the characteristics of the directions of digitalization of the company's commercial sales cycle are considered. The advantages and eﬃciency, which is formed through the use of digital technologies and ready-made IT solutions, are analyzed. Keywords: digitalization; digital technologies; sales; commercial sales cycle; ready-made IT solutions; digital marketing; digital sales. Коммерческая деятельность предприятий строительного сектора, в первую очередь, нацелена на чистый финансовый результат – то есть получение прибыли. Ее формирование происходит через реализационную деятельность, где продажа производимой продукции генерирует выручки и доходы организации. Анализируемая нами компания проводит не только строительные работы по возведению объектов жилой недвижимости, но и реализует построенные квартиры на первичном рынке регионов Российской Федерации. Соответственно, менеджменту организации важно применять современные технологии, инновации и готовые IT-решения, которые позволяют проводить цифровизацию коммерческого цикла продаж, включая автоматизацию бизнес-процессов отдела продаж. Кроме того, актуальность научного исследования на тематику «влияние цифровизации на эффективность коммерческого цикла продаж» обусловлена цифровой трансформацией строительного сектора российской экономики, из-за чего все большее число конкурирующих организаций внедряют новейшие технологии при управлении эффективностью своих продаж. По этой причине целью научной работы выступает проведение исследовательского анализа перспективных направлений совершенствования работы отдела продаж строительной компании при помощи цифровой трансформации бизнес-процессов и внедрения готовых IT-решений. Одним из наиболее перспективных направлений цифровизации работы отдела продаж строительной компании является применение цифровых технологий в маркетинге, что предполагает переход на омниканальность. Например, по нашему мнению, предприятиям сферы строительства объектов недвижимости и продажи квартир на первичном рынке России необходимо использование таких универсальных цифровых технологии, как Social Media Marketing (SMM) и Community Management. Social Media Marketing (SMM) – это коммуникации компании с аудиторией социальных сетей и форумов от имени бренда или организации [9]. Алгоритм Social Media Marketing состоит из следующих поэтапных действий: – как формируется SMM-стратегия продвижения компании и достижения бизнес-целей; – как происходит графическое оформление сообществ в социальных сетях; – как создаются контент-стратегии продвижения товаров и услуг; – как проводится рекламная кампания через таргетированные настройки предложений компании. Community Management (CM) – это маркетинговая стратегия продвижения, основанная на взаимоотношениях, сетях и взаимодействиях, ориентированных на долгосрочные взаимовыгодные отношения с отдельными потребителями. При этом ценность создается участвующими в отношениях сторонами совместно [2,10]. Том 9, № 7 (2021) Business Strategies стр 205 Алгоритм Community Management состоит из следующих поэтапных действий: – определяются ценности и миссия бренда, которые будут позиционировать при продвижении товаров и услуг; – выявляется функциональное ядро клиентского сообщества; – создается интегрированная площадка компании с социальными сетями через тот же SMM; – в рамках онлайн-сообщества проводится сотрудничество среди потребителей; – происходит прием обратной связи и ее оценка, а также организация мероприятий для клиентского сообщества. Еще одним из главных направлений совершенствования системы управления коммерческим циклом продаж анализируемой компании будет использование цифровых технологий Big Data при персонализации предложений клиентам, разделенных на подробные группы целевой аудитории. Конечной целью персонализации, на примере продаж квартир на первичном рынке, выступает повышение уровня релевантности, что крайне важно при онлайн-продажах через коммерческие сайты, лейдинговые странички или на страницах социальных сетей. При этом персонализация маркетингового продвижения в работе отдела продаж при взаимодействии через рекламные каналы достигается путем: – размещения медийной рекламы с использованием RTB и ретаргетинга; – использованием e-mail-рассылок; – созданием персонализированных рекламных сообщений и т. д. Однако при применении персонализации предложений при управлении эффективностью коммерческого цикла продаж может сформироваться ряд барьеров, тормозящих развитие данного подхода в управлении маркетингом [4]. Главная проблема – это отсутствие практического применения технологий и инструментов, способных проводить персонализацию предложений на автоматической основе, анализируя при этом огромный массив входящей и исходящей информации. Чтобы ее решить, руководству компании в рамках персонализации маркетингового продвижения рекомендуется использование технологий Big Data. На сегодняшний день одной из наиболее приоритетных цифровых технологий маркетинга персонализированных предложений в 2021 году выступает Big Data. Данная технология обладает следующими характеристиками [1]: – максимальный объем информации; – децентрализованный способ хранения информации; – полуструктурированная и неструктурированная система хранения данных; – горизонтальная модель хранения и обработки информации; – слабая взаимосвязь данных и информации. Ключевой же причиной применения Big Data при персонализации маркетингового продвижения является наличие следующих преимуществ [5; 6; 7]: – происходит цифровизация процессов сбора, обработки и анализа большего массива данных, на что не способен ни один человек; – увеличивается инструментарий подбора информации, поскольку одновременно могут использовать различные виды материалов, например текст, графика, видео, аудио и т. д.; – формируется возможность персонализации маркетингового предложения, поскольку данные технологии могут анализировать информацию отдельных целевых аудиторий, выделяя ее от всех остальных; – проводится оценка реального уровня удовлетворенности клиентов; – возможно проведение оценки соответствия качества металлургического продукта или клиентского обслуживания с ожиданиями потребителей. Для того чтобы подтвердить экономическую эффективность применения маркетинговых технологий персонализации продвижения коммерческих предложений по приобретению квартир на первичном рынке для строительной компании на основе Big Data, можно взять исследования Economist Intelligence Unit, где получены следующие результаты положительного опыта от использования данной технологии (см. рис. 1). Рис. 1. Результаты положительного опыта от использования Big Data в маркетинговых исследованиях, % респондентов [8] Том 9, № 7 (2021) Business Strategies стр 206 Так, исходя из рис. 1, можно заключить, что главным направлением применения технологии Big Data выступает улучшение клиентского сервиса и повышение уровня реагирования на запросы клиентов. Именно последний пункт отражает вероятность формирования маркетинговых инструментов персонализации. Кроме того, персонализация предложений при помощи использования цифровых технологий Big Data может использоваться и в рамках совершенствования политики SMM. Например, персонализация позволит разбить рекламные кампании в социальных сетях таким образом, чтобы виды квартир по планировке и объектам недвижимости, в которых они находятся (ведь их ценовая политика может различаться), изображалась наиболее подходящим способом, то есть указывались именно те квартиры, которые, вероятнее всего, больше всего интересуют потенциального покупателя. Следующим направлением повышения эффективности коммерческого цикла продаж строительной компании при помощи цифровых технологий является использование готового IT-решения в виде CRM-системы, под которым подразумевается прикладное программное обеспечение для организаций, предназначенное для автоматизации стратегий взаимодействия с клиентами, в частности, для повышения уровня продаж, оптимизации маркетинга и улучшения обслуживания клиентов [3]. С помощью использования технологии CRM-системы компания способна получить следующие результаты: – возможность не потерять потенциальных клиентов; – контролировать работу сотрудников с клиентами; – накапливать статистическую информацию и в дальнейшем анализировать ее; – проводить автоматизацию готовых решений на определенные группы ситуаций. CRM-система выступает, в первую очередь, активным цифровым инструментом и сервисом, позволяющим управлять всей маркетинговой деятельностью компании. По этой причине необходимо понимать, что автоматизация процесса взаимодействия с клиентами – это эффективный способ достижения данной цели, но сам по себе, без участия грамотного специалиста он бесполезен. Поэтому зачастую применение CRM-системы и предполагается как использование готового IT-решения, где за функциональность и бесперебойность программного обеспечения будет ответственна сервисная организация, предоставляющая профессиональные услуги IT-аутсорсинга. Таким образом, в заключение научной работы подытожим, что к перспективным направлениям цифровизации работы отдела продаж строительной компании относится применение цифровых технологий в маркетинге, что предполагает под собой переход на омниканальность, использование цифровых технологий Big Data при персонализации предложений клиентам, а также использование готового IT-решения в виде CRM-системы. Список литературы Веретенников А.В. BigData: анализ больших данных // Молодой ученый. 2017. № 32(166). С. 9–12. Есаков В.А., Конобеева А.Б. Экосоциальные проблемы создания эффективных рыночных механизмов экономики в России//Вестник Московского государственного университета культуры и искусств. 2013. № 6 (56). С. 222–225. [3] Клочкова А.В., Бебякина А.А. CRM-система как инновационный инструмент повышения конкурентоспособности организации // Экономика и экологический менеджмент. 2019. № 4. [4] Конобеева А.Б. Экологические проблемы экономики//Научное обозрение. Серия 1: Экономика и право. 2012. № 6. С. 308–317. [5] Конобеева А.Б. Реинжиниринг бизнес-процессов как метод повышения эффективности управления организацией//Маркетинг и логистика. 2020. № 3(29). С. 77–81. [6] Митрович С. Рынок «больших данных» и их инструментов: тенденции и перспективы в России // МИР (Модернизация. Инновации. Развитие). 2018. Т. 9. № 1. С. 74–85. [7] Прохоренков П.А., Гусарова О.М., Аверьянова Т.В. Современные информационные технологии маркетинга // Фундаментальные исследования. 2018. № 12–1. С. 158–162. [8] Соболева А.О. Big Data: возможности для бизнеса // Научное сообщество: сб. ст. по мат. XXXV междунар. студ. науч.-практ. конф. № 24 (35). URL: https://sibac.info/archive/meghdis/24(35).pdf (дата обращения: 04.06.2021). [9] Уманская М.В., Петров С.В. SMM как элемент стратегии развития предприятия // Международный журнал гуманитарных и естественных наук. 2018. № 6–2. [10] Федоренко А.Н. Клиентское сообщество как новый способ продвижения компаний // Вестник евразийской науки. 2017. № 5(42). [1] [2]
https://openalex.org/W4234653199	https://npg.copernicus.org/articles/25/241/2018/npg-25-241-2018.pdf	English	null	Utsu aftershock productivity law explained from geometric operations on the permanent static stress field of mainshocks	null	2,017	cc-by	8,608	1 Introduction Abstract. The aftershock productivity law is an exponential function of the form K ∝exp(αM), with K being the num- ber of aftershocks triggered by a given mainshock of mag- nitude M and α ≈ln(10) being the productivity parameter. This law remains empirical in nature although it has also been retrieved in static stress simulations. Here, we param- eterize this law using the solid seismicity postulate (SSP), the basis of a geometrical theory of seismicity where seis- micity patterns are described by mathematical expressions obtained from geometric operations on a permanent static stress ﬁeld. We ﬁrst test the SSP that relates seismicity den- sity to a static stress step function. We show that it yields a power exponent q = 1.96 ± 0.01 for the power-law spa- tial linear density distribution of aftershocks, once uniform noise is added to the static stress ﬁeld, in agreement with ob- servations. We then recover the exponential function of the productivity law with a break in scaling obtained between small and large M, with α = 1.5ln(10) and ln(10), respec- tively, in agreement with results from previous static stress simulations. Possible biases of aftershock selection, proven to exist in epidemic-type aftershock sequence (ETAS) simu- lations, may explain the lack of break in scaling observed in seismicity catalogues. The existence of the theoretical kink, however, remains to be proven. Finally, we describe how to estimate the solid seismicity parameters (activation density δ+, aftershock solid envelope r∗and background stress am- plitude range 1o∗) for large M values. Aftershocks, one of the most studied patterns observed in seismicity, are characterized by three empirical laws, which are functions of time, such as the modiﬁed Omori law (e.g., Utsu et al., 1995), space (e.g., Richards-Dinger et al., 2010; Moradpour et al., 2014) and mainshock magnitude (Utsu, 1970a, b; Ogata, 1988). The present study focuses on the latter relationship, i.e., the Utsu aftershock productivity law, which describes the total number of aftershocks K produced by a mainshock of magnitude M as K (M) = K0exp[α (M −m0)], (1) (1) with m0 the minimum magnitude cutoff (Utsu, 1970b; Ogata, 1988). Utsu aftershock productivity law explained from geometric operations on the permanent static stress ﬁeld of mainshocks p p Arnaud Mignan ETHZ, Institute of Geophysics, Swiss Federal Institute of Technology, Zurich, Switzerland Correspondence: Arnaud Mignan (arnaud.mignan@sed.ethz.ch) Received: 10 July 2017 – Discussion started: 18 July 2017 Revised: 27 February 2018 – Accepted: 28 February 2018 – Published: 29 March 2018 Nonlin. Processes Geophys., 25, 241–250, 2018 https://doi.org/10.5194/npg-25-241-2018 © Author(s) 2018. This work is distributed under the Creative Commons Attribution 4.0 License. Nonlin. Processes Geophys., 25, 241–250, 2018 https://doi.org/10.5194/npg-25-241-2018 © Author(s) 2018. This work is distributed under the Creative Commons Attribution 4.0 License. A. Mignan: Utsu aftershock productivity law explained from geometric operations 242 Båth’s parameter 1mB. The α value was in turn decoupled from the β value in later studies (e.g., Seif et al., 2017 and references therein). sible (i.e., a sort of hard labeling). Based on this postu- late, Mignan (2012) demonstrated the power-law behavior of precursory seismicity in agreement with the observed time- to-failure equation (Varnes, 1989), while Mignan (2016a) demonstrated both the observed parabolic spatiotemporal front and the linear relationship with injection ﬂow rate of induced seismicity (Shapiro and Dinske, 2009). It remains unclear whether the SSP has a physical origin or not. If not, it would still represent a reasonable approximation of the linear relationship between event production and static stress ﬁeld in a simple clock-change model (Hainzl et al., 2010; Fig. 1a). For the testing of the SSP on the observed spatial distribution of aftershocks, see Sect. 2.2. The power of Eq. (5) is that it allows seismicity patterns to be deﬁned in terms of “solids” described by the spatial envelope r∗= r (σ = ±1o∗), where r is the distance from the static stress source (e.g., main- shock rupture) and r∗is the distance r at which there is a change of regime (quiescence–background at σ = −1o∗or background–activation at σ = 1o∗). The spatiotemporal rate of seismicity is then a mathematical expression deﬁned by the density of events δ times the volume characterized by r∗(see previous demonstrations in Mignan et al., 2007 and Mignan, 2011, 2012, 2016a where simple algebraic expres- sions were obtained). Although it seems obvious that Eq. (1) can be explained geometrically if the volume of the aftershock zone is corre- lated to the mainshock surface area S with S (M) = 10M−4 = exp[ln(10)(M −4)] (4) (4) (Kanamori and Anderson, 1975; Yamanaka and Shimazaki, 1990; Helmstetter, 2003), there is so far no analytical, phys- ical expression of Eq. (1) available. Although Hainzl et al. (2010) retrieved the exponential behavior in numerical simulations where aftershocks were produced by the perma- nent static stress ﬁeld of mainshocks of different magnitudes, it remains unclear how K0 and α relate to the underlying physical parameters. The aim of the present article is to describe the Utsu after- shock productivity equation (Eq. 1) in terms of a geometri- cal theory of seismicity coined “solid seismicity”, where the Eq. (4) scaling is parameterized using the solid seismicity postulate (SSP). 2.1 Demonstration of the productivity law by geometric operations with 1σ0 < 0 the mainshock stress drop, c the crack radius and r the distance from the crack. Equation (6) is a simpliﬁed representation of stress change from slip on a planar surface in a homogeneous elastic medium. It takes into account both the square root singularity at crack tip and the 1/r3 falloff at higher distances (Dieterich, 1994; Fig. 1b). It should be noted that this radial static stress ﬁeld does not represent the geo- metric complexity of Coulomb stress ﬁelds (Fig. 2a). How- ever, we are here only interested in the general behavior of aftershocks with Eq. (6) retaining the ﬁrst-order characteris- tics of this ﬁeld (i.e., on-fault seismicity; Fig. 2b), which cor- responds to the case where the mainshock relieves most of the regional stresses and aftershocks occur on optimally ori- ented faults. It is also in agreement with observations, most aftershocks being located on and around the mainshock fault traces in southern California (Fig. 2c; see Sect. 3). The oc- casional cases where aftershocks occur off-fault (e.g., Ross et al., 2017) can be explained by the mainshock not relieving all of the regional stress (King et al., 1994; Fig. 2d). with 1σ0 < 0 the mainshock stress drop, c the crack radius and r the distance from the crack. Equation (6) is a simpliﬁed representation of stress change from slip on a planar surface in a homogeneous elastic medium. It takes into account both the square root singularity at crack tip and the 1/r3 falloff at higher distances (Dieterich, 1994; Fig. 1b). It should be noted that this radial static stress ﬁeld does not represent the geo- metric complexity of Coulomb stress ﬁelds (Fig. 2a). How- ever, we are here only interested in the general behavior of aftershocks with Eq. (6) retaining the ﬁrst-order characteris- tics of this ﬁeld (i.e., on-fault seismicity; Fig. 2b), which cor- responds to the case where the mainshock relieves most of the regional stresses and aftershocks occur on optimally ori- ented faults. It is also in agreement with observations, most aftershocks being located on and around the mainshock fault traces in southern California (Fig. 2c; see Sect. 3). The oc- casional cases where aftershocks occur off-fault (e.g., Ross et al., 2017) can be explained by the mainshock not relieving all of the regional stress (King et al., 1994; Fig. 2d). 2 Physical expression of the aftershock productivity law (6) A. Mignan: Utsu aftershock productivity law explained from geometric operations The SSP has already been shown to effec- tively explain other empirical laws of both natural and in- duced seismicity from simple geometric operations on a per- manent static stress ﬁeld (Mignan, 2012, 2016a). The theory is applied here for the ﬁrst time to describe aftershocks. In the case of aftershocks, we deﬁne the static stress ﬁeld of the mainshock by σ (r) = −1σ0   1 − c3 (r + c)3 −1 2 −1  , (6) 1 Introduction This relationship was originally proposed by Utsu (1970a, b) by combining two other empirical laws, the Gutenberg–Richter relationship (Gutenberg and Richter, 1944) and Båth’s law (Båth, 1965), respectively: N (≥m) = Aexp[−β (m −m0)] N (≥M −1mB) = 1 , (2) (2) (2) with N the average number of events above magnitude m, A a seismic activity constant, β the magnitude size ratio (or b = β/ln(10) in base-10 logarithmic scale) and 1mB the magnitude difference between the mainshock and its largest aftershock, such that K (M) = N (≥m0 \|M ) = exp(−β1mB)exp[β (M −m0)], (3) (3) = exp(−β1mB)exp[β (M −m0)], (3) with K0 = exp(−β1mB) and α ≡β. Equation (3) was only implicit in Utsu (1970a) and not exploited in Utsu (1970b), where K0 was ﬁtted independently of the value taken by Published by Copernicus Publications on behalf of the European Geosciences Union & the American Geophysical Union. Published by Copernicus Publications on behalf of the European Geosciences Union & the American Geophysical Union. A. Mignan: Utsu aftershock productivity law explained from geometric operations 243 Figure 1. Deﬁnition of the aftershock solid envelope in a perma- nent static stress ﬁeld: (a) event density stress step-function δ(σ) (Eq. 5) of the solid seismicity postulate (SSP) in comparison to the linear clock-change model; (b) static stress σ versus distance r for different effective crack radii c and rupture stress drops 1σ0 (Eq. 6); (c) linear relationship between effective crack radius c and aftershock solid envelope radius r∗for different 1σ∗/1σ0 ratios (Eq. 7); (d) relationship between mainshock magnitude M and ef- fective crack radius c for different seismogenic widths w0 (Eq. 8). Figure 2. Possible static stress ﬁelds and inferred aftershock spa- tial distribution: (a) right-lateral Coulomb stress ﬁeld for optimally oriented faults, where the mainshock relieves all of the regional stresses σr = 10 bar, with 1σ0 ≈−Gs/L ≈−10 bar (G = 3.3 × 105 bar the shear modulus, s = 0.6 m the slip, L = 20 km the fault length and w = 10 km the fault width); (b) radial static stress ﬁeld computed from Eq. (6) with 1σ0 = −10 bar and c = √(Lw)/π for consistency with panel (a); (c) aftershock distribution of the largest strike-slip events in the southern California relocated catalog, iden- tiﬁed here as all events occurring within 1 day of the mainshock ( d S 3 1) (d) i h l l C l b ﬁld f i Figure 2. Possible static stress ﬁelds and inferred aftershock spa- tial distribution: (a) right-lateral Coulomb stress ﬁeld for optimally oriented faults, where the mainshock relieves all of the regional stresses σr = 10 bar, with 1σ0 ≈−Gs/L ≈−10 bar (G = 3.3 × 105 bar the shear modulus, s = 0.6 m the slip, L = 20 km the fault length and w = 10 km the fault width); (b) radial static stress ﬁeld computed from Eq. (6) with 1σ0 = −10 bar and c = √(Lw)/π for consistency with panel (a); (c) aftershock distribution of the largest strike-slip events in the southern California relocated catalog, iden- tiﬁed here as all events occurring within 1 day of the mainshock (see data Sect. A. Mignan: Utsu aftershock productivity law explained from geometric operations 3.1); (d) right-lateral Coulomb stress ﬁeld for opti- mally oriented faults, where the mainshock relieves only a fraction of the regional stresses σr = 100 bar with 1σ0 = −10 bar (same rupture as in panel a) – the black contour represents 1 bar in pan- els (a), (b) and (d) and a 10 km distance from rupture in panel (c). Coulomb stress ﬁelds of panels (a) and (d) were computed using the Coulomb 3 software (Lin and Stein, 2004; Toda et al., 2005). Figure 1. Deﬁnition of the aftershock solid envelope in a perma- nent static stress ﬁeld: (a) event density stress step-function δ(σ) (Eq. 5) of the solid seismicity postulate (SSP) in comparison to the linear clock-change model; (b) static stress σ versus distance r for different effective crack radii c and rupture stress drops 1σ0 (Eq. 6); (c) linear relationship between effective crack radius c and aftershock solid envelope radius r∗for different 1σ∗/1σ0 ratios (Eq. 7); (d) relationship between mainshock magnitude M and ef- fective crack radius c for different seismogenic widths w0 (Eq. 8). For r∗= r (σ = 1o∗), Eq. (6) yields the aftershock solid envelope of the following form: r∗(c) =    1 1 − 1 −1σ∗ 1σ0 −2 1 3 −1    c = Fc (7) r∗(c) =    1 1 − 1 −1σ∗ 1σ0 −2 1 3 −1    c = Fc (7) (7) The aftershock productivity K(M) is then the activation density δ+ times the volume V∗(M) of the aftershock solid. For the case in which the mainshock relieves most of the regional stress, stresses are increased all around the rup- ture (King et al., 1994), which is topologically identical to stresses increasing radially from the rupture plane (Fig. 2a– b). It follows that the aftershock solid can be represented by a volume of contour r∗(M) from the rupture plane geometric primitive, i.e., a disk or a rectangle for small and large main- shocks, respectively. This is illustrated in Fig. 3a–b and can be generalized by function of the crack radius c and of the ratio between background stress amplitude range 1o∗and stress drop 1σ0 (Fig. 1c). With 1σ0 independent of earthquake size (Kanamori and Anderson, 1975; Abercrombie and Leary, 1993) and 1o∗assumed constant, r∗is directly proportional to c with proportionality constant, or stress factor, F (Eq. 7). 2.1 Demonstration of the productivity law by geometric operations “Solid seismicity”, a geometrical theory of seismicity, is based on the following postulate (Mignan et al., 2007; Mignan, 2008, 2012, 2016a). Solid seismicity postulate: Seismicity can be strictly catego- rized into three regimes of constant spatiotemporal densities δ – background δ0, quiescence δ−and activation δ+ (with δ−≪δ0 ≪δ+) – occurring respective to the static stress step function: δ (σ) =    δ−, σ < −1o∗ δ0, σ ≤\|±1o∗\| δ+, σ > 1o∗ , (5) (5) with σ the static stress (stress unit), 1o∗the background stress amplitude range (stress unit), a stress threshold value separating two seismicity regimes, and δ the spatial density of events (number of events per unit of volume) per seismicity regime. We mean by “strictly categorized” that any seismicity pop- ulation is either part of the background, quiescence or acti- vation regime (or class), with no other regime or class pos- www.nonlin-processes-geophys.net/25/241/2018/ Nonlin. Processes Geophys., 25, 241–250, 2018 A. Mignan: Utsu aftershock productivity Figure 1. Deﬁnition of the aftershock solid env nent static stress ﬁeld: (a) event density stress (Eq. 5) of the solid seismicity postulate (SSP) the linear clock-change model; (b) static stress r for different effective crack radii c and ruptur (Eq. 6); (c) linear relationship between effective aftershock solid envelope radius r∗for differen (Eq. 7); (d) relationship between mainshock ma fective crack radius c for different seismogenic w For r∗= r (σ = 1o∗), Eq. (6) yields the envelope of the following form: r∗(c) =    1 1 − 1 −1σ∗ 1σ0 −2 1 3 −1    c = function of the crack radius c and of th background stress amplitude range 1o∗ 1σ0 (Fig. 1c). With 1σ0 independent of (Kanamori and Anderson, 1975; Abercro 1993) and 1o∗assumed constant, r∗is dire to c with proportionality constant, or stress Geometrical constraints due to the seismog w0 then yield c(M) =    S(M) π 1 2 , S (M) ≤πw2 0 w0, S (M) > πw2 0 , with S the rupture surface area deﬁned by coming an effective crack radius (Kanamo 1975; Fig. 1d). Note that the factor of 2 (i.e., of w0/2) comes from the free surface effec and Anderson, 1975; Shaw and Scholz, 200 A. Mignan: Utsu aftershock productivity law explained from geometric operations A. Mignan: Utsu aftershock productivity law explained from geometric operations Geometrical constraints due to the seismogenic layer width w0 then yield function of the crack radius c and of the ratio between background stress amplitude range 1o∗and stress drop 1σ0 (Fig. 1c). With 1σ0 independent of earthquake size (Kanamori and Anderson, 1975; Abercrombie and Leary, 1993) and 1o∗assumed constant, r∗is directly proportional to c with proportionality constant, or stress factor, F (Eq. 7). Geometrical constraints due to the seismogenic layer width w0 then yield c(M) =    S(M) π 1 2 , S (M) ≤πw2 0 w0, S (M) > πw2 0 , (8) (8) V∗(M) = 2r∗(M)S (M) + π 2 r2 ∗(M)d, (9) (9) with S the rupture surface area deﬁned by Eq. (4) and c be- coming an effective crack radius (Kanamori and Anderson, 1975; Fig. 1d). Note that the factor of 2 (i.e., using w0 instead of w0/2) comes from the free surface effect (e.g., Kanamori and Anderson, 1975; Shaw and Scholz, 2001). where d is the distance traveled around the geometric prim- itive by the geometric centroid of the semicircle of radius where d is the distance traveled around the geometric prim- itive by the geometric centroid of the semicircle of radius where d is the distance traveled around the geometric prim- itive by the geometric centroid of the semicircle of radius Nonlin. Processes Geophys., 25, 241–250, 2018 r∗(M) (i.e., Pappus’s Centroid Theorem), or r∗(M) (i.e., Pappus’s Centroid Theorem), or r∗(M) (i.e., Pappus’s Centroid Theorem), or d =    2π c(M) + 4 3π r∗(M) , c(M) + r∗(M) ≤w0 2 2w0, c(M) + r∗(M) > w0 2 . (10) For the disk, the volume (Eq. 9) corresponds to the sum of a cylinder of radius c(M) and height 2r∗(M) (ﬁrst term) and of half a torus of major radius c(M) and minus radius r∗(M) (second term). For the rectangle, the volume is the sum of a cuboid of length l(M) (i.e., rupture length), width w0 and height 2r∗(M) (ﬁrst term) and of a cylinder of radius r∗(M) and height w0 (second term; see red and orange volumes, respectively, in Fig. 3a–c). Finally inserting Eqs. (7), (8) and (10) into Eq. (9), we obtain A. Mignan: Utsu aftershock productivity law explained from geometric operations A. Mignan: Utsu aftershock productivity law explained from geometric operations 244 Figure 3. Geometric origin of the aftershock productivity law: (a) sketch of the aftershock solid for a small mainshock rupture represented by a disk; (b) sketch of the aftershock solid for a large mainshock rupture represented by a rectangle; (c) relative role of the two terms of Eq. (9), here with w0 = 10 km and 1σ∗ 1σ0 = −0.1 (to ﬁrst estimate c and r∗from Eqs. 8 and 7, respectively); (d) after- shock productivity law (normalized by δ+) predicted by solid seis- micity (Eq. 11). This relationship is of the same form as the Utsu productivity law (Eq. 1) for large M (see text for an explanation of the lack of break in scaling in Eq. 1 for small M). Dotted vertical lines represent M for c(M) + r∗(M) = w0/2 and S (M) = πw2 0, respectively. K (M) = K (M) = δ+    2F √π + F 2√π 1 + 4 3π F S 3 2 (M), S(M) ≤ w0 √π 2(1 + F) 2 2F √π S 3 2 (M) + F 2w0S(M), w0 √π 2(1 + F) 2 < S(M) ≤πw2 0 2Fw0S (M) + πF 2w3 0, S (M) > πw2 0 (11) (11) which is represented in Fig. 3d. Considering the two main regimes only (small versus large mainshocks) and inserting Eq. (4) into (11), we get K (M) = (12) δ+    2F √π + F 2√π 1 + 4 3π F exp 3ln(10) 2 (M −4) , small M 2Fw0exp[ln(10)(M −4)] + πF 2w3 0, large M (12) Figure 3. Geometric origin of the aftershock productivity law: (a) sketch of the aftershock solid for a small mainshock rupture represented by a disk; (b) sketch of the aftershock solid for a large mainshock rupture represented by a rectangle; (c) relative role of the two terms of Eq. (9), here with w0 = 10 km and 1σ∗ 1σ0 = −0.1 (to ﬁrst estimate c and r∗from Eqs. 8 and 7, respectively); (d) after- shock productivity law (normalized by δ+) predicted by solid seis- micity (Eq. 11). This relationship is of the same form as the Utsu productivity law (Eq. 1) for large M (see text for an explanation of the lack of break in scaling in Eq. A. Mignan: Utsu aftershock productivity law explained from geometric operations 1 for small M). Dotted vertical lines represent M for c(M) + r∗(M) = w0/2 and S (M) = πw2 0, respectively. which is a closed-form expression of the same form as the original Utsu productivity law (Eq. 1). Note that K and δ+ are both, implicitly, functions of the selected minimum after- shock magnitude threshold m0. Here, we predict that the α value decreases from 3ln(10)/2 ≈3.45 to ln(10) ≈2.30 when switching regime from small to large mainshocks (or from 1.5 to 1 in a base-10 logarithmic scale). It should be noted that Hainzl et al. (2010) observed the same break in scaling in static stress transfer simulations, which corroborates our analyti- cal ﬁndings. Hainzl et al. (2010) simulated aftershocks using the clock-change model where events were advanced in time by the static stress change produced by a mainshock in a 3-D medium. They explained the scaling break observed in sim- ulation as a transition from 3-D to 2-D scaling regime when the mainshock rupture dimension approached w0, which is compatible with the present demonstration. For large M, the scaling is fundamentally the same as in Eq. (4). Since that re- lation also explains the slope of the Gutenberg–Richter law (see physical explanation given by Kanamori and Anderson, 1975), it follows that α ≡β, which is also in agreement with the original formulation of Utsu (1970a, b; Eq. 3). Nonlin. Processes Geophys., 25, 241–250, 2018 www.nonlin-processes-geophys.net/25/241/2018/ Nonlin. Processes Geophys., 25, 241–250, 2018 2.2 Testing of the SSP on the aftershock spatial distribution The SSP predicts a step-like behavior of the aftershock spatial density for an idealized smooth static stress ﬁeld (Fig. 4a–b), which is in disagreement with real aftershock observations. A number of studies have shown that the spa- tial linear density distribution of aftershocks ρ is well repre- Nonlin. Processes Geophys., 25, 241–250, 2018 www.nonlin-processes-geophys.net/25/241/2018/ 3 Observations and model ﬁtting 3 ρ (r) ∝r−q, ρ (r) ∝r−q, Figure 5a represents the spatial linear density distribution of aftershocks ρ(r) for the four largest strike-slip mainshocks in southern California: 1987 M = 6.6 Superstition Hills, 1992 M = 7.3 Landers, 1999 M = 7.1 Hector Mine and 2010 M = 7.2 El Mayor. The distance between mainshock and af- tershocks is calculated as r = p (x −x0)2 + (y −y0)2, with (x, y) the aftershock coordinates and (x0, y0) the coordinates of the nearest point to the mainshock fault rupture (as de- picted in Fig. 2c). The dashed black lines shown in Fig. 5a are visual guides to q = 1.96, showing that the SSP is com- patible with real aftershock observations. with r the distance from the mainshock and q the power- law exponent. This parameter ranges over 1.3 ≤q ≤2.5 (Felzer and Brodsky, 2006; Lipiello et al., 2009; Marsan and Lengliné, 2010; Richards-Dinger et al., 2010; Shearer, 2012; Gu et al., 2013; Moradpour et al., 2014; van der Elst and Shaw, 2015). Although Felzer and Brodsky (2006) sug- gested a dynamic stress origin for aftershocks, their results were later questioned by Richards-Dinger et al. (2010). Most of the studies cited above suggest that the q value is ex- plained from a static stress process. As for the examples of aftershocks shown to be dynamically triggered (e.g., Fan and Shearer, 2016), they are too few to alter the aftershock pro- ductivity law and too remote to be consistently deﬁned as aftershocks in cluster methods. Comparing Fig. 5a to Fig. 4d suggests that r∗can be roughly estimated from the spatial linear density plot, be- ing the maximum distance r at which the plateau ends, here leading to r∗≈1 km. This parameter is constant for different large M values since both w0 and 1σ0 are constant while 1σ∗is also a priori a constant. We can then estimate the ra- tio 1σ∗/1σ0 from Eq. (7). However, the result is ambiguous due to uncertainties in the width w0. For w0 = {5,10,15} km, we get 1σ∗/1σ0 = {−0.54,−1.01,−1.38}. In a more realistic setting, the static stress ﬁeld must be heterogeneous (due to the occurrence of previous events and other potential stress perturbations). We therefore simulate the static stress ﬁeld by adding a uniform random compo- nent bounded over ±1o∗following Mignan (2011) (see also King and Bowman, 2003). Note that any deviation above www.nonlin-processes-geophys.net/25/241/2018/ Nonlin. A. Mignan: Utsu aftershock productivity law explained from geometric operations A. Mignan: Utsu aftershock productivity law explained fro Figure 4. Spatial distribution of aftershocks following the SSP: (a) Smooth static stress ﬁeld as a function of distance r from the mainshock, with 1σ0 = −10 bar and c = 10 km (Eq. 6); (b) step-like aftershock spatial linear density ρ(r) with δ+ = 1000 event km−1, δ0 = 1 event km−1 and 1σ∗= −0.31σ0 (ad hoc ratio yielding r∗= 3.5 km; Eq. (7) – event distances sampled from the δ(r) distribution, repeated 100 times). Such distribution is not observed in nature; (c) same as panel (a) but with random uniform noise representative of spatial heterogeneities added to the regional stress ﬁeld; (d) power-law-like aftershock spatial linear density ρ(r) with power exponent MLE estimate q = 1.96, representative of real aftershock observations (see Fig. 5a), due to the addition of uniform noise to the static stress ﬁeld. A. Mignan: Utsu aftershock productivity law explained from geometric operations 245 1o∗would be ﬂattened to 1o∗over time by temporal dif- fusion (the so-called “historical ghost static stress ﬁeld” in Mignan, 2016a). Figure 4c shows the resulting stress ﬁeld and Fig. 4d the predicted aftershock spatial density. Adding uniform noise blurs the contour of the aftershock solid, switching the aftershock spatial density from a step func- tion (Fig. 4b) to a power law (Fig. 4d). We ﬁt Eq. (13) to the simulated data using the maximum likelihood estimation (MLE) method with rmin = r∗(Clauset et al., 2009) and ﬁnd q = 1.96 ± 0.01, in agreement with the aftershock literature. This result alone is, however, insufﬁcient to prove the validity of the SSP. 3.2 Aftershock spatial density distribution ρ (r) ∝r−q, (13) 3.1 Data Figure 4. Spatial distribution of aftershocks following the SSP: (a) Smooth static stress ﬁeld as a function of distance r from the mainshock, with 1σ0 = −10 bar and c = 10 km (Eq. 6); (b) step-like aftershock spatial linear density ρ(r) with δ+ = 1000 event km−1, δ0 = 1 event km−1 and 1σ∗= −0.31σ0 (ad hoc ratio yielding r∗= 3.5 km; Eq. (7) – event distances sampled from the δ(r) distribution, repeated 100 times). Such distribution is not observed in nature; (c) same as panel (a) but with random uniform noise representative of spatial heterogeneities added to the regional stress ﬁeld; (d) power-law-like aftershock spatial linear density ρ(r) with power exponent MLE estimate q = 1.96, representative of real aftershock observations (see Fig. 5a), due to the addition of uniform noise to the static stress ﬁeld. We consider the case of southern California and extract af- tershock sequences from the relocated earthquake catalog of Hauksson et al. (2012) deﬁned over the period 1981– 2011, using the nearest-neighbor method (Zaliapin et al., 2008; used with its standard parameters originally calibrated for southern California, considering only the ﬁrst aftershock generation). Only events with magnitudes greater than m0 = 2.0 are considered (a conservative estimate following results of Tormann et al., 2014; saturation effects immediately after the mainshock are negligible when considering entire after- shock sequences; Helmstetter et al., 2005). 3.3 Aftershock productivity law The observed number n of aftershocks of magnitude m ≥m0 produced by a mainshock of magnitude M (for a total of N mainshocks) in southern California is shown in Figs. 5b (for large M ≥6) and 6a (for the full range M ≥m0). We ﬁt Eq. (1) to the data using the MLE method with the log- likelihood function LL(θ;X = {ni;i = 1,...,N}) = XN i=1 [niln[Ki(θ)] −Ki(θ) −ln(ni!)] (15) (15) for a Poisson process, representing the stochasticity of the count K of aftershocks produced by a mainshock at any given time. Inserting Eq. (1) in Eq. (15) yields Figure 5. Estimating the solid seismicity parameters from the spa- tial distribution of aftershocks: (a) spatial linear density distribution ρ(r) of aftershocks for the four largest strike-slip mainshocks in southern California (with ﬁrst-generation aftershocks only; the den- sity distribution comprising all aftershocks generated by the Lan- ders mainshock is represented by the dotted curve to illustrate the type of spatial heterogeneity, such as the Big Bear cluster, not con- sidered in the present study – see also Fig. 2c). The solid seismicity parameters r∗= 1 km and δ+(m0 = 2) = 1.23 event km−3 can be retrieved from the observed plateau ρ (r < r∗), in agreement with the SSP (see Fig. 4d). Note that the spatial power-law decay at high r is similar to the one expected by the SSP in the case of a static stress ﬁeld with additive uniform noise (expected q = 1.96 repre- sented by the dashed black lines); (b) aftershock productivity K for M > 6. The curves represent the productivity law as deﬁned by solid seismicity (Eq. 17) for different w0 values (ﬁrst term only cor- responds to w0 = 0; Eq. 18). Figure 5. Estimating the solid seismicity parameters from the spa- tial distribution of aftershocks: (a) spatial linear density distribution ρ(r) of aftershocks for the four largest strike-slip mainshocks in southern California (with ﬁrst-generation aftershocks only; the den- LL(θ = {K0,α};X) = ln(K0) XN i=1ni + α XN i=1 [ni (Mi −m0)] −K0 XN i=1exp[α (Mi −m0)] − XN i=1ln(ni!) (16) (16) (note that the last term can be set to 0 during LL maxi- mization). 3.3 Aftershock productivity law For southern California, we obtain αMLE = 2.32 (1.01 in log10 scale) and K0 = 0.025 when considering large (M ≥6) mainshocks only to avoid the issues of scaling break and data dispersion at lower magnitudes. This result, repre- sented by the black solid line in Fig. 5b, is in agreement with previous studies in the same region (e.g., Helmstetter, 2003; Helmstetter et al., 2005; Zaliapin and Ben-Zion, 2013; Seif et al., 2017) and with α = ln(10) ≈2.30 predicted for large mainshocks in solid seismicity (Eq. 12). Moreover we ﬁnd a bulk βMLE = 2.34 (1.02 in log10 scale) (Aki, 1965), in agree- ment with α ≡β. As for the plateau value ρ (r < r∗), it provides an estimate of the aftershock activation density δ+, with Let us now rewrite the solid seismicity aftershock produc- tivity law (Eq. 12) by only considering the large M case and injecting r∗= Fw0 (by combining Eqs. 7–8). We get δ+ = ρ (M,r < r∗) exp[ln(10)(M −4)] (14) (14) K (M > Mbreak) = δ+ n 2r∗exp[ln(10)(M −4)] + πr2 ∗w0 o . (17) (17) a volumetric density, i.e., the linear density ρ normalized by the mainshock rupture area (Eq. 4). Due to the ﬂuctuations in ρ (r < r∗), δ+ will be estimated from the productivity law instead (see Sect. 3.3), and ρ (r < r∗) will then be estimated from Eq. (14) (horizontal dashed colored lines), as detailed below. The role of w0 is illustrated in Fig. 5b for different values (dashed and dotted curves) and shown to be insigniﬁcant for large M values. Therefore Eq. (17) can be approximated to K (M > Mbreak) ≈2δ+r∗exp[ln(10)(M −4)]. (18) By analogy with Eq. (1), we get δ+ = K0exp[ln(10)(4 −m0)] 2r∗ . (19) (18) It should be noted that we consider only the ﬁrst- generation aftershocks to avoid ρ heterogeneities from sec- ondary aftershock clusters occurring off-fault. An example of such heterogeneity and anisotropy is illustrated by the Landers–Big Bear case (Fig. 2c; dotted colored curve in Fig. 5a). Those cases are not systematic and therefore not considered in the aftershock productivity law. However, they are also due to static stress changes (e.g., King et al., 1994) with the anisotropic effects explainable by solid seismic- (19) With r∗≈1 km estimated from ρ(r) (Sect. 3.2) and K0 = 0.025, we obtain δ+ = 1.23 events km−3 for m0 = 2. A. Mignan: Utsu aftershock productivity law explained from geometric operations A. Mignan: Utsu aftershock productivity law explained from geometric operation 246 Figure 5. Estimating the solid seismicity parameters from the spa- tial distribution of aftershocks: (a) spatial linear density distribution ρ(r) of aftershocks for the four largest strike-slip mainshocks in southern California (with ﬁrst-generation aftershocks only; the den- sity distribution comprising all aftershocks generated by the Lan- ders mainshock is represented by the dotted curve to illustrate the type of spatial heterogeneity, such as the Big Bear cluster, not con- sidered in the present study – see also Fig. 2c). The solid seismicity parameters r∗= 1 km and δ+(m0 = 2) = 1.23 event km−3 can be retrieved from the observed plateau ρ (r < r∗), in agreement with the SSP (see Fig. 4d). Note that the spatial power-law decay at high r is similar to the one expected by the SSP in the case of a static stress ﬁeld with additive uniform noise (expected q = 1.96 repre- sented by the dashed black lines); (b) aftershock productivity K for M > 6. The curves represent the productivity law as deﬁned by solid seismicity (Eq. 17) for different w0 values (ﬁrst term only cor- responds to w0 = 0; Eq. 18). ity through the concept of historical ghost static stress ﬁeld (Mignan, 2016a). ity through the concept of historical ghost static stress ﬁeld (Mignan, 2016a). ρ (r) ∝r−q, Processes Geophys., 25, 241–250, 2018 3.3 Aftershock productivity law We then get back the plateau ρ (r < r∗) for different M values from A. Mignan: Utsu aftershock productivity law explained from geometric operations Aftershock productivity deﬁned as the number of after-    λ(t,x,y) = µ(t,x,y) + P i:tj <tK(Mi) f (t −ti)g (x −xi,y −yi \|Mi ) f (t) = cp−1(p −1)(t + c)−p g (x,y \|M ) = 1 π deγ (M−m0)q−1 x2 + y2 + deγ (M−m0)−q(q −1). (21) (21) Aftershock sequences are deﬁned by power laws, both in time and space (for an alternative temporal function, see Mignan, 2015, 2016b; the spatial power-law distribution is in agreement with solid seismicity in the case of a hetero- geneous static stress ﬁeld – see Sect. 2.2). The southern California background seismicity, as deﬁned by the nearest- neighbor method (with same t, x, y and m), is denoted by µ. We ﬁx the ETAS parameters to θ = {c = 0.011 day, p = 1.08, d = 0.0019 km2, q = 1.47, γ = 2.01, β = 2.29, K0 = 0.08}, following the ﬁtting results of Seif et al. (2017) for the southern California relocated catalog and m0 = 2 (see their Table 1). However, we deﬁne the productivity function K(M) from Eq. (20) with Mbreak = 5. Examples of ETAS simulations are shown in Fig. 6b for comparison with the ob- served southern California time series. Figure 6c allows us to verify that the simulated aftershock productivity is kinked at Mbreak, as deﬁned by Eq. (20). Eq. (14), as shown in Fig. 5a (horizontal dashed colored lines). Although based on limited data, this result suggests that the activation parameter δ+ is constant (at least for large M) in southern California. Note that if ρ (r < r∗) was well constrained, it could have been estimated jointly with r∗from Fig. 5a to predict the aftershock productivity law of Fig. 5b without further ﬁtting required (hence removing K0 from the equation, K0 having no physical meaning in solid seismic- ity). We then select aftershocks from the ETAS simulations with the nearest-neighbor method. Figure 4d represents the estimated aftershock productivity, which has lost the break in scaling originally implemented in the simulations (with an underestimated αMLE = 2.07 as observed in the real case for M ≥m0). Note that a similar result is obtained when us- ing a windowing method (Gardner and Knopoff, 1974). A. Mignan: Utsu aftershock productivity law explained from geometric operations This demonstrates that the theoretical break in scaling predicted in the aftershock productivity law can be lost in observations due to an aftershock selection bias, all declustering tech- niques assuming continuity over the entire magnitude range. Nonlin. Processes Geophys., 25, 241–250, 2018 Nonlin. Processes Geophys., 25, 241–250, 2018 A. Mignan: Utsu aftershock productivity law explained from geometric operations A. Mignan: Utsu aftershock productivity law explained from geometric operations 247 Figure 6. Aftershock productivity deﬁned as the number of after- shocks K(m0 = 2) per mainshock of magnitude M: (a) observed aftershock productivity in southern California with aftershocks se- lected using the nearest-neighbor method; (b) seismicity time series with distinction made between background events and aftershocks, observed (“obs”, in black) and ETAS-simulated (“sim”, colored); (c) true simulated aftershock productivity with kink, deﬁned from Eq. (20); (d) retrieved simulated aftershock productivity with after- shocks selected using the nearest-neighbor method – data points in panels (a), (c) and (d) are represented by grey dots; the model MLE ﬁts are represented by the dashed and dotted black lines for M ≥6 and M ≥m0, respectively; dashed and dotted grey lines are visual guides to α = 3/2ln(10) and ln(10), respectively.    K0 exp[ln(10)(Mbreak −m0)] exp h 3 2ln(10)(Mbreak −m0) iexp 3 2ln(10)(M −m0) M ≤Mbreak K0exp[ln(10)(M −m0)], M > Mbreak M > Mbreak but with the best MLE result obtained for Mbreak = m0, sug- gesting no break in scaling in the aftershock productivity data, as observed in Fig. 6a. Final parameter estimates are αMLE = 1.95 (0.85 in log10 scale) and K0 = 0.141 for the full mainshock magnitude range M ≥m0 (dotted line), sub- ject to high scattering at low M values. We now identify whether the lack of break in scaling in aftershock productivity observed in earthquake catalogues could be an artefact related to the aftershock selection method. We run epidemic-type aftershock sequence (ETAS) simulations (Ogata, 1988; Ogata and Zhuang, 2006), with the seismicity rate Figure 6. Aftershock productivity deﬁned as the number of after- shocks K(m0 = 2) per mainshock of magnitude M: (a) observed aftershock productivity in southern California with aftershocks se- lected using the nearest-neighbor method; (b) seismicity time series with distinction made between background events and aftershocks, observed (“obs”, in black) and ETAS-simulated (“sim”, colored); (c) true simulated aftershock productivity with kink, deﬁned from Eq. (20); (d) retrieved simulated aftershock productivity with after- shocks selected using the nearest-neighbor method – data points in panels (a), (c) and (d) are represented by grey dots; the model MLE ﬁts are represented by the dashed and dotted black lines for M ≥6 and M ≥m0, respectively; dashed and dotted grey lines are visual guides to α = 3/2ln(10) and ln(10), respectively. Figure 6. 4 Role of aftershock selection on productivity scaling break We tested the following piecewise model to identify any break in scaling at smaller M, as predicted by Eq. (12): K (M) = (20) (20) K (M) = A. Mignan: Utsu aftershock productivity law explained from geometric operations 248 While such a bias is possible, it does not prove that the break in scaling exists. The fact that a similar break in scaling was obtained in independent Coulomb stress simulations (Hainzl et al., 2010), however, provides high conﬁdence in our re- sults. second-order stress behaviors might explain part of the scat- tering observed around Eq. (1) (Fig. 6a), such as overpressure due to trapped high-pressure gas for example (Miller et al., 2004 – see also Mignan, 2016a, for an overpressure ﬁeld due to ﬂuid injection). Other σ(r) formulations could be tested in the future, the only constraint on generating so-called seis- micity solids being the use of the postulated static stress step function of Eq. (5) (i.e., the solid seismicity postulate). One other possible explanation for the lack of scaling break is that our demonstration assumes moment magnitudes while the southern California catalogue is in local magni- tudes. Deichmann (2017) demonstrated that while ML ∝Mw at large M, ML ∝1.5Mw at smaller M values. This could in theory cancel the kink in real data. However, the scal- ing break predicted by Deichmann (2017) occurs at several magnitude units below the geometric scaling break expected by solid seismicity, invalidating this second option for mid- range magnitudes M. Finally, the disappearance of the predicted scaling break in the aftershock productivity law once declustering is applied (Fig. 6) indicates that more work is required in that domain. Only a declustering technique that does not dictate a constant scaling at all M will be able to identify whether a scaling break really exists or not. Data availability. The seismicity data used in the present study is published (see Sect. 3.1) and publicly available via the Southern California Earthquake Data Center. 5 Conclusions In the present study, a closed-form expression deﬁned from geometric and static stress parameters was proposed (Eq. 12) to describe the empirical Utsu aftershock productivity law (Eq. 1). This demonstration is similar to the previous ones made by the author to explain precursory accelerating seis- micity and induced seismicity (Mignan, 2012, 2016a). In all these demonstrations, the main physical parameters remain the same, i.e., the activation density δ+ (also δ−and δ0), the background stress amplitude range 1o∗and the solid en- velope r∗which describes the geometry of the “seismicity solid” (Fig. 3a–b). Further studies will be needed to evaluate whether the δ+ and 1o∗parameters are universal or region- speciﬁc and if the same values apply to different types of seismicity at a same location. Competing interests. The author declares that he has no conﬂict of interest. Acknowledgements. I thank Nadav Wetzler and two anonymous re- viewers, as well as editor Ilya Zaliapin, for their valuable comments. Acknowledgements. I thank Nadav Wetzler and two anonymous re- viewers, as well as editor Ilya Zaliapin, for their valuable comments. Edited by: Ilya Zaliapin Reviewed by: two anonymous referees Edited by: Ilya Zaliapin Reviewed by: two anonymous referees Edited by: Ilya Zaliapin Reviewed by: two anonymous referees K (M) = www.nonlin-processes-geophys.net/25/241/2018/ www.nonlin-processes-geophys.net/25/241/2018/ Nonlin. Processes Geophys., 25, 241–250, 2018 A. Mignan: Utsu aftershock productivity law explained from geometric operations 249 Mignan, A.: Modeling aftershocks as a stretched expo- nential relaxation, Geophys. Res. Lett., 42, 9726–9732, https://doi.org/10.1002/2015GL066232, 2015. Felzer, K. R. and Brodsky, E. E.: Decay of aftershock density with distance indicates triggering by dynamic stress, Nature, 441, 735–738, https://doi.org/10.1038/nature04799, 2006. Gardner, J. K. and Knopoff, L.: Is the sequence of earthquakes in Southern California, with aftershocks removed, Poissonian?, B. Seismol. Soc. Am., 64, 1363–1367, 1974. Mignan, A.: Static behaviour of induced seismicity, Nonlin. 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https://openalex.org/W4320063092	https://journal.formosapublisher.org/index.php/ajae/article/download/1414/1145	Indonesian	null	Praktikalitas Pengembangan Modul PPKn Berbasis Contextual Teaching Learning untuk Siswa Kelas V SDN 101835 Sibolangit	Asian Journal of Applied Education	2,022	cc-by	3,044	Asian Journal of Applied Education (AJAE) Vol. 1, No. 1, 2022: 33-42 A B S T R A K A R T I C L E I N F O Kata Kunci: Praktikalitas, Modul, PPKn, Sekolah Dasar Received : 05 September Revised : 04 Oktober Accepted: 10 Oktober ©2022 Bukit, Perangin-angin, Murad: This is an open-access article distributed under the terms of the Creative Commons Atribusi 4.0 Internasional. A B S T R A K Penelitian bertujuan menganalisis tingkat praktikalitas modul PPKn berbasis Contextual Teaching Learning. Modul PPKn berbasis CTL adalah bahan ajar mandiri yang dikembangkan berdasarkan komponen-komponen pendekatan kontekstual, yaitu konstruktivisme, bertanya, menemukan, masyarakat belajar, pemodelan, refleksi, dan penilaian autentik. Pengembangan modul PPKn berbasis CTL dengan model 4D yang oleh Thiagarajan dalam empat tahap: Definition, Design, Development, dan Disseminate. Modul PPKn berbasis CTL diperuntukkan bagi siswa kelas 5 SD. Penelitian ini menggunakan metode analisis. Data dikumpulkan dari hasil angket respon 1 orang guru kelas 5 SDN 101835 Sibolangit dan 25 orang siswa kelas 5 SDN 101835 Sibolangit terhadap kepraktisan penggunaan modul. Hasil penelitian menunjukkan tingkat kepraktisan modul PPKn oleh guru 90% kategori sangat Praktis dan kepraktisan oleh siswa 83,13% kategori Sangat Praktis. A R T I C L E I N F O Kata Kunci: Praktikalitas, Modul, PPKn, Sekolah Dasar Received : 05 September Revised : 04 Oktober Accepted: 10 Oktober ©2022 Bukit, Perangin-angin, Murad: This is an open-access article distributed under the terms of the Creative Commons Atribusi 4.0 Internasional. A R T I C L E I N F O Kata Kunci: Praktikalitas, Modul, PPKn, Sekolah Dasar Received : 05 September Revised : 04 Oktober Accepted: 10 Oktober ©2022 Bukit, Perangin-angin, Murad: This is an open-access article distributed under the terms of the Creative Commons Atribusi 4.0 Internasional. Kata Kunci: Praktikalitas, Modul, PPKn, Sekolah Dasar ( DOI prefik: 10.55927 https://journal.formosapublisher.org/index.php/ajae 33 Bukit, Perangin-angin, Murad PENDAHULUAN Guru dan siswa adalah komponen aktif dalam proses pembelajaran di sekolah. Guru selaku pendidik memliki peranan penting untuk merancang pembelajaran, melaksanakan pembelajaran, dan melakukan evaluasi terhadap proses pembelajaran. Dalam merancang pembelajaran, guru harus menyusun rencana pelaksananaan pembelajaran (RPP) sesuai yang memuat komponen inti yaitu tujuan pembelajaran, langkah-langkah pembelajaran, dan penilaian pembelajaran. Dalam pelaksanaan pembelajaran pembelajaran guru harus memiliki sumber belajar yang praktis untuk digunakan. Guru, siswa, dan sumber belajar akan membentuk interaksi aktif selama pembelajaran berlangsung. Sumber belajar adalah semua sumber baik dalam bentuk data, orang, metode, media, tempat berlangsungnya pembelajaran, yang digunakan untuk memudahkan siswa dalam belajar (Samsinar, 2019). Dengan adanya sumber belajar yang mampu memudahkan siswa dalam belajar, maka proses pembelajaran akan lebih menarik dan menyenangkan. Sebagai pendidik, guru harus merancang proses pembelajaran yang menarik dan menyenangkan bagi seluruh siswa agar tercapai tujuan pendidikan nasional (Bukit et al, 2022). Kemampuan guru tersebut diatas merupakan salah satu kompetensi pedagogik yang harus ada pada guru. Dengan demikian, guru perlu membuat sumber belajar yang praktis untuk digunakan oleh siswa dalam pencapaian kompetensi dasar. Salah satu sumber belajar yang perlu dikembangkan oleh guru adalah bahan ajar. Guru harus mengembangkan bahan ajar untuk memfasilitasi siswa dalam proses pembelajaran. Hal ini sesuai dengan UU Nomor 14 tahun 2005 tentang guru dan dosen Pasal 20, diisyaratkan bahwa guru diharapkan dapat mengembangkan materi pembelajaran. Bahan ajar memiliki peranan sebagai sebagai materi pembelajaran selama pembelajaran berlangsung (Nisa, 2019). Sementara Kosasih (2021, p. 1) menyatakan bahwa bahan ajar adalah segala sesuatu yang digunakan guru atau peserta didik untuk memudahkan proses pembelajaran. Salah satu bahan ajar yang dapat dikembangkan guru adalah modul. Modul adalah media pembelajaran yang dapat digunakan siswa sebagai sumber belajar dalam pembelajaran (Rikizaputra et al, 2021). Demikian halnya menurut (Negara et al., 2019) modul merupakan salah satu bentuk bahan ajar yang sistematis untuk membantu siswa menguasai tujuan belajar. Sehingga modul bisa dimanfaatkan secara mandiri oleh siswa (Permatasari & Yerimadesi, 2020). Dari hasil wawancara dengan kepala SDN 101835 Sibolangit diperoleh informasi bahwa guru belum pernah mengembangkan bahan ajar seperti modul. Dalam proses pembelajaran, guru hanya memanfaatkan buku tematik guru dan siswa. Sekolah belum menyediakan buku referensi pelajaran bagi guru dan siswa. Sekolah hanya memiliki beberapa buku teks bacaan yang ada di perpustakaan namun belum tentu berkaitan dengan materi pembelajaran. 34 Asian Journal of Applied Education (AJAE) Vol. 1, No. PENDAHULUAN 1, 2022: 33-42 Sehinga dapat disimpulkan sekolah belum mampu memfasilitasi sumber belajar lain yang berkaitan bagi siswa dan guru seperti modul pembelajaran. Padalah dengan adanya modul pembelajaran dapat membantu siswa lebih mandiri dalam mengerjakan tugas karena sudah dilengkapi dengan petunjuk belajar dan kegiatan belajar di dalamnya. Dari hasil wawancara dengan guru diperoleh informasi bahwa guru belum pernah mengembangkan bahan ajar seperti modul. Guru hanya pernah menyusun lembar kerja peserta didik (LKPD) seperti yang dilaksanakan dalam kegiatan kelompok kerja guru (KKG). Sehingga guru belum mengetahui sistematika penyusunan modul pembelajaran. Selama ini guru masih menggunakan buku tematik saja dalam pembelajaran baik dalam memberikan tugas maupaun teks-teks pelajaran. Demikian halnya untuk muatan pelajaran PPKn. PPKn sebagai muatan pelajaran wajib di sekolah dasar memiliki peranan dalam pembentukan potensi siswa menjadi manusia yang beriman dan bertaqwa kepada Tuhan, sehat, berilmu, berakhlak mulia, kreatif, mandiri, dan menjadi warga Negara yang demokratis. Melalui pembelajaran PPKn diharapkan dapat membentuk pribadi siswa yang mandiri, kreatif, cerdas, dan bertaqwa kepada Tuhan dalam menghadapi kehidupan sosial (Parhan & Sukaenah, 2020). Salah satu sikap yang perlu dibentuk dalam diri siswa adalah mandiri. Siswa yang mandiri akan terlihat menjadi pribadi yang memiliki rasa ingin tahu, tanggung jawab terhdap tugas, memiliki sikap kerja dan tidak bergantung pada orang lain. Oleh karena itu melalui penggunaan modul pembelajaran diharapkan mampu menumbuhkan kemandirian belajar siswa. Dari hasil wawancara dengan siswa kelas V SDN 101835 Sibolangit diperoleh informasi bahwa jika hanya menggunakan buku tematik, siswa tidak mampu membedakan materi PPKn dengan muatan pelajaran lainnya. Demikian juga dengan membedakan tugas-tugas PPKn dengan tugas muatan pelajarn lainnya. Sehingga pada akhirnya, siswa merasa kesulitan dalam pembelajaran PPKn. Beranjak dari situasi ini, sebagian siswa mulai mencari sumber belajar lain terkait materi pelajaran PPKn baik di buku-buku perpustakaan ataupaun di buku-buku KTSP PPKn yang ada mereka peroleh dari teman-temannya. Hal ini menunjukkan bahwa, siswa kelas V SDN 101835 memiliki inisiatif untuk mencari sumber belajar yang dapat membantu mereka dalam belajar. Berdasarkan situasi ini menujukkan bahwa siswa membutuhkan sumber belajar baru untuk menambah semangat belajar mereka. Oleh karena itu, dibutuhkan upaya untuk melengkapi kebutuhan bahan ajar siswa dengan mengembangkan modul pembelajaran khususnya muatan pelajaran PPKn. Dengan penggunaan modul pembelajaran dapat memudahkan siswa dalam memahami materi pelajaran (Diani, 2015). Hal ini dikarenakan materi-materi 35 Bukit, Perangin-angin, Murad pembelajaran yang disajikan berurut dan lengkap dan mudah menggunakannya (Danuri, 2014). PENDAHULUAN Pengembangan modul PPKn yang telah disusun oleh peneliti untuk melengkapi kebutuhan bahan ajar siswa kelas V SDN 101835 Sibolangit berbasis CTL. Kemudian disebut dengan Modul PPKn berbasis CTL untuk Siswa Kelas V SD. Pembelajaran yang dikaitkan dengan kehidupan nyata siswa akan mendorong siswa berpikir tentang peristiwa yang terjadi dalam kehidupan dan memahaminya dengan baik (Novianska et al., 2021). Bahkan dengan pengembangan modul PPKn berbasis CTL akan membantu siswa memperoleh pengetahuan dan keterampilan untuk mengkontruksikan diri, sehingga mampu untuk memecahkan permasalahan di dalam kehidupan sosial (Riska & Rahmawati, 2022). Modul PPKn berbasis CTL yang dikembangkan sudah dinyatakan sangat layak oleh ahli materi, ahli desain pembelajaran, dan ahli bahasa memiliki keunggulan dalam kelengkapan materi pelajaran, desain modul yang berwarna sesuai dengan kebutuhan siswa sekolah dasar, dan tata bahasa yang baik dan mudah dimengerti oleh siswa sekolah dasar. Berikut ini adalah tabel hasil kelayakan modul PPKn berbasis CTL oleh tim ahli: Tabel. 1 Hasil Penilaian Kelayakan Modul PPKn berbasis CTL untuk Sisw Tabel. 1 Hasil Penilaian Kelayakan Modul PPKn berbasis CTL untuk Siswa y Kelas V SDN 101835 Sibolangit oleh Tim Ahli Validator/ Ahli Persentase Kriteria Ahli Materi 90,63% Sangat Layak Ahli Desain 93,75% Sangat Layak Ahli Bahasa 96,88% Sangat Layak Rerata 93,75% Sangat Layak Kelas V SDN 101835 Sibolangit oleh Tim Ahli g y Berdasarkan hasil penilaian kelayakan oleh tim ahli, maka dibutuhkan analasis tingkat praktikalisasi modul PPKn berbasis CTL ini untuk digunakan oleh siswa kelas V SD dan guru kelas V SD. Praktikalitas merupakan kemudahan produk yang dihasilkan pada saat digunakan (Rikizaputra, 2021). Demikian juga menurut Hamdunah (2015) menjelaskan praktikalitas merupakan tingkat keterpakaian perangkat pembelajaran dengan melakukan uji coba menggunakan modul. Oleh karena itu tujuan penelitian ini adalah untuk menganalsis tingkat praktikalisasi modul PPKn berbasis CTL yang telah dinyatakan sangat layak oleh ahli materi, ahli desain pembelajaran, dan ahli bahasa untuk digunakan oleh siswa dan guru. METODE Metode penelitian ini adalah Research and Development (R&D). Penelitian R & D merupakan penelitian untuk menghasilkan produk yang layak dan praktis untuk digunakan (Saputro, 2017, p. 7). Model pengembangan modul ini adalah 4D yang disarankan oleh Sivasailam Thiagarajan, Dorothy S, 36 Asian Journal of Applied Education (AJAE) Vol. 1, No. 1, 2022: 33-42 Asian Journal of Applied Education (AJAE) Vol. 1, No. 1, 2022: 33-42 Semmel & Melvyn (1974, p. 5). Terdapat empat tahapan pengembangan pada model 4D ini yaitu Definisi, Desain, Develop, dan Disseminate. Alat pengumpul data penelitian adalah wawancara tak berstruktur dan angket respon guru dan siswa terhadap kepraktisan modul PPKn berbasis CTL. Wawancara tak berstruktur merupakan proses wawancara bebas di mana peneliti tidak menggunakan pedoman wawancara yang tersusun secara sistematis untuk pengumpulan datanya (Sugiyono, 2013, p. 233). Wawancara dilakukan kepada kepala sekolah, guru kelas V SD, dan siswa kelas V SDN 101835 Sibolangit. Angket respon guru dan siswa merupakan angket yang memuat butir-butir penilaian terhadap modul dijadikan sebagai alat pengumpul data penelitian ini. Angket akan diisi oleh 1 orang guru kelas V SD dan 25 oran siswa kelas V SDN 101835 Sibolangit sebagai subjek dalam penelitian ini. Teknik analisis data secara deskriptif kuantitatif dengan menghitung persentase indikator untuk setiap butir respon terhadap modul yang berdasarkan rumus berikut: Hasil persentase indikator kemudian ditafsirkan dengan kalimat kualitatif seperti pada tabel dibawah ini: Hasil persentase indikator kemudian ditafsirkan dengan kalimat kualitatif seperti pada tabel dibawah ini: Tabel. 2 Kriteria Praktikalitas Modul oleh Siswa dan Guru Persentase Skor Kategori 81 % - 100 % Sangat Praktis 61 % - 80 % Praktis 41 % - 60 % Cukup Praktis 21 % - 40 % Kurang Praktis 0 % - 20 % Tidak Praktis Sumber : (Sugiyono, 2013, p. 118) Tabel. 2 Kriteria Praktikalitas Modul oleh Siswa dan Guru Hasil Penelitian Berikut ini adalah tabel respons guru kelas V SDN 101835 Sibolangit terhadap kepraktisan penggunaan modul PPKn berbasis CTL: Tabel 3. Hasil Respon Guru terhadap Kepraktisan Penggunaan Modul PPKn berbasis CTL Tabel 3. Hasil Respon Guru terhadap Kepraktisan Penggunaan Modul PPKn berbasis CTL Butir Penilaian SKOR Ketepatan Bahasa 3 Warna unsur tata letak harmonis dan memperjelas fungsi 3 Kelengkapan Materi 4 37 Bukit, Perangin-angin, Murad Keakuratan konsep dan definisi 4 Mendorong rasa ingin tahu 4 Jumlah 18 Persentase 90% Kategori Sangat Praktis Dari hasil respon guru terhadap tingkat kepraktisan penggunaan modul PPKn berbasis CTL untuk siswa kelas V SDN 101835 Sibolangit diperoleh persentase 90% dengan kategori sangat praktis untuk digunakan dalam proses pembelajaran PPKn. Dengan penggunaan modul ini dapat memfasilitasi guru dalam melengkapi sumber belajar lainnya bagi seluruh siswa keas V SDN 101835 Sibolangit. Berikut ini adalah tabel hasil respon siswa kelas V SDN 101835 Sibolangit terhadap kepraktisan penggunaan modul PPKn berbasis CTL: Tabel 4. Hasil Respon Siswa terhadap Kepraktisan Penggunaan Modul PPKn berbasis CTL Tabel 4. Hasil Respon Siswa terhadap Kepraktisan Penggunaan Modul PP Tabel 4. Hasil Respon Siswa terhadap Kepraktisan Penggunaan Modul PPKn Tabel 4. Hasil Respon Siswa terhadap Kepraktisan Penggunaan Modul PPKn berbasis CTL Butir Penilaian Indikator Jumlah Skor Kriteria Desain Modul a. Tampilan modul menarik 83,33 Sangat Baik b. Gambar pada modul menarik 86,67 Sangat Baik c. Warna modul menarik 75 Baik Isi Modul d. Materi modul dapat dimengerti 80 Baik e. Tugas terdapat pada modul 85 Sangat Baik f. Tugas modul dapat dikerjakan 83,33 Sangat Baik Manfaat Modul g.Menggunakan modul membuat saya ingin tahu terhadap materi pelajaran 83,33 Sangat Baik h.Menggunakan modul membuat saya percaya diri mengerjakan tugas 88,33 Sangat Baik Persentase Klasikal 83.13% Kriteria Persentase Sangat Praktis Tabel 4. Hasil Respon Siswa terhadap Kepraktisan Penggunaan Modul PPKn berbasis CTL Pembahasan Respon Guru Terhadap Kepraktisan Penggunan Modul PPKn berbasis CTL Guru memberikan respon berupa penilaian kepraktisan penggunaan modul PPKn berbasis CTL. Guru memberikan hasil penilaian terhadap modul PPKn berbasis CTL bahwa pada dalam hal tata bahasa yang digunakan sudah sesuai dengan tingkat perkembangan siswa kelas V SD. Bahasa yang digunakan mudah dimengerti siswa dan sudah sesuai dengan ejaan bahasa Indonesia yang 38 baik dan benar. Hal ini sejalan dengan pendapat Arum (2016) bahwa bahasa yang digunakan pada modul perlu disesuaikan dengan bahasa siswa sekolah dasar. Dalam hal kejelasan warna yang digunakan pada modul sudah dipadukan dengan warna yang cerah, sehingga menarik bagi siswa untuk menggunakannya. Bahkan dalam materi pelajaran sudah membuat stimulus untuk merangsang rasa ingin tahu siswa terhadap materi pelajaran yang disajikan. Siswa dengan rasa ingin tahu yang tinggi akan menunjukkan kemauan dari dalam diri untuk mencari sesuatu hal yang baru yang belum didapatkan dari pembelajaran melalui berbagai sumber yang tersedia (Nehru & Irianti, 2020). Misalnya. materi pelajaran dikaitkan dengan kehidupan nyata siswa. Pembelajaran yang mengaitkan materi pelajaran dengan kahidupan nyata menunjukkan adanya pendekatan pembelajaran berbasis kontekstual (Komalasari, 2015, p. 46). Sebagai contoh siswa diminta untuk menunjukkan sikap-sikap dalam menjaga kebersihan lingkungan rumah dan lingkungan sekolah melalui bermain peran seperti yang terdapat pada modul PPKN berbasis CTL. Bahkan terdapat materi yang berkaitan dengan rasa bangga terhadap penggunaan bahasa Indonesia dalam kehidupan sehari-hari. Melalui pendekatan CTL pada modul ini, siswa akan berpikir dan mengeksplor materi dengan apa yang ada pada kehidupannya (El-Majid dalam Bukit, 2022). Hasil respon guru menunjukkan tingkat kepraktisan sangat praktis dengan persentase 90%. Sehingga modul PPKn berbasis CTL sudah layak digunakan guru sebagai sumber belajar yang baru bagi siswa. Respon Siswa Terhadap Kepraktisan Penggunan Modul PPKn berbasis CTL Siswa memberikan respon berupa kepraktisan penggunaan modul PPKn berbasis CTL. Guru memberikan penilaian bahwa modul PPKn berbasis CTL memiliki warna yang menarik. Karena ada warna cerah untuk dilihat. Bahkan dilengkapi dengan gambar-gambar yang ada hubungannya dengan kehidupan sehari-hari mereka. Seperti gambar siswa yang mmbuat tulisan bangga menggunakan bahasa Indonesia. Siswa merasa senang terhdap penggunaan modul karena tugas-tugas dilengkapi dengan petunjuk pengerjaan. Materi PPKn di dalam modul mudah ditemukan dan dipahamai. Siswa juga menyatakan bahwa tugas-tugas yang ada dapat dikerjakan dengan mudah karena teks bacaan berhubungan dengan tugas. Sehingga siswa memiliki rasa ingin tahu yang lebih untuk membaca modul. Dan pada akhirnya siswa dapat bekerja keras untuk mengerjakan tugas tanpa bergantung pada orang lain. Pembahasan Hal ini sejalan dengan pendapat (Yuniarti & YL Sukestiyarno, 2020) dengan adanya sikap kerja keras dalam diri siswa menunjukkan adanya 39 Bukit, Perangin-angin, Murad kesungguhan dalam mengatasi berbagai kesulitan belajar dan menyelesaikan tugas dengan sebaik-baiknya. kesungguhan dalam mengatasi berbagai kesulitan belajar dan menyelesaikan tugas dengan sebaik-baiknya. KESIMPULAN Berdasarkan hasil respon guru diperoleh tingkat kepraktisan penggunaan modul PPKn berbasis CTL adalah sangat praktis dengan persentase 90%. Sementara hasi respon siswa terhadap kepraktisan penggunaan modul PPKn berbasis CTL menunjukkan tingkat kepraktisan sebesar 83,13% dengan kategori sangat praktis. 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https://openalex.org/W3137230333	https://peerj.com/articles/10706v0.3/submission	English	null	Peer Review #1 of "Myofascial release versus Mulligan sustained natural apophyseal glides’ immediate and short-term effects on pain, function, and mobility in non-specific low back pain (v0.2)"	null	2,021	cc-by	11,463	Myofascial release versus Mulligan sustained natural apophyseal glides' immediate and short-term eﬀects on pain, function, and mobility in non-speciﬁc low back pain Vignesh Bhat P 1 , Vivek Dineshbhai Patel Corresp., 1 , Charu Eapen 1 , Manisha Shenoy 2 , Steve Milanese 3 1 Department of Physiotherapy, Kasturba Medical College, Manipal Academy of Higher Education, Mangalore, Karnataka, India 2 Department of Physical Therapy, Hamad Medical Corporation, Doha, Qatar 3 International Centre for Allied Health Evidence, University of South Australia, Adelaide, South Australia, Australia Corresponding Author: Vivek Dineshbhai Patel Email address: vivek.patel@manipal.edu 1 Department of Physiotherapy, Kasturba Medical College, Manipal Academy of Higher Education, Mangalore, Karnataka, India 2 Department of Physical Therapy, Hamad Medical Corporation, Doha, Qatar 3 International Centre for Allied Health Evidence, University of South Australia, Adelaide, South Australia, Australia Corresponding Author: Vivek Dineshbhai Patel Email address: vivek.patel@manipal.edu 1 Department of Physiotherapy, Kasturba Medical College, Manipal Academy of Higher Education, Mangalore, Karnataka, I 2 Department of Physical Therapy, Hamad Medical Corporation, Doha, Qatar 3 International Centre for Allied Health Evidence, University of South Australia, Adelaide, South Australia, Australia Corresponding Author: Vivek Dineshbhai Patel E il dd i k t l@ i l d 3 International Centre for Allied Health Evidence, University of South Australia, Adelaide, South Australia, Australia Corresponding Author: Vivek Dineshbhai Patel Email address: vivek.patel@manipal.edu Corresponding Author: Vivek Dineshbhai Patel Email address: vivek.patel@manipal.edu Background: Myofascial release (MFR) and Mulligan Sustained Natural Apophyseal Glides (SNAGs) are manual therapy techniques routinely practiced in the management of non-speciﬁc low back pain (NSLBP)., As a solo intervention or along with other therapies, both methods have reported positive results for individuals with NSLBP. However, which technique improves NSLBP-related pain, restricted range of motion (ROM) and disability, warrants further research. Objective: To study the comparative eﬀects of MFR and SNAGs on pain, disability, functional ability, and lumbar ROM in NSLBP. Method: A parallel-group study was conducted at tertiary care hospitals. Sixty-ﬁve Sub-acute or chronic NSLBP patients were allocated to receive strengthening exercises along with either MFR (n=33) or SNAGs (n=32) for six treatment sessions over one week. An independent assessor evaluated outcome measures such as the Visual Analog Scale (VAS), Patient-Speciﬁc Function Scale (PSFS), and ROM at baseline, immediate (after 1 st treatment), and short-term (post-sixth day of the intervention). The Modiﬁed Oswestry disability index (MODI) was assessed at baseline and short-term. Results: Within-group analysis found clinically and statistically signiﬁcant (p<0.05) changes for VAS and PSFS at immediate and short-term for both the groups. The lumbar extension also showed improvement immediately and in the short-term. Improvement in Lumbar ﬂexion was seen only in the SNAGs group over the short-term. Manuscript to be reviewed Myofascial release versus Mulligan sustained natural apophyseal glides' immediate and short-term eﬀects on pain, function, and mobility in non-speciﬁc low back pain Vignesh Bhat P 1 , Vivek Dineshbhai Patel Corresp., 1 , Charu Eapen 1 , Manisha Shenoy 2 , Steve Milanese 3 A statistically signiﬁcant improvement was seen for MODI in both the groups but was not clinically signiﬁcant in the MFR group. The analysis observed no statistically signiﬁcant diﬀerence (p > 0.05) between the groups at both the immediate and short-term. Conclusions: Pain and restricted function associated with NSLBP can be improved using SNAGs or MFR, along with strengthening exercises. For limited lumbar ﬂexion ROM, Mulligan SNAGs have a better outcome than MFR over the short-term. Hence, both manual therapy techniques can be incorporated along with exercises for immediate and short-term management of sub-acute to chronic NSLBP. [(https://ctri.nic.in) number- CTRI/2018/12/016787] PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) Manuscript to be reviewed 1 Myofascial release versus Mulligan sustained natural apophyseal glides' immediate and 2 short-term effects on pain, function, and mobility in non-specific low back pain. 2 short-term effects on pain, function, and mobility in non-specific low back pain. 3 Vignesh Bhat P1, Vivek D Patel2, Dr. Charu Eapen3, Manisha P Shenoy4, Steve Milanese5 4 1Post graduate, Department of Physiotherapy, Kasturba Medical College, Manipal Academy o 5 Higher Education, Mangalore 6 2Assistant Professor, Department of Physiotherapy, Kasturba Medical College, Manipal 7 Academy of Higher Education, Mangalore 8 3 Professor and HOD, Department of Physiotherapy, Kasturba Medical College, Manipal 9 Academy of Higher Education, Mangalore, 10 4Physiotherapy specialist, Hamad Medical Corporations, Doha, Qatar 11 5Associate Professor, International Centre for Allied Health Evidence, University of South 12 Australia, Adelaide, SA, Australia 13 14 Corresponding Author 15 Mr. Vivek D Patel 16 Assistant Professor, Department of Physiotherapy, Kasturba Medical College, Manipal Acade 17 of Higher Education, Mangalore 18 Tel +91-8050338884 19 vivek.patel@manipal.edu 20 PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) Bhat P1, Vivek D Patel2, Dr. Charu Eapen3, Manisha P Shenoy4, Steve Milanese5 4 1Post graduate, Department of Physiotherapy, Kasturba Medical College, Manipal Academy of 5 Higher Education, Mangalore 6 2Assistant Professor, Department of Physiotherapy, Kasturba Medical College, Manipal 7 Academy of Higher Education, Mangalore 6 2Assistant Professor, Department of Physiotherapy, Kasturba Medical College, Manipal 7 Academy of Higher Education, Mangalore PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) Manuscript to be reviewed 21 22 Abstract 23 Background: Myofascial release (MFR) and Mulligan Sustained Natural Apophyseal Glides 24 (SNAGs) are manual therapy techniques routinely practiced in the management of non-specific 25 low back pain (NSLBP)., As a solo intervention or along with other therapies, both methods have 26 reported positive results for individuals with NSLBP. However, which technique improves 27 NSLBP-related pain, restricted range of motion (ROM) and disability, warrants further research. 28 Objective: To study the comparative effects of MFR and SNAGs on pain, disability, functional 29 ability, and lumbar ROM in NSLBP. PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) 22 Abstract 23 Background: Myofascial release (MFR) and Mulligan Sustained Natural Apophyseal Glides 24 (SNAGs) are manual therapy techniques routinely practiced in the management of non-specific 25 low back pain (NSLBP)., As a solo intervention or along with other therapies, both methods have 26 reported positive results for individuals with NSLBP. However, which technique improves 27 NSLBP-related pain, restricted range of motion (ROM) and disability, warrants further research. 28 Objective: To study the comparative effects of MFR and SNAGs on pain, disability, functional 29 ability, and lumbar ROM in NSLBP. 30 Method: A parallel-group study was conducted at tertiary care hospitals. Sixty-five Sub-acute 31 or chronic NSLBP patients were allocated to receive strengthening exercises along with either 32 MFR (n=33) or SNAGs (n=32) for six treatment sessions over one week. An independent 33 assessor evaluated outcome measures such as the Visual Analog Scale (VAS), Patient-Specific 34 Function Scale (PSFS), and ROM at baseline, immediate (after 1st treatment), and short-term 35 (post-sixth day of the intervention). The Modified Oswestry disability index (MODI) was 36 assessed at baseline and short-term. 37 Results: Within-group analysis found clinically and statistically significant (p<0.05) changes 38 for VAS and PSFS at immediate and short-term for both the groups. The lumbar extension also 39 showed improvement immediately and in the short-term. Improvement in Lumbar flexion was 40 seen only in the SNAGs group over the short-term. A statistically significant improvement was 41 seen for MODI in both the groups but was not clinically significant in the MFR group. The PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) Manuscript to be reviewed 42 analysis observed no statistically significant difference (p > 0.05) between the groups at both the 43 immediate and short-term. 42 analysis observed no statistically significant difference (p > 0.05) between the groups at both the 43 immediate and short-term. 42 analysis observed no statistically significant difference (p > 0.05) between the groups at both the 43 immediate and short-term. 43 immediate and short-term. 44 Conclusions: Pain and restricted function associated with NSLBP can be improved using 45 SNAGs or MFR, along with strengthening exercises. For limited lumbar flexion ROM, Mulligan 46 SNAGs have a better outcome than MFR over the short-term. Hence, both manual therapy 47 techniques can be incorporated along with exercises for immediate and short-term management 48 of sub-acute to chronic NSLBP. 49 [(https://ctri.nic.in) number- CTRI/2018/12/016787] 50 51 Keywords: Mulligan SNAGs; Myofascial release; Non-specific low back pain; strengthening 52 exercises 53 54 Implications for practice 55 - Manual therapy techniques such as MFR and SNAGs can be considered for short-term 56 management of NSLBP related pain, disability, and functional improvement. 57 - Restricted sagittal plane lumbar ROM can be addressed better with the Mulligan SNAGs 58 technique than the MFR technique in NSLBP. 59 60 Introduction 44 Conclusions: Pain and restricted function associated with NSLBP can be improved using 45 SNAGs or MFR, along with strengthening exercises. For limited lumbar flexion ROM, Mulligan 46 SNAGs have a better outcome than MFR over the short-term. Hence, both manual therapy 47 techniques can be incorporated along with exercises for immediate and short-term management 48 of sub-acute to chronic NSLBP. 44 Conclusions: Pain and restricted function associated with NSLBP can be improved using 45 SNAGs or MFR, along with strengthening exercises. For limited lumbar flexion ROM, Mulligan 46 SNAGs have a better outcome than MFR over the short-term. Hence, both manual therapy 47 techniques can be incorporated along with exercises for immediate and short-term management 48 of sub-acute to chronic NSLBP. 49 [(https://ctri.nic.in) number- CTRI/2018/12/016787] 55 - Manual therapy techniques such as MFR and SNAGs can be considered for short-term 56 management of NSLBP related pain, disability, and functional improvement. 57 - Restricted sagittal plane lumbar ROM can be addressed better with the Mulligan SNAGs 58 technique than the MFR technique in NSLBP. 57 - Restricted sagittal plane lumbar ROM can be addressed better with the Mulligan SNAGs 58 technique than the MFR technique in NSLBP. 57 - Restricted sagittal plane lumbar ROM can be addressed better with the Mulligan SNAGs 58 technique than the MFR technique in NSLBP. PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) Manuscript to be reviewed 61 Low back pain (LBP) is a debilitating health condition, ranked first in musculoskeletal disease 62 burden worldwide[1,2]. It is reported to have an 18.3% mean point prevalence and 30.8% one- 63 month prevalence.[3] According to the Global Burden of Disease study, LBP emerged as a 64 primary cause for years lived with disability (YLD) for all age groups in both sexes.[1] From 65 1990 to 2007, YLDs due to LBP increased by 30%, with a further increase of 17% in the last 66 decade.[1] 67 Approximately 10% of LBP cases have an identifiable pathology, while the remaining 90% are 68 non-specific LBP (NSLBP), reflecting LBP of unknown underlying pathology, characterized by 69 pain, muscle tension, and stiffness between 12th rib and inferior gluteal fold.[3]. Based on 70 duration, LBP can be categorized as acute (less than six weeks), sub-acute (six to twelve weeks), 71 and chronic (more than twelve weeks).[4] 67 Approximately 10% of LBP cases have an identifiable pathology, while the remaining 90% are 68 non-specific LBP (NSLBP), reflecting LBP of unknown underlying pathology, characterized by 69 pain, muscle tension, and stiffness between 12th rib and inferior gluteal fold.[3]. Based on 70 duration, LBP can be categorized as acute (less than six weeks), sub-acute (six to twelve weeks), 71 and chronic (more than twelve weeks).[4] 72 One proposed mechanism underpinning NSLBP involves changes in lumbosacral proprioception 73 and core muscle recruitment patterns due to atrophy of the lumbar stabilizers[5] and gluteus 74 maximus[6] along with other hip muscles weakness.[7] The gradual decrease in motor control 75 leads to uncontrolled and abnormal tissue loading on the myofascial complex,[8,9] stressing the 76 lumbar spine leading to pain.[5] 77 The primary line of management for NSLBP includes analgesics and physical therapy 78 interventions[3,10]. The routine physical therapy interventions are transcutaneous electrical 79 nerve stimulation, low-level LASER therapy, manual therapy,[11] back schools, exercise, and 80 timely review.[10,11] Despite the range of interventions available, NSLBP leads to chronic loss 81 of health by limiting activity participation and loss of function, potentially resulting in prolonged 82 work disability.[4] Manual therapies such as Mulligan mobilization,[12–18] McKenzie PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) Manuscript to be reviewed 83 exercises,[14] Maitland mobilization,[15,16], and Myofascial release therapy (MFR)[8,9,19–22] 84 are used routinely in clinical practice for the management of NSLBP. 83 exercises,[14] Maitland mobilization,[15,16], and Myofascial release therapy (MFR)[8,9,19–22] 84 are used routinely in clinical practice for the management of NSLBP. 83 exercises,[14] Maitland mobilization,[15,16], and Myofascial release therapy (MFR)[8,9,19–22] 84 are used routinely in clinical practice for the management of NSLBP. 84 are used routinely in clinical practice for the management of NSLBP. 85 The Mulligan concept is based on the theory that minor position faults of articulating joints' 86 surfaces following injury or strain result in a painful and restricted, range of motion 87 (ROM).[11,12] Mulligan Sustained Natural Apophyseal Glides (SNAGs) technique adds a 88 passive accessory glide, parallel to the joint plane using a vertebral spinous process or transverse 89 process, during which the patient performs the previously painful or restricted active 90 movement.[12–18,23] Stimulation of mechanical receptors by spinal mobilization activates 91 large-diameter nerve fibers leading to activation of the pain gate mechanism.[24] At the central 92 level, descending pain pathways may be facilitated via the midbrain's periaqueductal grey 93 matter.[24] These descending neurons may release the primary mediators' opioids, nor- 94 adrenaline, and serotonin, modulating pain, reducing the muscle spasm, and improving restricted 95 lumbar movements.[16,24] 96 A systematic review indicated moderate level evidence of Mulligan technique for short-term 97 effect on LBP associated pain and disability [11]. Studies in this review delivered SNAGs in 98 addition to conventional therapy, including stretching and back extensor strengthening, 96 A systematic review indicated moderate level evidence of Mulligan technique for short-term 97 effect on LBP associated pain and disability [11]. Studies in this review delivered SNAGs in 98 addition to conventional therapy, including stretching and back extensor strengthening, 99 [12,15,16], and thoracic postural exercises.[15,17] Immediate and short term benefits of SNAGs 100 as a standalone treatment is reported in patients with NSLBP[13] as well as healthy 101 individuals[23] compared to sham SNAGs. Mulligan SNAGs found superior to McKenzie 102 extension exercises to improve lumbar ROM but not for pain and disability in patients with 103 NSLBP.[14] 99 [12,15,16], and thoracic postural exercises.[15,17] Immediate and short term benefits of SNAGs 100 as a standalone treatment is reported in patients with NSLBP[13] as well as healthy 101 individuals[23] compared to sham SNAGs. Manuscript to be reviewed Mulligan SNAGs found superior to McKenzie 102 extension exercises to improve lumbar ROM but not for pain and disability in patients with 103 NSLBP.[14] 104 Myofascial release (MFR) is a manual technique that utilizes a superintend force in a 105 predetermined direction to stretch or optimize the myofascial complex's length and gliding 104 Myofascial release (MFR) is a manual technique that utilizes a superintend force in a 105 predetermined direction to stretch or optimize the myofascial complex's length and gliding Manuscript to be reviewed 106 properties.[8,9] MFR improves myofascial restriction by breaking intermolecular cross-links and 107 redistributing internal fluids.[8,9,19–22,24] The prolonged-release in MFR superimposes stretch 108 over joint and muscle mechanoreceptors.[22] These mechanoreceptors activate the sympathetic 109 system by somatic efferent and periaqueductal grey matter modulating the descending pain 110 pathway.[19,22] 111 Two Systematic reviews suggested emerging evidence of MFR for chronic LBP[25]; however, 112 the observed effect was not clinically significant[26]. In these reviews, MFR was given as an 113 adjunct to specific back exercises[8] and occupational therapy[19] or compared to sham 114 intervention[9] in NSLBP patients. Improvement was observed for pain, fascial mobility, and 115 functional abilities following MFR intervention among non-specific neck and back pain 116 patients[9]. MFR as a standalone treatment improved pain, performances of daily activities, and 117 fear of pain [20] and lumbar ROM in patients with NSLBP over the short-term.[21] 118 Both MFR and SNAGs have shown beneficial effects in managing NSLBP. However, a dearth of 119 evidence about the comparative effect of MFR and SNAGs as an adjunct to strengthening 120 exercises in NSLBP warrants further research. Hence, this study sought to compare the 121 (immediate and short term) effects of MFR and SNAGs as adjunct treatments in patients with 122 NSLBP. 111 Two Systematic reviews suggested emerging evidence of MFR for chronic LBP[25]; however, 112 the observed effect was not clinically significant[26]. In these reviews, MFR was given as an 113 adjunct to specific back exercises[8] and occupational therapy[19] or compared to sham 114 intervention[9] in NSLBP patients. Improvement was observed for pain, fascial mobility, and 115 functional abilities following MFR intervention among non-specific neck and back pain 116 patients[9]. MFR as a standalone treatment improved pain, performances of daily activities, and 117 fear of pain [20] and lumbar ROM in patients with NSLBP over the short-term.[21] Manuscript to be reviewed 129 CTRI/2018/12/016787. Written and oral instructions about the study procedures, interventions, 130 and possible benefits and risks were given to the patients. Written informed consent was taken 131 from all the patients. Patients were assigned to either an intervention group in an alternate 132 sequence at a 1:1 ratio. The therapist, who delivered the intervention, did a non-concealed 133 allocation of the patients. As it is an inherent issue to manual therapy trials, the therapist could 134 not be blinded to the patient's group allocation. However, patients were blinded to the other 135 intervention group. 123 124 Materials & Methods 125 The parallel-group study was carried out at tertiary care hospitals from November 2018 to 126 March 2020. Institutional Ethics Committee, Kasturba Medical College, Mangalore granted 127 ethical approval (IEC KMC MLR 11-18/429) to carry out the study. The study design was 128 registered under the clinical trial registry of India, https://ctri.nic.in with identifier 124 Materials & Methods 125 The parallel-group study was carried out at tertiary care hospitals from November 2018 to 126 March 2020. Institutional Ethics Committee, Kasturba Medical College, Mangalore granted 127 ethical approval (IEC KMC MLR 11-18/429) to carry out the study. The study design was 128 registered under the clinical trial registry of India, https://ctri.nic.in with identifier Manuscript to be reviewed 150 An independent blinded assessor collected all the outcome data from the patients at baseline, 151 immediately post first treatment session except MODI questionnaire, and after the sixth day of 152 intervention (short term). 150 An independent blinded assessor collected all the outcome data from the patients at baseline, 151 immediately post first treatment session except MODI questionnaire, and after the sixth day of 152 intervention (short term). 153 Pain levels were assessed with the VAS. It is a 100mm horizontal scale with 'no pain' and 'worst 154 possible pain' labels at the line's extremes. The VAS has demonstrated good test-retest reliability, 155 which is higher among literate (r= 0.94, p< 0.001) than illiterate (r= 0.71, p<0.001) subjects.[28] 153 Pain levels were assessed with the VAS. It is a 100mm horizontal scale with 'no pain' and 'worst 154 possible pain' labels at the line's extremes. The VAS has demonstrated good test-retest reliability, 155 which is higher among literate (r= 0.94, p< 0.001) than illiterate (r= 0.71, p<0.001) subjects.[28] 156 Patient-Specific Function scale (PSFS) was used to assess functional ability. The patient was 157 asked to write down three activities that were the most restricted or challenging to perform. All 158 the activities were scored on a scale of zero to ten, where 'zero' is unable to perform/challenging 159 to do, and 'ten' can do as before. Previous research on the PSFS has reported moderate to good 160 reliability with an intraclass correlation coefficient (ICC) of 0.713, and a minimal detectable 161 change (MDC) of three, and minimal important difference (MID) of 1.2.[29] 162 Disability assessment was measured using the Modified Oswestry Disability Index (MODI) 163 questionnaire, which has ten sections and provides information on LBP's effect on the patient’s 164 ability to manage everyday life. A total score was converted to percentage points. Fritz and 165 Irrgang (2001) reported a high test-retest reliability of the MODI in 67 LBP patients with an ICC 166 of 0.90 and a minimum clinically important difference (MCID) of six percentage points.[30] 167 Range of motion (ROM) was assessed using a bubble inclinometer, as described by Norkin CC 168 et al. [31]. 136 Patients 137 Patients referred by orthopedic surgeons for physiotherapy were recruited. Patients with 138 localized back pain with restricted/painful lumbar spine movements were screened for eligibility 139 The inclusion criteria were sub-acute to chronic NSLBP, either gender, 18-60 years old, and a 140 minimum baseline Visual Analog Scale (VAS) score of four.[27] Patients with contraindications 141 to manual therapy interventions excluded if they presented with lumbar radiculopathy, spinal 142 pathology (fracture or tumors) or history of any spinal surgery, lumbar canal stenosis, 143 osteoporosis, pregnancy-related back pain, and spinal deformities like scoliosis or 144 kyphosis[12,24,27]. Sixty-five patients with sub-acute to chronic NSLBP were included in the 145 study after screening for eligibility criteria. After screening for eligibility, patients were 146 examined for active lumbar ROM to identify involved painful/restricted segment, which was 147 confirmed using passive accessory intervertebral movement examination in a prone position. 148 Patients' flow is highlighted in the consort flow diagram (figure 1). 149 Outcome measures 137 Patients referred by orthopedic surgeons for physiotherapy were recruited. Patients with 138 localized back pain with restricted/painful lumbar spine movements were screened for eligibility. 139 The inclusion criteria were sub-acute to chronic NSLBP, either gender, 18-60 years old, and a 140 minimum baseline Visual Analog Scale (VAS) score of four.[27] Patients with contraindications 141 to manual therapy interventions excluded if they presented with lumbar radiculopathy, spinal 142 pathology (fracture or tumors) or history of any spinal surgery, lumbar canal stenosis, 143 osteoporosis pregnancy related back pain and spinal deformities like scoliosis or PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) Manuscript to be reviewed A study on the within and between-day bubble inclinometer reliability in determining 169 standing lumbar spine ROM (Flexion, extension, and lateral flexion) in healthy individuals and 170 chronic NSLBP patients found ICCs ranged from 0.908 to 0.982.[32] Inferiorly S2 and PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) Manuscript to be reviewed 171 superiorly T12 spinous process was used for double inclinometer measurement technique. The 172 patient was instructed to perform active lumbar movements without bending knees. 173 Intervention 174 A total of six intervention sessions in a week were delivered to all participants in both groups. 175 Procedure for SNAGs[33] 176 The flexion or extension glide application was decided based on the movement examination for 177 restricted lumbar ROM and pain response. The SNAGs were applied in the sitting position with 178 the patient’s pelvic stabilized with a Mulligan belt at the level of the anterior superior iliac spine 179 (ASIS). The therapist’s hand's ulnar aspect was used over the spinous process of the superior 180 vertebra of the involved segment for flexion glide and the inferior vertebra's spinous process for 181 extension glide. A passive accessory glide was administered and maintained until the patient 182 completed a full movement arc, which was restricted or painful earlier. The glides were given for 183 six repetitions for three sets every session. (Figure 2). The glide was administered over the 184 spinous process, where the force's amplitude was upheld within the patient’s comfort, as 185 Mulligan has previously described that SNAGs should not provoke any pain. 176 The flexion or extension glide application was decided based on the movement examination for 177 restricted lumbar ROM and pain response. The SNAGs were applied in the sitting position with 178 the patient’s pelvic stabilized with a Mulligan belt at the level of the anterior superior iliac spine 179 (ASIS). The therapist’s hand's ulnar aspect was used over the spinous process of the superior 180 vertebra of the involved segment for flexion glide and the inferior vertebra's spinous process for 181 extension glide. A passive accessory glide was administered and maintained until the patient 182 completed a full movement arc, which was restricted or painful earlier. The glides were given for 183 six repetitions for three sets every session. (Figure 2). The glide was administered over the 184 spinous process, where the force's amplitude was upheld within the patient’s comfort, as 185 Mulligan has previously described that SNAGs should not provoke any pain. 187 The patient was positioned comfortably in prone lying. Direct MFR was administered to the 188 lower back muscles with the therapists' knuckles, and the stretch held into the end range for up to 189 120 seconds or until the therapist felt giving away the taut tissues before being released. MFR 190 was delivered for ten repetitions, with a total of 20 minutes of intervention. Manuscript to be reviewed 192 Strengthening exercises were prescribed for all the patients, according to the referring orthopedic 193 surgeon's direction. Both groups received strengthening exercises with two sets of ten repetitions 194 without any additional resistance. Abdominal draw-in manoeuvre to activate transverse 195 abdominis was performed in crook lying. Cat and camel exercises were carried out for lumbar 196 multifidus training in a quadruped position. Strengthening of gluteal muscles (hip abductors and 197 extensors) was performed in a side-lying and prone lying positions with straight leg raise 198 exercises without any additional resistance. Patients were also briefed about ergonomic advice 199 on posture and lifting techniques to incorporate during routine activities. 173 Intervention (Figure 3) 191 Strengthening exercises[6,7,34] 187 The patient was positioned comfortably in prone lying. Direct MFR was administered to the 188 lower back muscles with the therapists' knuckles, and the stretch held into the end range for up to 189 120 seconds or until the therapist felt giving away the taut tissues before being released. MFR 190 was delivered for ten repetitions, with a total of 20 minutes of intervention. (Figure 3) 191 Strengthening exercises[6 7 34] PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) 200 Power calculation 201 The sample size was calculated using the GPower analysis software (version 3.0.10). The effect 202 size for VAS was estimated, d=20mm, and standard deviation (σ) =26.5mm from a previous 203 study. [14] With a power of 85% and α level of 0.05 total sample size estimated to be seventy 204 (35 in each group) considering a 10% dropout rate. 201 The sample size was calculated using the GPower analysis software (version 3.0.10). The effect 202 size for VAS was estimated, d=20mm, and standard deviation (σ) =26.5mm from a previous 203 study. [14] With a power of 85% and α level of 0.05 total sample size estimated to be seventy 204 (35 in each group) considering a 10% dropout rate. 201 The sample size was calculated using the GPower analysis software (version 3.0.10). The effect 202 size for VAS was estimated, d=20mm, and standard deviation (σ) =26.5mm from a previous 203 study. [14] With a power of 85% and α level of 0.05 total sample size estimated to be seventy 204 (35 in each group) considering a 10% dropout rate. 206 Statistical analysis 207 An independent statistician analyzed data using SPSS Version 25.0 (SPSS Inc, Chicago, IL, 208 USA). Data were assessed for normality using skewness and kurtosis values and observation of 209 Q-Q plots, which indicated that non-parametric tests were required. The demographic 210 characteristics of the patients were summarized with median and interquartile ranges. Data for 211 the lost to follow-up patients were analyzed using the intention to treat analysis by carrying 212 forward the value of outcome measures assessed at the last follow-up. p values less than 0.05 213 were considered statistically significant. The Friedman rank-sum test was used to determine the 207 An independent statistician analyzed data using SPSS Version 25.0 (SPSS Inc, Chicago, IL, 208 USA). Data were assessed for normality using skewness and kurtosis values and observation of 209 Q-Q plots, which indicated that non-parametric tests were required. The demographic 210 characteristics of the patients were summarized with median and interquartile ranges. Data for 211 the lost to follow-up patients were analyzed using the intention to treat analysis by carrying 212 forward the value of outcome measures assessed at the last follow-up. p values less than 0.05 213 were considered statistically significant. The Friedman rank-sum test was used to determine the PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) Manuscript to be reviewed 214 within-group differences from baseline to post-treatment sessions for VAS, PSFS, and lumbar 215 ROM. Post-hoc analysis using the Wilcoxon signed-rank test with Bonferroni correction was 216 performed for timegroup differences. Mann- Whitney U test was used to explore the two 217 groups' differences from baseline to immediate, and short-term. 218 219 Results 220 Throughout the trial phases, patients' flow is highlighted in the consort flowchart (figure 1). One 221 hundred and sixty-seven patients were screened for eligibility, of which 102 patients were 222 excluded based on exclusion criteria. Sixty-five patients could be recruited within the study 223 period and allocated to either MFR (n=33) and SNAGs (n=32) groups. Eight patients dropped 224 out before the sixth session, either because they dramatically improved and discharged or 225 migrated. Intention to treat analysis was considered to accommodate dropouts. The demographic 226 data of all the participants are shown in Table 1. Baseline characteristics of all the outcome 227 measures were homogenous and statistically insignificant between the groups. (Table 2) The 228 within-group analysis identified statistically significant differences for VAS, PSFS, and 229 extension ROM for both the groups and only for flexion ROM in the SNAGs group. (Table 3) 230 Modified Oswestry disability index also showed statistically significant (p<0.05) improvement 231 from sixteen and fourteen points at baseline in MFR and SNAGs groups respectively to twelve 232 and eight points over the short-term. 233 Timegroup: For both the groups, VAS and PSFS showed immediate and short-term 234 improvement. Lumbar extension improved immediately and in the short term in both the groups; 214 within-group differences from baseline to post-treatment sessions for VAS, PSFS, and lumbar 215 ROM. Post-hoc analysis using the Wilcoxon signed-rank test with Bonferroni correction was 216 performed for timegroup differences. Mann- Whitney U test was used to explore the two 217 groups' differences from baseline to immediate, and short-term. 214 within-group differences from baseline to post-treatment sessions for VAS, PSFS, and lumbar 215 ROM. Post-hoc analysis using the Wilcoxon signed-rank test with Bonferroni correction was 216 performed for timegroup differences. Mann- Whitney U test was used to explore the two 217 groups' differences from baseline to immediate, and short-term. 220 Throughout the trial phases, patients' flow is highlighted in the consort flowchart (figure 1). One 221 hundred and sixty-seven patients were screened for eligibility, of which 102 patients were 222 excluded based on exclusion criteria. PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) 240 Discussion 241 This study was aimed to determine the comparative effects of MFR and SNAGs in combination 242 with strengthening exercises on pain, disability, ROM, and functional ability in NSLBP patients. 243 However, both MFR and SNAGs groups demonstrated statistically significant (p< 0.05) 244 improvements for outcomes VAS, ROM, and PSFS, immediately and in the short-term, 245 including MODI, there were no significant differences between the groups (p>0.05). 246 The clinically significant improvement was observed on VAS for pain in both SNAGs and MFR 247 group with strengthening exercises. Considering the MCID value of 20mm for VAS in chronic 248 pain [35], SNAGs groups demonstrated a significant change of 30.5mm immediately after the 1st 249 treatment session and 41mm over the short-term. In comparison, an MFR group improved by 250 21mm immediately and 42mm in the short-term. 241 This study was aimed to determine the comparative effects of MFR and SNAGs in combination 242 with strengthening exercises on pain, disability, ROM, and functional ability in NSLBP patients. 243 However, both MFR and SNAGs groups demonstrated statistically significant (p< 0.05) 244 improvements for outcomes VAS, ROM, and PSFS, immediately and in the short-term, 245 including MODI, there were no significant differences between the groups (p>0.05). 241 This study was aimed to determine the comparative effects of MFR and SNAGs in combination 242 with strengthening exercises on pain, disability, ROM, and functional ability in NSLBP patients. 243 However, both MFR and SNAGs groups demonstrated statistically significant (p< 0.05) 244 improvements for outcomes VAS, ROM, and PSFS, immediately and in the short-term, 245 including MODI, there were no significant differences between the groups (p>0.05). 246 The clinically significant improvement was observed on VAS for pain in both SNAGs and MFR 247 group with strengthening exercises. Considering the MCID value of 20mm for VAS in chronic 248 pain [35], SNAGs groups demonstrated a significant change of 30.5mm immediately after the 1st 249 treatment session and 41mm over the short-term. In comparison, an MFR group improved by 250 21mm immediately and 42mm in the short-term. 251 Similarly to this study's findings, when Waqqar et al. Manuscript to be reviewed Sixty-five patients could be recruited within the study 223 period and allocated to either MFR (n=33) and SNAGs (n=32) groups. Eight patients dropped 224 out before the sixth session, either because they dramatically improved and discharged or 225 migrated. Intention to treat analysis was considered to accommodate dropouts. The demographic 226 data of all the participants are shown in Table 1. Baseline characteristics of all the outcome 227 measures were homogenous and statistically insignificant between the groups. (Table 2) The 228 within-group analysis identified statistically significant differences for VAS, PSFS, and 229 extension ROM for both the groups and only for flexion ROM in the SNAGs group. (Table 3) 230 Modified Oswestry disability index also showed statistically significant (p<0.05) improvement 231 from sixteen and fourteen points at baseline in MFR and SNAGs groups respectively to twelve 232 and eight points over the short-term. 233 Timegroup: For both the groups, VAS and PSFS showed immediate and short-term 233 Timegroup: For both the groups, VAS and PSFS showed immediate and short-term 234 improvement. Lumbar extension improved immediately and in the short term in both the groups; 235 however, lumbar flexion showed improvement only in the SNAGs group over the short-term but 233 Timegroup: For both the groups, VAS and PSFS showed immediate and short-term 234 improvement. Lumbar extension improved immediately and in the short term in both the groups; 235 however, lumbar flexion showed improvement only in the SNAGs group over the short-term but PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) Manuscript to be reviewed 236 not immediately. Lateral flexion ROM did not show any significant change for both the groups 237 (Table 4). 236 not immediately. Lateral flexion ROM did not show any significant change for both the groups 237 (Table 4). 238 Comparison between the groups for VAS, PSFS, and ROM showed no statistically significant 239 difference (p>0.05) immediately and also in the short-term, including MODI (Table 5). 238 Comparison between the groups for VAS, PSFS, and ROM showed no statistically significant 239 difference (p>0.05) immediately and also in the short-term, including MODI (Table 5). 240 Discussion compared SNAGs with other manual 252 therapy techniques like McKenzie exercises, they found both have a similar effect for pain and 253 disability.[14] Other studies also found SNAGs [17] or MFR[8,919,20,21], when administered 254 as an adjunct to occupational therapy, stretching, back strengthening exercises, and thoracic 255 postural correction exercises, have short-term beneficial effects on NSLBP. This study's findings 256 indicate that both techniques with exercise can be used to address NSLBP. Though both groups 257 improved similarly over the short-term in the present study, SNAGs were clinically superior to PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) Manuscript to be reviewed 281 flexion ROM. However, other studies have reported that MFR as an adjunct to back school 282 along with exercises[19] and work station modifications [22] improved lumbar flexion ROM. 283 These outcomes could be related to more MFR sessions, which helps break down the scar matrix 284 and intermolecular crosslinks, redistribute internal fluids, and improve collagen extensibility. 285 These effects of MFR may help in enhancing fascial mobility and soft-tissue extensibility [22]. 286 In this study, MODI demonstrated statistical (p<0.05) significant difference for both MFR and 287 SNAGs groups from baseline to short-term. However, only the SNAGs group shown clinically 288 significant improvement. The MCID value for the MODI has been estimated to be six 289 points.[30] Over the short-term, the MFR group showed a gain of four points, while a change of 290 six points was observed in the SNAGs group. Rezkhallah et al. reported similar findings with the 291 SNAGs group improved more than the MFR group in non-specific neck pain patients. The more 292 significant gain observed in pain and ROM can explain the improvement in disability amongst 293 the SNAGs group compared to the MFR group. 289 points.[30] Over the short-term, the MFR group showed a gain of four points, while a change of 290 six points was observed in the SNAGs group. Rezkhallah et al. reported similar findings with the 291 SNAGs group improved more than the MFR group in non-specific neck pain patients. The more 292 significant gain observed in pain and ROM can explain the improvement in disability amongst 293 the SNAGs group compared to the MFR group. 294 Both the groups demonstrated improved function immediately and over short-term, but between 295 the group, there was no significant difference for PSFS. The change for the PSFS was 2.67 in 296 both MFR and SNAGs groups over the short-term, which was not clinically significant. Abbott 297 and Schmidt[36] and Vliet et al.[37] reported MCID values of 3.3 and 4.3, respectively, for more 298 considerable clinical change of PSFS in chronic mechanical LBP. 299 Manual therapy interventions like MFR and SNAGs stimulate mechanoreceptors located in the 300 soft tissues and the lumbar spine's facet joints. The activity of these receptors constantly feeds 301 CNS for neuro-reflexive muscle activation. Manuscript to be reviewed 258 improve pain immediately. A study by Rezkhallah et al. has reported similar findings in non- 259 specific neck pain patients. In their research, SNAGs improved pain with more percentage points 260 than MFR [24]. 258 improve pain immediately. A study by Rezkhallah et al. has reported similar findings in non- 259 specific neck pain patients. In their research, SNAGs improved pain with more percentage points 260 than MFR [24]. 261 Both MFR and SNAGs help in correcting anomalies within the elements of the movement 262 system by stimulating mechanoreceptors, which might improve the activation pattern of para- 263 spinal muscles, improving pain-free ROM. However, in the current study, only the SNAGs 264 group improved with lumbar flexion ROM, while lumbar extension improved for both the 265 groups. This finding contradicted the result in non-specific neck pain patients in which both 266 MFR and SNAGs improved neck ROM in all the planes [24]. 267 In line with the finding of this study, SNAGs application had an immediate and short term effect 268 on lumbar flexion ROM among healthy individuals[23] and patients with NSLBP.[14–18] 269 Passively administered spinal accessory glide breaks adhesions, leading to increased facet joint 270 vascular supply and necessary nutrients, enhancing the soft tissue healing around the injury 271 site.[24] The application of the glides over the spinous process concentrated on correcting 272 flexion and extension positional faults and promoting pain-free physiological lumbar spine 273 movement.[11] In this study, Mulligan mobilization was delivered with a contact of the spinous 274 process, which glides both facets in the same direction. During mobilization, patients also 275 performed only sagittal plane movements of lumbar flexion or extension, as it was primarily 276 restricted movement. We hypothesize, this reason for no observed improvement in lateral 277 flexion, as it requires ipsilateral facet to move in extension with contralateral facet moving to 278 flexion and can be better-improved giving mobilization using unilateral transverse process. 279 MFR found to normalize flexion relaxation phenomenon in individuals with NSLBP[21], which 280 contradicted the observation of this study in which the MFR group did not improve with lumbar 279 MFR found to normalize flexion relaxation phenomenon in individuals with NSLBP[21], which 280 contradicted the observation of this study in which the MFR group did not improve with lumbar PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) Manuscript to be reviewed A possible explanation that both MFR and SNAGs 302 groups showed similar improvement could be related to these techniques' effect on seducing 303 CNS by balancing these receptors' activity and re-establish dynamic control. Global , 321 regional , and national incidence , prevalence , and years lived with disability for 354 322 diseases and injuries for 195 countries and territories , 1990 – 2017 : a systematic analysis 323 for the Global Burden of Disease Study 2017. Lancet 2018;392(10159):1789–858. 324 doi:10.1016/S0140-6736(18)32279-7. Manuscript to be reviewed 304 Limitation: 305 Severe limitation of this trial was the non-random allocation of patients to treatment arms due to 306 the trial's lack of insurance cover. 307 The study could not complete the estimated sample size recruitment within the data collection 308 time-frame. 309 310 Conclusions 311 The current study results suggest that strengthening exercises with SNAGs or MFR techniques 312 can be considered for immediate and short-term management of pain, function, and lumbar 313 extension ROM in sub-acute to chronic NSLBP. Future trials should consider assessing the 314 long-term effects of SNAGs and MFR for improvement in lumbar ROM. Varying duration of 304 Limitation: 305 Severe limitation of this trial was the non-random allocation of patients to treatment arms due to 306 the trial's lack of insurance cover. 307 The study could not complete the estimated sample size recruitment within the data collection 308 time-frame. 309 310 Conclusions 311 The current study results suggest that strengthening exercises with SNAGs or MFR techniques 312 can be considered for immediate and short-term management of pain, function, and lumbar 313 extension ROM in sub-acute to chronic NSLBP. Future trials should consider assessing the 314 long-term effects of SNAGs and MFR for improvement in lumbar ROM. Varying duration of 315 MFR hold also needs to be studied with a long-term follow-up. 316 Conflict of interest 317 The authors declare there are no conflict of interest 318 319 References 320 [1] GBD 2017 Disease and Injury Incidence and Prevalence Collaborators (2018). Global , 307 The study could not complete the estimated sample size recruitment within the data collection 308 time-frame. 307 The study could not complete the estimated sample size recruitment within the data collection 308 time-frame. 309 310 Conclusions 311 The current study results suggest that strengthening exercises with SNAGs or MFR techniques 312 can be considered for immediate and short-term management of pain, function, and lumbar 313 extension ROM in sub-acute to chronic NSLBP. Future trials should consider assessing the 314 long-term effects of SNAGs and MFR for improvement in lumbar ROM. Varying duration of 315 MFR hold also needs to be studied with a long-term follow-up. 316 Conflict of interest 317 The authors declare there are no conflict of interest 318 319 References 320 [1] GBD 2017 Disease and Injury Incidence and Prevalence Collaborators (2018). 310 Conclusions 311 The current study results suggest that strengthening exercises with SNAGs or MFR techniques 312 can be considered for immediate and short-term management of pain, function, and lumbar 313 extension ROM in sub-acute to chronic NSLBP. Future trials should consider assessing the 314 long-term effects of SNAGs and MFR for improvement in lumbar ROM. Varying duration of 315 MFR hold also needs to be studied with a long-term follow-up. 311 The current study results suggest that strengthening exercises with SNAGs or MFR techniques 312 can be considered for immediate and short-term management of pain, function, and lumbar 313 extension ROM in sub-acute to chronic NSLBP. Future trials should consider assessing the 314 long-term effects of SNAGs and MFR for improvement in lumbar ROM. Varying duration of 315 MFR hold also needs to be studied with a long-term follow-up. 320 [1] GBD 2017 Disease and Injury Incidence and Prevalence Collaborators (2018). Global , 321 regional , and national incidence , prevalence , and years lived with disability for 354 322 diseases and injuries for 195 countries and territories , 1990 – 2017 : a systematic analysis 323 for the Global Burden of Disease Study 2017. Lancet 2018;392(10159):1789–858. 324 doi:10.1016/S0140-6736(18)32279-7. PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) Manuscript to be reviewed Manuscript to be reviewed 325 [2] Hoy D, March L, Brooks P, Blyth F, Woolf A, Bain C, Williams G, Smith E, Vos T, 326 Barendregt J, Murray C, Burstein R, Buchbinder R. The global burden of low back pai 327 Estimates from the Global Burden of Disease 2010 study. 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Manuscript to be reviewed The effects of gluteus muscle strengthening 337 exercise and lumbar stabilization exercise on lumbar muscle strength and balance in 338 chronic low back pain patients. J Phys Ther Sci 2015;27(12):3813–6. doi: 339 10.1589/jpts.27.3813. 336 [6] Jeong UC, Kim CY, Hwang-Bo G NC. The effects of gluteus muscle strengthening 337 exercise and lumbar stabilization exercise on lumbar muscle strength and balance in 338 chronic low back pain patients. J Phys Ther Sci 2015;27(12):3813–6. doi: 340 [7] de Sousa CS, de Jesus FLA, Machado MB, Ferreira G, Ayres IGT, de Aquino LM, 341 Fukuda TY, Gomes-Neto M. Lower limb muscle strength in patients with low back pain: a 342 systematic review and meta-analysis. J Musculoskelet Neuronal Interact. 2019 Mar 343 1;19(1):69-78. 344 [8] Ajimsha MS, Daniel B, Chithra S. Effectiveness of Myofascial release in the management 345 of chronic low back pain in nursing professionals. 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Inclusion and exclusion criteria used in non-specific low back 408 pain trials: a review of randomised controlled trials published between 2006 and 2012. 409 BMC Musculoskelet Disord 2018;19(1):113. doi:10.1186/s12891-018-2034-6. 410 [28] Hawker GA Mian S Kendzerska T French M Measures of adult pain: Visual Analog Manuscript to be revie Manuscript to be reviewed 371 [16] Elrazik RKA, Samir SM, Zaki LA, Koura GA. Mobilisation with movement versus 372 postero-anterior mobilisation in chronic non specific low back pain. Int J PharmTech Res 373 2016;9(6):9–16. 374 [17] Tul Ain SQ, Shakil Ur Rehman S, Maryam M, Kiani SK. Effects of Sustained Natural 375 Apophyseal Glides with and without thoracic posture correction techniques on mechanical 376 back pain: a randomized control trial. J Pak Med Assoc 2019;69(11):1584–7. 374 [17] Tul Ain SQ, Shakil Ur Rehman S, Maryam M, Kiani SK. Effects of Sustained Natural 375 Apophyseal Glides with and without thoracic posture correction techniques on mechanical 376 back pain: a randomized control trial. J Pak Med Assoc 2019;69(11):1584–7. 378 [18] Muhanna NA. Effectiveness of Snags Mobilization in Chronic Mechanical Low Back 379 Pain. J Adv Sch Res Allied Educ 2018;15:153–8. doi:10.29070/15/57740. 380 [19] Saratchandran R DS. Myofascial release as an adjunct to conventional occupational 381 therapy in mechanical low back pain. Indian J Occup Ther. 2013;45(2) 3-7 382 [20] Arguisuelas MD, Lisón JF, Sánchez-Zuriaga D, Martínez-Hurtado I, Doménech- 383 Fernández J. Effects of Myofascial Release in Nonspecific Chronic Low Back Pain. Spine 384 2017;42(9):627–34. doi:10.1097/BRS.0000000000001897. 385 [21] Arguisuelas MD, Lisón JF, Doménech-Fernández J, Martínez-Hurtado I, Salvador 386 Coloma P, Sánchez-Zuriaga D. Effects of myofascial release in erector spinae myoelectric 387 activity and lumbar spine kinematics in non-specific chronic low back pain: Randomized 388 controlled trial. Clin Biomech 2019;63:27–33. doi:10.1016/j.clinbiomech.2019.02.009. 389 [22] Balasubramaniam A, Ghandi V, Sambandamoorthy A. Role of myofascial release therapy 389 [22] Balasubramaniam A, Ghandi V, Sambandamoorthy A. Role of myofascial release therapy PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) 390 on pain and lumbar range of motion in mechanical back pain: an exploratory investigatio 391 of desk job workers. Ibnosina J Med Biomed Sci 2013;6(2):75–80. 392 [23] Moutzouri M, Billis E, Strimpakos N, Kottika P, Oldham JA. The effects of the Mulliga 393 Sustained Natural Apophyseal Glide (SNAG) mobilisation in the lumbar flexion range o 394 asymptomatic subjects as measured by the Zebris CMS20 3-D motion analysis system. 395 BMC Musculoskelet Disord 2008;9(1):1–9. doi:10.1186/1471-2474-9-131. 396 [24] Rezkallah SS, Abdullah GA. Comparison between sustained natural apophyseal glides 397 (SNAG’s) and myofascial release techniques combined with exercises in non specific 398 neck pain. Physiother Pr Res 2018;39(2):135–45. doi:10.3233/PPR-180116. 399 [25] Ajimsha MS, Al-Mudahka NR, Al-Madzhar JA. Effectiveness of myofascial release: 400 Systematic review of randomized controlled trials. J Bodyw Mov Ther 2015;19(1):102–1 401 doi:10.1016/j.jbmt.2014.06.001. 412 Questionnaire (MPQ), Short-Form McGill Pain Questionnaire (SF-MPQ), Chronic Pain 413 Grade Scale (CPGS), Short Form-36 Bodily Pain Scale (SF-36 BPS) and measure of 414 Intermittent and Constant Osteoarthritis Pain (ICOAP). Arthritis Care Res 2011;63:240– 415 52. doi:10.1002/acr.20543. 416 [29] Hefford C. The Patient-Specific Functional Scale: Validity, Reliability, and 417 Responsiveness in Patients With Upper Extremity Musculoskeletal Problems. J Orthop 418 Sports Phys Ther. 2012;42(2):56–65. doi:10.2519/jospt.2012.3953. 419 [30] Fritz JM, Irrgang JJ. A Comparison of a Modified Oswestry Low Back Pain Disability 420 Questionnaire and the Quebec Back Pain Disability Scale. Phys Ther 2001;81(2):776–88 421 doi:10.1093/ptj/81.2.776. 422 [31] Norkin CC, White DJ. Measurement of Joint Motion: A Guide to Goniometry. 4th ed. 423 Philadelphia, 424 [32] PA: F. A. Davis Company, 2009. Sadeghi R, Mosallanezhad Z, Nodehi-Moghadam A, 425 Nourbakhsh M, Biglarian A, Ezati K. The Reliability of Bubble Inclinometer and Tape 426 Measure in Determining Lumbar Spine Range of Motion in Healthy Individuals and 427 Patients Phys Treat Specif Phys Ther 2015;15(3):137–44. 428 [33] Mulligan B. Manual Therapy, NAGS, SNAGS MWMS etc. 5th ed. Wellington, New: 429 Zealand: Plane View Services Limited; 2010. 430 [34] Sadler S, Cassidy S, Peterson B, Spink M, Chuter V. Gluteus medius muscle function in 431 people with and without low back pain : a systematic review. BMC Musculoskelet Disord Manuscript to be revie Manuscript to be reviewed 390 on pain and lumbar range of motion in mechanical back pain: an exploratory investigation 391 of desk job workers. Ibnosina J Med Biomed Sci 2013;6(2):75–80. 390 on pain and lumbar range of motion in mechanical back pain: an exploratory investigation 391 of desk job workers. Ibnosina J Med Biomed Sci 2013;6(2):75–80. 392 [23] Moutzouri M, Billis E, Strimpakos N, Kottika P, Oldham JA. The effects of the Mulligan 393 Sustained Natural Apophyseal Glide (SNAG) mobilisation in the lumbar flexion range of 394 asymptomatic subjects as measured by the Zebris CMS20 3-D motion analysis system. 395 BMC Musculoskelet Disord 2008;9(1):1–9. doi:10.1186/1471-2474-9-131. 396 [24] Rezkallah SS, Abdullah GA. Comparison between sustained natural apophyseal glides 397 (SNAG’s) and myofascial release techniques combined with exercises in non specific 398 neck pain. Physiother Pr Res 2018;39(2):135–45. doi:10.3233/PPR-180116. 399 [25] Ajimsha MS, Al-Mudahka NR, Al-Madzhar JA. Effectiveness of myofascial release: 400 Systematic review of randomized controlled trials. J Bodyw Mov Ther 2015;19(1):102–12. 401 doi:10.1016/j.jbmt.2014.06.001. 402 [26] Laimi K, Mäkilä A, Bärlund E, Katajapuu N, Oksanen A, Seikkula V, Karppinen J, 402 [26] Laimi K, Mäkilä A, Bärlund E, Katajapuu N, Oksanen A, Seikkula V, Karppinen J, 403 Saltychev M Effectiveness of myofascial release in treatment of chronic musculoskeletal 404 pain: a systematic review. Clin Rehabil 2018;32:440–50. 405 doi:10.1177/0269215517732820. 406 [27] Amundsen PA, Evans DW, Rajendran D, Bright P, Bjørkli T, Eldridge S, Buchbinder R, 407 Underwood M, Froud R. Inclusion and exclusion criteria used in non-specific low back 408 pain trials: a review of randomised controlled trials published between 2006 and 2012. 409 BMC Musculoskelet Disord 2018;19(1):113. doi:10.1186/s12891-018-2034-6. 406 [27] Amundsen PA, Evans DW, Rajendran D, Bright P, Bjørkli T, Eldridge S, Buchbinder R, 407 Underwood M, Froud R. Inclusion and exclusion criteria used in non-specific low back 408 pain trials: a review of randomised controlled trials published between 2006 and 2012. 409 BMC Musculoskelet Disord 2018;19(1):113. doi:10.1186/s12891-018-2034-6. 410 [28] Hawker GA, Mian S, Kendzerska T, French M. Measures of adult pain: Visual Analog 411 Scale for Pain (VAS Pain), Numeric Rating Scale for Pain (NRS Pain), McGill Pain PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) Manuscript to be reviewed 412 Questionnaire (MPQ), Short-Form McGill Pain Questionnaire (SF-MPQ), Chronic Pain 413 Grade Scale (CPGS), Short Form-36 Bodily Pain Scale (SF-36 BPS) and measure of 414 Intermittent and Constant Osteoarthritis Pain (ICOAP). Arthritis Care Res 2011;63:240– 415 52. doi:10.1002/acr.20543. 416 [29] Hefford C. The Patient-Specific Functional Scale: Validity, Reliability, and 417 Responsiveness in Patients With Upper Extremity Musculoskeletal Problems. J Orthop 418 Sports Phys Ther. 2012;42(2):56–65. doi:10.2519/jospt.2012.3953. 419 [30] Fritz JM, Irrgang JJ. A Comparison of a Modified Oswestry Low Back Pain Disability 420 Questionnaire and the Quebec Back Pain Disability Scale. Phys Ther 2001;81(2):776–88. 421 doi:10.1093/ptj/81.2.776. 422 [31] Norkin CC, White DJ. Measurement of Joint Motion: A Guide to Goniometry. 4th ed. 423 Philadelphia, 424 [32] PA: F. A. Davis Company, 2009. Sadeghi R, Mosallanezhad Z, Nodehi-Moghadam A, 425 Nourbakhsh M, Biglarian A, Ezati K. The Reliability of Bubble Inclinometer and Tape 426 Measure in Determining Lumbar Spine Range of Motion in Healthy Individuals and 427 Patients Phys Treat Specif Phys Ther 2015;15(3):137–44. 428 [33] Mulligan B. Manual Therapy, NAGS, SNAGS MWMS etc. 5th ed. Wellington, New: 429 Zealand: Plane View Services Limited; 2010. 430 [34] Sadler S, Cassidy S, Peterson B, Spink M, Chuter V. Gluteus medius muscle function in 431 people with and without low back pain : a systematic review. BMC Musculoskelet Disord 432 2019;4:1–17. doi.org/10.1186/s12891-019-2833-4 430 [34] Sadler S, Cassidy S, Peterson B, Spink M, Chuter V. Gluteus medius muscle function in 431 people with and without low back pain : a systematic review. BMC Musculoskelet Disord 432 2019;4:1–17. doi.org/10.1186/s12891-019-2833-4 PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) Manuscript to be reviewed Manuscript to be reviewed 433 [35] Vela LI, Haladay DE, Denegar C. Clinical Assessment of Low-Back-Pain Treatment 434 Outcomes in Athletes. J Sport Rehabil 2011;20(1):74–88. doi: 10.1123/jsr.20.1.74 435 [36] Abbott JH Minimum Important Differences for the Patient-Specific Functional Scale 4 435 [36] Abbott JH. Minimum Important Differences for the Patient-Specific Functional Scale, 4 436 Region-Specific Outcome Measures, and the Numeric Pain Rating Scale. J Orthop Sports 437 Phys Ther. 2014;44(8):560–4. doi:10.2519/jospt.2014.5248. 438 [37] Vliet DVAN, Hefford PTC, Abbott MJH. The Patient-Specific Functional Scale: 438 [37] Vliet DVAN, Hefford PTC, Abbott MJH. The Patient-Specific Functional Scale: 439 Psychometrics, Clinimetrics, and Application as a Clinical Outcome Measure. J Orthop 440 Sport Phys Ther 2012;42:30–42. doi:10.2519/jospt.2012.3727 443 PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) Figure 1 CONSORT Flow Diagram Manuscript to be reviewed CONSORT Flow Diagram PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) Manuscript to be reviewed PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) Figure 2 Mulligan SNAGs Technique Mulligan SNAGs Technique PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) Manuscript to be reviewed PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) Figure 3 Manuscript to be reviewed Figure 3 Myofascial Release Technique Myofascial Release Technique PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) Manuscript to be reviewed Table 1(on next page) Table 1(on next page) Manuscript to be reviewed Table 2(on next page) Table 2(on next page) Table 1(on next page) Demographic details of the participants PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) 1 2 3 Table 1. Demographic details of sixty-five participants Variables MFR (n=33) SNAGs (n=32) Age (years, Mean ± SD) 25±7.11 24.34±5.37 Male 15 5 Gender Female 18 27 Sub-acute (6-12 weeks) 2 5 Duration of LBP (weeks) Chronic (>12 weeks) 31 27 Manuscript to be reviewed Manuscript to be reviewed 2 3 Table 1. Demographic details of sixty-five participants 4 n- Total number of participants, SD- Standard Deviation, LBP- Low back pain 5 Variables MFR (n=33) SNAGs (n=32) Age (years, Mean ± SD) 25±7.11 24.34±5.37 Male 15 5 Gender Female 18 27 Sub-acute (6-12 weeks) 2 5 Duration of LBP (weeks) Chronic (>12 weeks) 31 27 Table 1. Demographic details of sixty-five participants 3 4 n- Total number of participants, SD- Standard Deviation, LBP- Low bac 10 PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) Table 2(on next page) Baseline characteristics of VAS, MODI, PSFS and ROM for MFR and SNAGs groups p<0.05 statistical signiﬁcant p<0.05 statistical signiﬁcant PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) Manuscript to be reviewed 1 Table 2. Baseline characteristics of VAS, MODI, PSFS and ROM for MFR and SNAGs 2 groups 4 IQR- Inter quartile Range, MFR: Myofascial release, SNAGs: 5 Sustained Natural Apophyseal Glides, VAS: Visual Analog Scale, 6 mm- millimetre, MODI: Modified Oswestry Disability Index, PSFS: Patient Specific Functional Scale, 7 3 * p<0.05 statistical significance MEDIAN (IQR) Variables MFR SNAGs p-value VAS (mm) 6.2 (5.2-7.2) 6.1(4.5-4.7) 0.11 MODI (%) 16(12-25) 14(1.5-25) 0.545 PSFS 4.33(3.83-5.33) 4.33(3.33-5.33) 0.232 Flexion (degrees) 50(45-57.5) 50 (44.25-57.75) 0.712 Extension(degrees) 18 (10-20) 16.5 (10.25-25) 0.889 Left lateral flexion(degrees) 20 (15-25) 20 (15-28.5) 0.595 Right lateral flexion(degrees) 20 (13-25) 20 (15-25) 0.633 Baseline characteristics of VAS, MODI, PSFS and ROM for MFR and SNAG groups 8 8 Manuscript to be reviewed Table 3(on next page) PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) Table 3(on next page) Within-group analysis of VAS, PSFS, and ROM for MFR and SNAGs groups at Baseline, immediate and short-term p<0.05 statistical signiﬁcant PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) Manuscript to be reviewed Manuscript to be reviewed 1 Table 3. Within-group analysis of VAS, PSFS, and ROM for MFR and SNAGs groups at 2 Baseline, immediate and short-term 4 IQR- Inter quartile Range, VAS: Visual Analog Scale, mm- millimetre, MFR: 5 Myofascial release, SNAGs: Sustained Natural Apophyseal Glides, PSFS: Patient Specific Functional Scale, MEDIAN (IQR) Variable Group Baseline Immediate Short-term Friedman Chi- square p-value MFR 6.2 (5.2-7.2) 4.1(1.9-5.2) 2(1.0-3.45) 49.465 <0.001 VAS (mm) SNAGs 6.1(4.5-4.7) 3.05(2.05-4.37) 2(0.85-3.65) 53.382 <0.001* MFR 4.33(3.83-5.33) 5.66(4.49-6.16) 7(5.83-7.58 55.197 <0.001* PSFS SNAGs 4.33(3.33-5.33) 5.33(5-6.66) 6.83(6.12- 7.62) 49.589 <0.001* MFR 50(45-57.5) 50 (45-59) 52 (45.5-60) 3.227 0.199 Flexion(degrees) SNAGs 50 (44.25-57.75) 52.5 (45-59.5) 55.5 (50-60) 6.660 0.035* MFR 18 (10-20) 20 (15-30) 25 (19-30) 30.624 <0.001* Extension(degrees) SNAGs 16.5 (10.25-25) 21.5 (18.25-29.5) 25 (20-31.5) 22.505 <0.001* MFR 20 (15-25) 22 (15-30) 20 (20-25) 0.890 0.640 Left lateral flexion (degrees) SNAGs 20 (15-28.5) 20 (15-25) 24.5 (15-28) 2.347 0.309 MFR 20 (13-25) 20 (17.5-26) 20 (17.5-26) 5.957 0.050 Right lateral flexion(degrees) SNAGs 20 (15-25) 20 (15-27.75) 20 (15.75-28) 3.588 0.166 p<0.05 statistical significance 6 IQR- Inter quartile Range, VAS: Visual Analog Scale, mm- millimetre, MFR l significance NAGs: Sustained Natural Apophyseal Glides, PSFS: Patient Specific Functional Scale, Manuscript to be reviewed Table 4(on next page) Table 4(on next page) PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) Sustained Natural Apophyseal Glides, PSFS: Patient Specific Functional Scale, g y 4 Sustained Natural Apophyseal Glides, PSFS: Patient Specific Functional Scale, p 0.05 statistical significance VAS: Visual Analog Scale, mm- millimetre, MFR: Myofascial release, SNAGs: e Table 4(on next page) Timegroup analysis for VAS, PSFS and ROM for MFR and SNAGs groups p<0.05 statistical signiﬁcant p<0.05 statistical signiﬁcant PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) Manuscript to be reviewed 3 VAS: Visual Analog Scale, mm- millimetre, MFR: Myofascial release, SNAGs: 4 Sustained Natural Apophyseal Glides, PSFS: Patient Specific Functional Scale, *p<0.05 statistical significance Manuscript to be reviewed Table 5(on next page) Table 5(on next page) Table 5(on next page) Manuscript to be reviewed 1 Table 4 time group analysis for VAS, PSFS, and ROM for MFR and SNAGs groups 3 VAS: Visual Analog Scale, mm- millimetre, MFR: Myofascial release, SNAGs: 4 Sustained Natural Apophyseal Glides, PSFS: Patient Specific Functional Scale, Variables Group Factors Mean Difference Std. Error p-value 95% Confidence Interval Pre * Immediate 2.503 0.344 0.0001** 1.63 3.37 MFR Pre * Post 3.806 0.309 0.0001** 3.02 4.58 Pre * Immediate 2.484 0.0322 0.0001** 1.66 3.3 VAS(mm) SNAGs Pre * Post 3.544 0.315 0.0001** 2.74 4.34 Pre * Immediate -1 0.175 0.0001** -1.441 -0.559 MFR Pre * Post -2.156 0.197 0.0001** -2.655 -1.658 Pre * Immediate -1.495 0.212 0.0001** -2.031 -0.959 PSFS SNAGs Pre * Post -2.751 0.278 0.0001** -3.454 -2.047 Pre * Immediate -0.545 1.112 1 -3.35 2.26 MFR Pre * Post -1.879 0.894 0.13 -4.13 0.379 Pre * Immediate -1.875 1.24 0.422 -5.01 1.26 Flexion (degrees) SNAGs Pre * Post -4.594 1.489 0.013** -8.36 -0.82 Pre * Immediate -6.03 1.234 0.0001** -9.14 -2.91 MFR Pre * Post -7.455 1.258 0.0001** -10.63 -4.27 Pre * Immediate -5.03 1.522 0.007** -8.88 -1.17 Extension (degrees) SNAGs Pre * Post -7.219 1.42 0.0001** -10.81 -3.62 Pre * Immediate -1.27 1.366 1 -4.72 2.17 MFR Pre * Post -0.97 1.413 1 -4.54 2.6 Pre * Immediate -0.375 0.912 1 -2.68 1.93 Left lateral flexion (degrees) SNAGs Pre * Post -1.688 1.283 0.594 -4.93 1.56 Pre * Immediate -2.242 1.059 0.126 -4.91 0.43 MFR Pre * Post -2.758 1.416 0.181 -6.33 0.82 Pre * Immediate -1.06 1.033 0.934 -3.67 1.55 Right lateral flexion (degrees) SNAGs Pre * Post -1.594 1.19 0.57 -4.6 1.41 *p<0.05 statistical significance 5 5 PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) Table 5(on next page) Between-group analysis of VAS, MODI, PSFS, and ROM : Immediate and over short-term p<0.05 statistical signiﬁcant p<0.05 statistical signiﬁcant PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) IQR- Inter quartile Range, VAS: Visual Analog Scale, mm- millimetre, MFR: Myofascial release, SNAGs: Sustained Natural Apophyseal Manuscript to be reviewed 1 Table 5. Between group analysis of outcomes: immediate and over short-term 3 IQR- Inter quartile Range, VAS: Visual Analog Scale, mm- millimetre, 4 MFR: Myofascial release, SNAGs: Sustained Natural Apophyseal 5 Glides, PSFS: Patient Specific Functional Scale, MODI: Modified Oswestry Disability Index 2 p<0.05 statistical significance MEDIAN (IQR) Variable MFR SNAGs p-value Immediate VAS (mm) 4.1(1.9-5.2) 3.05(2.05-4.37) 0.328 Flexion (degrees) 50 (45-59) 52.5 (45-59.5) 0.937 Extension(degrees) 20 (15-30) 21.5 (18.25-29.5) 0.889 Left lateral flexion (degrees) 22 (15-30) 20 (15-25) 0.595 Right lateral flexion (degrees) 20 (17.5-26) 20 (15-27.75) 0.889 PSFS 5.66(4.49-6.16) 5.33(5-6.66) 0.388 Short-term VAS (mm) 2(1.0-3.45) 2(0.85-3.65) 0.674 Flexion (degrees) 52 (45.5-60) 55.5 (50-60) 0.380 Extension(degrees) 25 (19-30) 25 (20-31.5) 0.731 Left lateral flexion (degrees) 20 (20-25) 24.5 (15-28) 0.754 Right lateral flexion (degrees) 20 (17.5-26) 20 (15.75-28) 0.931 MODI (%) 12(6-16) 8(6-15.5) 0.472 PSFS 7(5.83-7.58 7(6.12-7.62) 0.385 Table 5. Between group analysis of outcomes: immediate and over short-term 1 1 6 PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020) PeerJ reviewing PDF \| (2020:08:51682:2:0:NEW 10 Dec 2020)
https://openalex.org/W2562181707	https://link.springer.com/content/pdf/10.1617/s11527-016-0982-6.pdf	English	null	Factors affecting hydraulic conductivity of asphalt mixture	Materials and structures	2,016	cc-by	10,271	M. Aboufoul A. Garcia (&) Department of Civil Engineering, Nottingham Transportation Engineering Centre [NTEC], University of Nottingham, Nottingham NG7 2RD, UK e-mail: alvaro.garcia@nottingham.ac.uk Factors affecting hydraulic conductivity of asphalt mixture M. Aboufoul . A. Garcia Received: 13 July 2016 / Accepted: 30 November 2016 / Published online: 22 December 2016 The Author(s) 2016. This article is published with open access at Springerlink.com Keywords Hydraulic conductivity Air void content Asphalt mixture X-ray CT Weibull model Functional equations Abstract In this paper, the topological properties of air voids in asphalt mixture: air void content, average void diameter, Euler number, genus, enclosed cavities, percolation number, aspect ratio, circularity and tortuosity were analysed using X-ray tomography scans and related to the hydraulic conductivity of a wide range of asphalt mixtures representative of those commonly used in practice. Moreover, a model for the hydraulic conductivity of asphalt mixture that is valid for the whole range of air void content was proposed. The model is based on statistical and physical considerations that lead to a system of functional equations. Finally, the model was related to experi- mental and literature data. It was observed that the range of asphalt mixtures studied hydraulic conduc- tivity is related mostly to the air void content, while the topological parameters (e.g. tortuosity or aspect ratio) are not the primary factors affecting hydraulic con- ductivity. For this reason, the hydraulic conductivity of asphalt mixture commonly used in practice can be predicted using a simple hyperbolic equation with ﬁxed, known, parameters. Materials and Structures (2017) 50:116 DOI 10.1617/s11527-016-0982-6 ORIGINAL ARTICLE 1 Introduction Hydraulic conductivity of asphalt mixture has a sub- stantial impact on the ability of roads to inﬁltrate the surface water to the sublayers. Asphalt with high permeability will reduce the surface runoff quantity [1] and increase trafﬁc security. However, an excess of water retained inasphalt may result inpavementdistress through moisture damage, i.e. losing the bond between aggregates and the binder [2]. Moisture damage is associated with stripping, excessive permanent defor- mation and cracking [3, 4]. Therefore, predicting the hydraulic conductivity and understanding the factors inﬂuencing it will help to establish a balance between deterioration and drainage performance. Hydraulic conductivity of saturated asphalt mixture is deﬁned as the rate of discharge ﬂow of water through the cross unit area under laminar ﬂow conditions [5]. Its value provides an indication of the drainage capacity of asphalt mixture pavements. In addition, hydraulic conductivity is considered to be anisotropic; consequently, the hydraulic conductivity in the vertical and horizontal directions usually presents signiﬁcative differences [6], although the reason for this could be that the methods to measure vertical and horizontal hydraulic conductivities differ. Materials and Structures (2017) 50:116 116 Page 2 of 16 Besides, different authors have stated that hydraulic conductivity increases with the air void content and pore connectivity [7], while it is reduced with the aspect ratio of air void and tortuosity [8]. The relationship between all these topological parameters and hydraulic conductivity is not clear yet. multiple tests and under different conditions. The conclusion obtained from Fig. 1 is that the hydraulic conductivity has a clear relationship with the air void content, following a positive trend. On the other hand, results present a high dispersion, possibly for the variety of techniques used to measure hydraulic conductivity and air void content. On the other hand, previous studies have outlined the inﬂuence of macropores on the hydraulic conduc- tivity of soils. Representative examples are Katuwal et al. [9, 10], who found that pore connectivity and pore size distribution are vital for the ﬂow of air and water through soils. Luo et al. [11–13] found that macroporosity (air void content) and number of connected paths are the best predictors for the hydraulic conductivity of soils. Moreover, Naveed et al. [14] found a linear relationship between air permeability and macroporosity of soils. The main objective of this research is to understand the effect of air void topology on the hydraulic conductivity of asphalt mixture. 1 Introduction With this purpose, permeability tests have been conducted on asphalt test samples with a wide range of air void content, ranging from 8.7 to 26%, built by changing aggregate grada- tion and using different compaction levels. We have aimed that these materials are representative of asphalt mixture in practice. Furthermore, CT-scans have been used to assess the structure of air voids, and their topological properties have been related to the hydraulic conductivity of asphalt mixture. Finally, a Weibull model for hydraulic conductivity has been proposed and used to explain the relationship between hydraulic conductivity and air voids in commonly used asphalt road materials. Today, there are different equations to predict the hydraulic conductivity of asphalt mixture, such as: (1) Kozeny–Carman equation [15], which does not apply to asphalt mixtures with hydraulic conductivities less than 1 9 10-1 cm/s [7]. (2) Exponential [16] power [17] and hyperbolic [18] models, which are arbitrarily selected and do not have a physical or fundamental statistical basis. (3) Norambuena–Garcia model [19] that was deduced from physical principles and is not representative for mixtures with very low hydraulic conductivities [16]. 2.1 Description of materials Moreover, Fig. 1 presents a review of the primary experimental results [19–32] on the saturated hydrau- lic conductivity of asphalt mixtures, measured using Three sets of asphalt mixtures were produced; see Table 1 for the aggregate gradation and binder content 1.0E-03 1.0E-02 1.0E-01 1.0E+00 1.0E+01 1.0E+02 1.0E+03 1.0E+04 1.0E+05 1.0E+06 0 5 10 15 20 25 30 35 Hydraulic conductivity x 10-5 (cm/s) Air void content (%) Norambuena et al. (2013) Literature data in Norambuena et al. (2013) Sprinkel and Apeagyei (2012) Kutai et al. (2007) Kanitpong et al. (2014) Gogula et al. (2004) Charbeneau et al. (2010) Mallick et al. (2003) Yeongsun et al. (2013) Setyawan (2005) Putman (2012) Romero (2000) Cooley (2002) Birgisson (2003) Sprinkel and Apeagyei (2013) Fig. 1 Hydraulic conductivity versus air void content of the asphalt mixtures in the literature [19–32] 1.0E-03 1.0E-02 1.0E-01 1.0E+00 1.0E+01 1.0E+02 1.0E+03 1.0E+04 1.0E+05 1.0E+06 0 5 10 15 20 25 30 35 Hydraulic conductivity x 10-5 (cm/s) Air void content (%) Norambuena et al. (2013) Literature data in Norambuena et al. (2013) Sprinkel and Apeagyei (2012) Kutai et al. (2007) Kanitpong et al. (2014) Gogula et al. (2004) Charbeneau et al. (2010) Mallick et al. (2003) Yeongsun et al. 2.3 Air void content (Avc) The air void content of asphalt mixture was calculated based on the maximum and bulk densities, as it is shown in Eq. (1). Vm ¼ qm qb qm 100% ð1Þ ð1Þ where Vm is the air void content in the mixture (%), qm is the maximum density of the mixture (kg/m3) and qb is the bulk density of the test sample in (kg/m3). 2.1 Description of materials (2013) Setyawan (2005) Putman (2012) Romero (2000) Cooley (2002) Birgisson (2003) Sprinkel and Apeagyei (2013) Page 3 of 16 116 Page 3 of 16 116 Materials and Structures (2017) 50:116 116 Table 1 Asphalt mixture composition Target air void content sieve size (mm) Cumulative aggregate weight % passing Set 1 and 2 Set 3 10% 13% 17% 21% 26% (17%) Max size 14 mm (17%) Max size 28 mm 63 100 100 100 100 100 100 100 40 100 100 100 100 100 100 100 31.5 100 100 100 100 100 100 100 20 99 99 99 100 99 100 92 16 91 91 91 96 91 100 84 14 83 83 82 88 78 93 76 10 59 56 51 53 28 50 51 8 49 44 39 36 18 37 38 6.3 41 34 29 23 14 29 29 4 30 25 21 18 11 22 21 2.8 25 21 19 17 11 19 19 2 21 18 16 14 9 16 16 1 15 13 11 10 7 12 11 0.5 11 10 9 8 5 9 9 0.25 9 8 7 6 4 7 7 0.125 7 6 6 5 4 6 6 0.063 6 5 5 4 3 5 5 Binder content (%) 4.5 4.2 3.8 3.3 3.2 3.8 3.8 used in the mixtures. Asphalt mixture in the three sets aimed to represent commonly used asphaltic materials used for roads. First, ten slabs of 300 mm 9 300 mm 9 50 mm were made with 60/40 penetration grade bitumen and limestone aggregates with 20 mm maximum size. These materials were mixed at 160 C, and roller-compacted at 140 C to the target air void contents: 10, 13, 17, 21, and 26%. Second, a new set of four asphalt slabs were built using the previous aggregate gradation and bitumen content of asphalt mixture with air void content 17%. These materials were also roller compacted to the target air void contents: 10, 13, 21 and 26%. Third, two new slabs with target air void content 17% were designed with maximum aggregate size 14 mm and 28 mm. Finally, 80 cores of 10 cm in diameter and 5 cm in height were extracted from the 16 slabs, ﬁve cores per slab. used in the mixtures. Asphalt mixture in the three sets aimed to represent commonly used asphaltic materials used for roads. 2.1 Description of materials First, ten slabs of 300 mm 9 300 mm 9 50 mm were made with 60/40 penetration grade bitumen and limestone aggregates with 20 mm maximum size. These materials were mixed at 160 C, and roller-compacted at 140 C to the target air void contents: 10, 13, 17, 21, and 26%. Second, a new set of four asphalt slabs were built using the previous aggregate gradation and bitumen content of asphalt mixture with air void content 17%. These materials were also roller compacted to the target air void contents: 10, 13, 21 and 26%. Third, two new slabs with target air void content 17% were designed with maximum aggregate size 14 mm and 28 mm. Finally, 80 cores of 10 cm in diameter and 5 cm in height were extracted from the 16 slabs, ﬁve cores per slab. test samples). In addition, the bulk density of the test specimens was determined according to BS EN 12697, part 6 (2012) [34] by measuring the Bulk Density- Sealedspecimen(drymassdividedbythevolumeoftest specimens calculated as dry minus submerged mass). 2.3 Air void content (Avc) 2.3 Air void content (Avc) 2.2 Density The hydraulic conductivity test was done at room temperature (20 C), following the Florida Method [35] that is a falling head method. Asphalt mixture density was determined according to BS EN 12697, part 5 (2009) [33] by the mathematical method (dry mass divided by the measured volume of 116 Page 4 of 16 Materials and Structures (2017) 50:116 Figure 2 shows the apparatus and experimental setup used to measure hydraulic conductivity of asphalt concrete cores. First, the test samples were saturated for 12 h byplacing themunder water. Then,the samples side was tightly wrapped using a latex membrane (10 cm). Finally, to conﬁne the test samples and prevent the breakage of the latex membrane, the cores were conﬁned in a hollow rubber cylinder and the gaps were sealed using silicon. Then, the rubber cylinder was placed in a hollow metal cylinder with a hole in the bottom to allow water movement, as shown in Fig. 2. operated with an acceleration voltage of 290 kV and a current of 1300 lA. The X-ray microtomography scans were carried out on the micro computed tomography Hounsﬁeld facil- ity at the University of Nottingham. The samples were mounted on a rotational table at a distance of 906.84 mm from the X-ray source. The pixel size obtained was of 96 lm; the scans had an isotropic resolution, meaning that the slice thickness was also 96 lm. The images were originally 16-bit (.tiff format) and the voxel value represented the X-ray attenuation. The images were processed with the software tools Avizo 8.1 and ImageJ, Version 1.49 [37], the images were converted to 8-bit grayscale resolution and cropped to a region of interest (ROI) of 6 cm 9 6 cm 9 4 cm. 3D Gaussian and Median ﬁlters (1 9 1 9 1 Kernel size) were used to reduce the noise in the images. Finally, a graduated acrylic tube was placed on top of the rubber cylinder for measuring the time that 500 ml of water needed to pass through the test samples. The permeability coefﬁcient (k) was calculated based on Darcy’s law [36] as: k ¼ aL At ln h1 h2 ð2Þ ð2Þ Reconstructions of the microstructure were pre- pared by segmenting the materials in the ROI, based on grayscale thresholding. This allows separating aggregates, bitumen and air voids. With this purpose ImageJ was used. 2.2 Density where a is the inner cross-sectional area of the graduated tube (cm2), L is the test sample thickness (cm), A is the test sample cross-sectional area (cm2), t is the time elapsed between the initial head and the ﬁnal head (s), h1 is the initial head across the test specimen (cm), h2 the is ﬁnal head across the test specimen (cm). The thresholded images of the air voids were stacked in ImageJ. Therefore, it is possible to generate surfaces that encase each group of neighboring pixels belonging to a common void space. As it is known that small isolated clusters of voids or grain voxels may correspond to small isolated pores or to noise effects [38], these were removed from the image prior further analyses. All features less than 0.5 mm in diameter were removed from the binary segmented data to prevent being classiﬁed as pores. 2.5 X-ray computed tomography (CT scans) Asphalt cores were scanned under dry condition using a Phoenix v\|tome\|xL scanner; the X-ray source was h1 h2 Asphalt core Graduated Acrylic Tube Base metal cylinder Plastic sink to collect passed water Rubber cylinder 500 ml 10 cm Fig. 2 Schematic representation of the hydraulic conductivity test setup h1 h2 Asphalt core Graduated Acrylic Tube Base metal cylinder Plastic sink to collect passed water Rubber cylinder 500 ml 10 cm 3.1 Relationship between topological properties of air voids 3.1 Relationship between topological properties of air voids The topological constants for all the test samples analysed using X-ray computed tomography have been represented in Table 2. In addition, Table 3 shows the Pearson’s correlation coefﬁcient for all the topological properties analysed. It can be seen that the average void diameter is directly related to the volume of the biggest air void in asphalt mixture (r = 0.90), and that the Euler number has a very high correlation to the number of air voids (r = 0.85) and percolation number (r = 0.84). For that reason, the main topo- logical properties studied will be the average void diameter and Euler number. Furthermore, the average void diameter and Euler number have a very high correlation to the macroporosity, r = 0.91 and 0.69, respectively. As an example, see Fig. 3a that shows the relationship between the Euler number and macroporosity (which is consistent with previous research, see Ref. [42]), and Fig. 3b that relates the average void diameter to the macroporosity. x ¼ N C þ H ð4Þ ð4Þ x ¼ N C þ H The macropores tortuosity was calculated as the ratio of the total macropore length to the total shortest distance between the ends of all macropores in the ROI. Macropore length was obtained with the Skele- tonize 2D/3D and Analyse Skeleton modules in ImageJ [41], with no pruning of the dead ends. In addition, the percolation of air voids was deﬁned as the relationship between the volume of the biggest air void (Vb) and the total volume of air voids in the ROI. This parameter was called Percolation Number (PN). When this value approaches 1, it means that all the air voids are connected [42]. Also, the air void shape properties, i.e. circularity, roundness and aspect ratio, were calculated using the Analyze Particles plugin in ImageJ, by activating the shape descriptors. When the circularity, roundness, and aspect ratio of air voids are close to 1, that represents a circular air void shape, while if the aspect ratio is higher than 1 and the value of circularity and roundness are approaching to 0, that indicates increas- ingly elongated air void shapes. Moreover, in Fig. 3a it can be observed that test samples with air void content lower than 13.7% had positive Euler number, i.e. 3.1 Relationship between topological properties of air voids air voids percolated in the ROI when the air void content was above 13.7%. See for example the difference in the air void size between test samples #2 (Avc, 10.6%) and #5 (Avc, 13.7%) in Fig. 4. Furthermore, the average percolation number increased from 65% to 93%, when the average air void content went from 9.8 to 15.1% (see Table 2). Above 15.1% air void content, the percolation number increased continuously, until it reached a value of practically 100% at 23.8% air void content. This mean that almost all the air voids are connected. 2.6 Topology of air voids The macropore characteristics that were quantiﬁed based on CT Scans included macroporosity, average void diameter, connectivity, and tortuosity. These are commonly used properties to analyze the topol- ogy or soils and porous media [10]. The macrop- orosity was calculated as the volume of macropores per unit of volume in the ROI. Note that from now on, when we refer to air void content results will have been obtained using the mathematical method (dry mass divided by the measured volume of test samples). Moreover, when we speak about macrop- orosity we will refer to results obtained using CT Scan images in the ROI. Rubber cylinder 10 cm Base metal cylinder Asphalt core Plastic sink to collect passed water Fig. 2 Schematic representation of the hydraulic conductivity test setup Fig. 2 Schematic representation of the hydraulic conductivity test setup Page 5 of 16 116 Materials and Structures (2017) 50:116 116 The average void diameter was calculated using a thickness algorithm within the Particle Analyser plugin in ImageJ [26]. Furthermore, the average void diameter, Avd (mm) was calculated as: This correlation coefﬁcient has been considered in absolute value, with values between 0 and 1; 0 being no correlation at all, and 1 a perfect correlation. As an indication, it can be said that when the Pearson’s correlation coefﬁcient ranges from 0 to 0.4, there is a little or very low correlation; from 0.4 to 0.7, there is a moderate correlation and from 0.7 to 1 there is a high or very high correlation [42]. Avd ¼ Pn i¼1 diVi Pn i¼1 Vi ð3Þ ð3Þ where di and Vi are the diameter and volume of each macropore within the ROI. The connectivity of pore networks is commonly expressed based on the Euler number (v). v is a function of the number of isolated air voids (N), the number of redundant connections in the air paths, or genus (C), and the number of completely enclosed cavities (H) [39, 40]. When the Euler number is negative, is an indication that the air voids are percolated [29]. These parameters were determined with the BoneJ particle analyser plugin (Version 1.3.11) in ImageJ [41]. 2.7 Statistics The correlation between the various parameters stud- ied was analysed with linear regression and described using Pearson’s correlation coefﬁcients (r). 2.7 Statistics Besides, the average tortuosity of air voids was approximately 1.21 for test samples with 26% air void 116 Page 6 of 16 Materials and Structures (2017) 50:116 Table 2 Topological constants of asphalt mixture Air void target and max aggregate size Sample number Air void content (%) Macroporosity (%) Average void diameter (mm) Euler number Volume of the biggest air void (mm3) Percolation number Tortuosity Voids perimeter (%) Voids Aspect ratio (%) Voids circularity (%) Voids roundness (%) Hydraulic conductivity (9 10-5 cm/ s) Set 1 10% 1 9.4 10.7 1.13 336 10,415.1 0.67 1.29 7.01 1.89 0.7 0.66 78 2 10.6 12.6 1.14 10 15,164.1 0.83 1.29 235 3 8.7 10 1.11 473 4250.5 0.28 1.29 16 4 10.6 11.7 1.14 239 14,101.6 0.82 1.3 50 13% 5 13.7 13.5 1.72 -189 18,011.7 0.88 1.25 13.75 2.03 0.59 0.69 2407 6 15.4 15.2 1.85 -130 21,442.2 0.92 1.25 4674 7 15.1 15.8 1.75 -399 23,904.6 0.95 1.24 2279 8 16 17.3 1.83 -413 25,284.8 0.96 1.24 3238 17%, 20 mm 9 17.2 16.8 1.78 -315 24,735.7 0.94 1.25 16.05 2.04 0.57 0.68 3533 10 16.4 15.9 2.09 -164 22,038.3 0.91 1.24 5600 11 18.5 18.3 2.14 -326 26,993.8 0.98 1.23 13,068 12 18.3 18.1 2.06 -374 27,524.2 0.98 1.23 9811 21% 13 21.5 21.6 2.05 -627 32,224.3 0.99 1.23 19.23 2.06 0.55 0.72 24,113 14 19.6 19.6 2.12 -457 29,005.2 0.99 1.24 16,421 15 22.9 23.3 2.44 -471 34,191.6 1.00 1.22 26,332 16 21.2 20.6 1.87 -645 30,248.3 0.99 1.24 15,275 26% 17 21.3 26.3 2.61 -610 38,624 1.00 1.22 36.28 2.05 0.49 0.9 42,806 18 24.1 29.4 3.04 -469 42,090.9 1.00 1.21 48,818 19 24.8 28.8 2.6 -531 40,673.4 1.00 1.21 45,535 20 26 30.4 3.14 -429 43,451.2 1.00 1.22 51,136 Set 2 10% 21 12.1 11.2 1.71 -248 16,047.2 0.89 1.25 14.68 2.09 0.55 0.72 2189 22 14.6 14.9 1.98 -527 22,676.3 0.95 1.25 2023 23 12.8 12.3 1.58 -288 18,624.1 0.93 1.24 2831 13% 24 13.3 11.3 1.52 -89 15,070 0.8 1.25 13.9 2.05 0.56 0.71 1015 25 14.6 14.5 1.73 -377 21,788.1 0.93 1.24 5983 21% 26 18.7 10.6 1.58 40 12,869.3 0.85 1.24 15.68 2.03 0.55 0.7 11,822 27 19.6 17.6 1.89 -348 25,136.8 0.97 1.23 14,892 28 18.3 16.4 1.73 -301 21,866.8 0.87 1.24 4729 29 19.7 18.5 2.02 -418 27,610 0.96 1.23 15,815 78 235 16 50 2407 4674 2279 3238 3533 5600 13,068 9811 24,113 16,421 26,332 15,275 42,806 48,818 45,535 51,136 2189 2023 2831 1015 5983 11,822 14,892 4729 15,815 1.13 1.14 1.11 1.14 1.72 1.85 1.75 1.83 1.78 2.09 2.14 2.06 2.05 2.12 2.44 1.87 2.61 3.04 2.6 3.14 1.71 1.98 1.58 1.52 1.73 1.58 1.89 1.73 2.02 Materials and Structures (2017) 50:116 Page 7 of 16 116 116 Air void target and max aggregate size Sample number Air void content (%) Macroporosity (%) Average void diameter (mm) Euler number Volume of the biggest air void (mm3) Percolation number Tortuosity Voids perimeter (%) Voids Aspect ratio (%) Voids circularity (%) Voids roundness (%) Hydraulic conductivity (9 10-5 cm/ s) 26% 30 22.4 22 1.93 -757 32,683.2 0.98 1.23 21.39 2.06 0.52 0.75 24,440 31 23.8 23.6 2.44 -499 37,252.2 0.99 1.22 28,139 32 22.9 22 1.98 -657 33,101.5 0.99 1.22 24,526 Set 3 17%, 14 mm 33 17.9 18.3 1.89 -502 30,583.5 0.99 1.23 16.95 2.04 0.54 0.74 9688 34 15.8 16.8 1.65 -494 26,873.1 0.96 1.24 5173 35 18.3 19.7 1.95 -658 33,182.2 0.99 1.23 12,772 17%, 28 mm 36 17.7 17.5 2.07 -452 27,830.6 0.95 1.23 17.7 2.04 0.54 0.74 11,422 37 18.2 18 1.86 -584 28,074.4 0.97 1.23 6285 38 17.1 16.8 1.68 -445 25,341.5 0.95 1.24 3712 content and increased in asphalt mixture with lower air void content, until it reached the average value of 1.24 in test samples with air void content 13.7%. 2.7 Statistics Below this air void content, the tortuosity increased suddenly to 1.29 in test samples with 10% air void content (see Table 2). This is another indication of the percolation threshold at 13.7% air void content. 3.2 Air void shape properties The average values of the air void shape properties (i.e. air void perimeter, aspect ratio, circularity and roundness) are shown in Table 2. Moreover, Fig. 5 illustrates the relationship between air void perimeter and air void content for the three sets of test samples studied. The most signiﬁcant feature is that the average aspect ratio for test samples with air void content above 10.6% was approximately 2.05 and independent of the air void content, level of com- paction, aggregate gradation, binder content and maximum aggregate size. As a result, the air voids were longer in the horizontal plane than in the vertical one. This effect is similar to that reported in Ref. [43] for asphalt mixture. On the other hand, the aspect ratio averaged 1.89 for test samples with air void content lower than 10.6% (see Table 2). Authors hypothesize that this happened because the air in the pores was subjected to conﬁning pressure during compaction in mixtures with non-connected air voids. Furthermore, Fig. 5 shows the increase in the air void perimeter with the air void content. It can be observed that above 23.8% air void content, there is a sudden increase in the air void perimeter. Further- more, in Fig. 3a the Euler number of test samples above 23.8% air void content does not seem to increase, although more experiments are needed to validate this result. This is an indication that above 23.8% air void content, the connectivity is not improving anymore (see for example the percolation number in Table 2 for mixtures with target air void content 26%). In addition, Table 2 shows that the air void circularity decreases when the air void content increases. Furthermore, it shows also that there is a step reduction in the circularity when the air void content is above 10.6%, as the circularity falls from 0.7 to 0.59. 3.2 Air void shape properties At this point, the roundness and air voids perimeter increased suddenly from approximately 3.3 Hydraulic conductivity of asphalt mixture Table 3 Pearson’s correlation coefﬁcients between all the parameters studied Characteristics of air voids Avc M Avd X C H N Vb PN T k Air void content Avc 1 Macroporosity M 0.91 1 Average void diameter Avd 0.86 0.91 1 Euler number X 0.74 0.69 0.63 1 Genus C 0.30 0.18 0.38 0.08 1 Number of enclosed cavities H 0.24 0.27 0.15 0.03 0.56 1 Number of air voids N 0.33 0.68 0.77 0.85 0.43 0.13 1 Volume of the biggest air void Vb 0.91 0.97 0.90 0.82 0.15 0.20 0.79 1 Percolation number PN 0.68 0.60 0.63 0.84 0.60 0.05 0.85 0.76 1 Tortuosity T 0.87 0.76 0.83 0.82 0.37 0.11 0.94 0.84 0.76 1 Hydraulic conductivity k 0.86 0.93 0.88 0.50 0.24 0.27 0.54 0.85 0.44 0.69 1 R² = 0.6357 -1000 -800 -600 -400 -200 0 200 400 600 (a) (b) 0 5 10 15 20 25 30 35 Euler number Macroporosity (%) Eluer No. Set 1 Eluer No. Set 2 Euler No. Set 3 #5 Fully connected #2 R² = 0.8934 0 0.5 1 1.5 2 2.5 3 3.5 0 5 10 15 20 25 30 35 Avd (mm) Macroporosity (%) Avd Set 1 Avd Set 2 Avd Set 3 #5 #2 Fig. 3.2 Air void shape properties 3 Relationship between macroporosity with a Euler number for all samples studied, b average void diameter Table 3 Pearson’s correlation coefﬁcients between all the parameters studied Characteristics of air voids Avc M Avd X C H N Vb PN T k Air void content Avc 1 Macroporosity M 0.91 1 Average void diameter Avd 0.86 0.91 1 Euler number X 0.74 0.69 0.63 1 Genus C 0.30 0.18 0.38 0.08 1 Number of enclosed cavities H 0.24 0.27 0.15 0.03 0.56 1 Number of air voids N 0.33 0.68 0.77 0.85 0.43 0.13 1 Volume of the biggest air void Vb 0.91 0.97 0.90 0.82 0.15 0.20 0.79 1 Percolation number PN 0.68 0.60 0.63 0.84 0.60 0.05 0.85 0.76 1 Tortuosity T 0.87 0.76 0.83 0.82 0.37 0.11 0.94 0.84 0.76 1 Hydraulic conductivity k 0.86 0.93 0.88 0.50 0.24 0.27 0.54 0.85 0.44 0.69 1 Table 3 Pearson’s correlation coefﬁcients between all the parameters studied R² = 0.6357 -1000 -800 -600 -400 -200 0 200 400 600 (a) 0 5 10 15 20 25 30 35 Euler number Macroporosity (%) Eluer No. Set 1 Eluer No. Set 2 Euler No. Set 3 #5 Fully connected #2 (b) R² = 0.8934 0 0.5 1 1.5 2 2.5 3 3.5 0 5 10 15 20 25 30 35 Avd (mm) Macroporosity (%) Avd Set 1 Avd Set 2 Avd Set 3 #5 #2 Fig. 3 Relationship between macroporosity with a Euler number for all samples studied, b average void diameter 3.2 Air void shape properties Furthermore, the air void roundness increased constantly with the air void content until it reached Materials and Structures (2017) 50:116 116 Page 8 of 16 Table 3 Pearson’s correlation coefﬁcients between all the parameters studied Characteristics of air voids Avc M Avd X C H N Vb PN T k Air void content Avc 1 Macroporosity M 0.91 1 Average void diameter Avd 0.86 0.91 1 Euler number X 0.74 0.69 0.63 1 Genus C 0.30 0.18 0.38 0.08 1 Number of enclosed cavities H 0.24 0.27 0.15 0.03 0.56 1 Number of air voids N 0.33 0.68 0.77 0.85 0.43 0.13 1 Volume of the biggest air void Vb 0.91 0.97 0.90 0.82 0.15 0.20 0.79 1 Percolation number PN 0.68 0.60 0.63 0.84 0.60 0.05 0.85 0.76 1 Tortuosity T 0.87 0.76 0.83 0.82 0.37 0.11 0.94 0.84 0.76 1 Hydraulic conductivity k 0.86 0.93 0.88 0.50 0.24 0.27 0.54 0.85 0.44 0.69 1 R² = 0.6357 -1000 -800 -600 -400 -200 0 200 400 600 (a) (b) 0 5 10 15 20 25 30 35 Euler number Macroporosity (%) Eluer No. Set 1 Eluer No. Set 2 Euler No. Set 3 #5 Fully connected #2 R² = 0.8934 0 0.5 1 1.5 2 2.5 3 3.5 0 5 10 15 20 25 30 35 Avd (mm) Macroporosity (%) Avd Set 1 Avd Set 2 Avd Set 3 #5 #2 Fig. 3 Relationship between macroporosity with a Euler number for all samples studied, b average void diameter 116 Page 8 of 16 Materials and Structures (2017) 50:116 23.8%. 3.3 Hydraulic conductivity of asphalt mixture 23.8%. At this point, the roundness and air voids perimeter increased suddenly from approximately 0.75 to 0.9 and from 25 to 40 mm, respectively. This sudden increase of air void perimeter and roundness for test samples 18, 19 and 20 could happen because asphalt mixture with 26% air void content had a much higher connectivity than the rest, which is caused by the lower amount of ﬁne aggregates and bitumen (see Table 1). In the future more tests will be needed to validate this hypothesis. 23.8%. At this point, the roundness and air voids perimeter increased suddenly from approximately 0.75 to 0.9 and from 25 to 40 mm, respectively. This sudden increase of air void perimeter and roundness for test samples 18, 19 and 20 could happen because asphalt mixture with 26% air void content had a much higher connectivity than the rest, which is caused by the lower amount of ﬁne aggregates and bitumen (see Table 1). In the future more tests will be needed to validate this hypothesis. Figure 6 shows the hydraulic conductivity versus the air void content of all the asphalt mixtures studied. It can be seen that the hydraulic conductivity increased with the air void content, ranging from 15.9 9 10-5 cm/s at 8.7% Avc to 0.51 cm/s at 26% Avc. Furthermore, Fig. 7 shows that experimental hydraulic conductivity data for test specimens with air void contents ranging between 9–11, 14–16 and Page 9 of 16 116 Materials and Structures (2017) 50:116 # 2 AVC 10.6% M 12.6% Avd 1.14 k 235 # 5 AVC 13.7% M 13.5% Avd 1.72 k 2061 (a) (b) Fig. 4 3D reconstruction of macropores from samples number # 2 and # 5 (a) (b) (b) (a) Fig. 4 3D reconstruction of macropores from samples number # 2 and # 5 Moreover, in Table 3 it can be seen that the main factors affecting hydraulic conductivity of asphalt mixture are (1) the average void diameter, (2) macroporosity (or air void content) and (3) volume of the biggest air void, as the Pearson’s correlations are 0.88, 0.93 and 0.85 respectively. A similar result was obtained by Kumar et al. [44], although for soils, who found that saturated hydraulic conductivity had an excellent correlation with air void content. 3.3 Hydraulic conductivity of asphalt mixture In a previous section, it has been commented that the air void content, diameter and volume of the biggest air void have a high correlation (see Table 3). For this reason, it can be concluded that hydraulic conductivity depends mainly on the macroporosity or air void content. 0 5 10 15 20 25 30 35 40 45 5 10 15 20 25 30 35 voids perimeter Air void content (%) R² = 0.5854 Set 1 Set 2 Set 3 #18 Percolation #20 #19 Fully connected Fig. 5 Relationship between 2D voids perimeter properties and air void content for set 1, 2 and 3 0 5 10 15 20 25 30 35 40 45 5 10 15 20 25 30 35 voids perimeter Air void content (%) R² = 0.5854 Set 1 Set 2 Set 3 #18 Percolation #20 #19 Fully connected Fig. 5 Relationship between 2D voids perimeter properties and air void content for set 1, 2 and 3 3.4 Effect of aggregate gradation and compaction level on air voids topology and hydraulic conductivity 19–21% can be successfully ﬁtted to a Weibull distribution function. This shows that differences in hydraulic conductivity for asphalt with similar air voids are based on statistical factors. Table 2 compares the average void diameter of asphalt from set 2 (i.e. built using different compaction forces) to the average void diameter from mixtures with the Materials and Structures (2017) 50:116 116 Page 10 of 16 most similar air void content in set 1 (i.e. with different aggregate gradations). It can be seen that set 2 always had lower average void diameter than set 2. The conclusion is that increasing or decreasing the com- 1.00E-03 1.00E-02 1.00E-01 1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 0 5 10 15 20 25 30 35 Hydraulic conductivity x 10 -5 (cm/s) Air void content (%) 10% 13% 17% 21% 26% Set 2 - 10% Set 2 - 13% Set 2 - 21% Set 2 - 26% Set 3 - Max size 14 mm Set 3 - Max size 28 mm Fig. 6 Experimentally measured hydraulic conductivity versus air void content of the asphalt mixtures 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1 10 100 1000 10000 100000 Probability Hydraulic conductivity x 10-5 (cm/s) Weibull Distribution Air void (14-16%) Air void (9-11%) Air void (19-21%) Experiment Data Fig. 3.3 Hydraulic conductivity of asphalt mixture 7 Probability distribution plot of hydraulic conductivity in three air void content intervals 0 10000 20000 30000 40000 50000 0 10000 20000 30000 40000 50000 Hydraulic conductivity (x 10-5) - Set 2 Hydraulic conductivity (x 10-5) - Set 1 Fig. 8 Comparison of hydraulic conductivity between mix- tures in set 1 and set 2 116 Page 10 of 16 Materials and Structures (2017) 50:116 1.00E-03 1.00E-02 1.00E-01 1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 0 5 10 15 20 25 30 35 Hydraulic conductivity x 10 -5 (cm/s) Air void content (%) 10% 13% 17% 21% 26% Set 2 - 10% Set 2 - 13% Set 2 - 21% Set 2 - 26% Set 3 - Max size 14 mm Set 3 - Max size 28 mm Fig. 6 Experimentally measured hydraulic conductivity versus air void content of the asphalt mixtures 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1 10 100 1000 10000 100000 Probability Hydraulic conductivity x 10-5 (cm/s) Weibull Distribution Air void (14-16%) Air void (9-11%) Air void (19-21%) Experiment Data Fig. 7 Probability distribution plot of hydraulic conductivity in three air void content intervals 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1 10 100 1000 10000 100000 Probability Hydraulic conductivity x 10-5 (cm/s) Weibull Distribution Air void (14-16%) Air void (9-11%) Air void (19-21%) Experiment Data 0 10000 20000 30000 40000 50000 0 10000 20000 30000 40000 50000 Hydraulic conductivity (x 10-5) - Set 2 Hydraulic conductivity (x 10-5) - Set 1 Air void (19-21%) Air void (14-16%) Fig. 7 Probability distribution plot of hydraulic conductivity in three air void content intervals most similar air void content in set 1 (i.e. with different aggregate gradations). It can be seen that set 2 always had lower average void diameter than set 2. The conclusion is that increasing or decreasing the com- paction force for any mixture does not produce higher average void diameter than changing the gradation for mixtures with the same air void content. Furthermore, Table 2 shows that increasing or decreasing the level of compaction did not affect the air void aspect ratio for mixtures with similar air void content. Further- more, Fig. 8 compares the hydraulic conductivities of test samples of set 1 and set 2. 4.2 Statistical derivation of the model The ﬁrst step to develop a model for the hydraulic conductivity of saturated asphalt mixture is to identify the relevant variables involved.The vertical movement of water through an asphalt mixture layer of certain height will depend on (i) an arbitrarily chosen speed of water through the mixture, k0, e.g. the maximum speed possible, when the air void content is a maximum; (ii) the speed at which water crosses the mixture, kt; (iii) the air void content of the asphalt mixture, Avc; (iv) an arbitrary variable, Avc0, which could the maximum possible air void content in the mixture (e.g. 100%) and (v), the probability, p, of having a mixture with certain hydraulic conductivity and air void content: First, let us imagine a very wide set of asphalt mixture test samples, with various air void contents. The mixtures are saturated and water is circulating through the air voids. A vc and K are two random variables that depend on the distribution of air voids through the mixture. In this set of asphalt mixtures, consider those with the same air void content, a. The probability distribution of K, gives the approximate percentage of the mixtures with a similar composition. In addition, their hydraulic conductivity will be K B k. Similarly, if we select a series of asphalt mixtures with a ﬁxed k; A vc would also be a random variable. In this case, the air void content will be A vc B a. Thus, it is possible to write that f(A vc, k) = f(a, K) = p. With this in mind, it is feasible to afﬁrm that f A vc a 3 K ¼ k and f K k 3 A vc ¼ a . m kt; k0; Avc; Avc0; p f g ð5Þ ð5Þ The dimensional matrix is represented in Table 4. Although other variables could have been chosen, the variables in Eq. (5) have been selected for their convenience and easiness of work. If webuildmanytestsamplesof mixtureswithvarious airvoidcontents,underidealﬁxedconditions,e.g.always the same type of aggregates (set A), the relationship K; A vc will look similar to the relationship in Fig. 6. Now, let us assume that if we build a second set of mixtures (set B), by changing the gradation curve or the compaction method slightly, these mixtures will generate a slightly different K; A vc relationship. 3.3 Hydraulic conductivity of asphalt mixture It can be observed that when the values are compared, they align and show very similar values, which means that hydraulic Fig. 8 Comparison of hydraulic conductivity between mix- tures in set 1 and set 2 conductivity is not affected by the compaction mode of the asphalt mixture. Finally, as it can be observed in Table 2, the air void content and diameter of the third set of test samples (i.e. constant air void content and different maximum size of aggregates) showed values inde- pendent of the maximum aggregate size. That is an indication that the maximum aggregate size had no signiﬁcant effect on the hydraulic conductivity of Materials and Structures (2017) 50:116 Page 11 of 16 116 116 asphalt mixture, although more tests have still to be conducted to conﬁrm this point. whole volume of asphalt mixture is occupied with air. Moreover, the dimensionless hydraulic conductivity, K, can be deﬁned as kt/ko. Finally, Eq. (6) can be written as whole volume of asphalt mixture is occupied with air. Moreover, the dimensionless hydraulic conductivity, K, can be deﬁned as kt/ko. Finally, Eq. (6) can be written as 4 Statistical model of hydraulic conductivity p ¼ f K; A vc ð7Þ p ¼ f K; A vc ð7Þ 4.1 Dimensional analysis 4.1 Dimensional analysis 4.2 Statistical derivation of the model The set A curve will not intersect with the curve generated by the Set B of mixture but it will be very close to it. As the K; A vc relationships for set A and set B are not intersecting, it is possible to afﬁrm that The well-known Buckingham P-Theorem allows the representation of these variables in terms of a reduced set of dimensionless variables [45]. In Table 4 it can be seen that the initial set of variables leads to a matrix of dimensions, where L and T refer to the fundamental magnitudes of length and time, respec- tively. Thus, the initial set of ﬁve variables reduces to a set of three dimensionless variables: f kt k0 ; Avc Avc0 ; p ¼ 0 ð6Þ ð6Þ f A vc a 3 K ¼ k ¼ f K k 3 A vc ¼ a : ð8Þ In addition, we can deﬁne Avc=Avc0 as the dimen- sionless air void content, A vc. A vc cannot be negative and its value will be between 0 and 1. A vc will be 0 when asphalt mixture is impermeable and 1, when the ð8Þ Second, we permit that K and A vc in Eq. (7) are random variables if the other is kept ﬁxed. This is the same we have done in the previous paragraph. But now, we let the curves intersect. For this we impose the feasibility assumption: Table 4 Dimensional analysis of the initial set of variables involved in the hydraulic conductivity problem kt kmax Avc Avc0 p L 1 1 3 3 0 T 1 1 0 0 0 f K1; A vc1 ¼ f K2; A vc2 ¼ p; or; A vc1 A vc2 ð9Þ if and only if; K1 K2; 116 Page 12 of 16 Materials and Structures (2017) 50:116 where a(A vci) and d(K) are the Weibull parameters of scale, b(A vci) and e(K) are parameters deﬁning threshold value of hydraulic conductivity and air void content, respectively and c(A vci) and f(K) are Weibull shape parameters of the cumulative distribution function. If we deﬁne A as the set of mixtures for which A vc B a, when K = k, and B is the set of mixtures for which K B k, when A vc = a, Eq. 4.2 Statistical derivation of the model (8) will be equiva- lent to A = B. Now, if a 2 A then, f A vca; K ¼ pa and A vca a; K ¼ k: ð10Þ ð10Þ Equation (13) is a functional equation, in which the unknowns are the six functions a(x), b(x), c(x), d(y), e(y) and f(y). x representing A vci and y representing K. Equation (13) can also be written as In addition, when f A vc; Ka ¼ pa; and A vc ¼ a; ð11Þ ð11Þ i Equation (13) can also be written as Based on the feasibility assumption (9) and on the Eqs. (10) and (11), we can conclude that for a, Ka B k if A vc = a. That is a 2 b. At the end, A = B, which validates expression (8). aðxÞy þ bðxÞ ½ cðxÞ¼ dðyÞx þ eðyÞ ½ fðyÞ: ð14Þ ð14Þ This functional equation has three families of solutions, already obtained by [46]: Third, imagine a hypothetical mixture with all the air voids connected (e.g. mixtures with Avc higher than 23%). There will be a single water conduit in the mixture, which connects both extremes of the material. The mixture is composed of n interconnected parallel sections. The hydraulic conductivity of the mixture, Y, will equal that of the section with lowest Ki, 1 B i B n. Moreover, if we let theithelementbetheonewithlowestairvoidcontent,and thus, Ki = Y. Let A vci be the value of the regressor variable which leads to a hydraulic conductivity Y, for the ith element. Then, A vci = X, and f x; y ð Þ ¼ 1 exp E Rx þ S ð ÞC logðayþbÞ h iE ; ð15Þ f x; y ð Þ ¼ 1 exp C x A ð Þ y B ð Þ þ D ½ E; ð16Þ f x; y ð Þ ¼ 1 exp E x A ð ÞC y B ð ÞD h i : ð17Þ ð15Þ ð16Þ ð17Þ where A, B, C, D, E, R, S, a and b are constants. where A, B, C, D, E, R, S, a and b are constants. 1 exp aðA vciÞK þ bðA vciÞ h icðA vciÞ ¼ 1 exp dðKÞA vci þ eðKÞ h ifðKÞ ; ð13Þ Finally, the asymptotic value of the hyperbole when the hydraulic conductivity has a vesry low value is ð13Þ ð19Þ lim y!1 x ¼ C; where B\0; because x 0: ð19Þ Page 13 of 16 116 of 16 116 Materials and Structures (2017) 50:116 116 1.00E-03 1.00E-02 1.00E-01 1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 0 5 10 15 20 25 30 35 Hydraulic conductivity x 10-5 (cm/s) Air Void content (%) Fitted Model Data from literature Study experiment data =7.561+(−0.8608)/( −0.0557) R2= 0.84 R2= 0.99 Fig. 9 Fitting of Eq. (18) to the experimental data represented in Fig. 6 and literature data 1.00E-03 1.00E-02 1.00E-01 1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 0 5 10 15 20 25 30 35 Hydraulic conductivity x 10-5 (cm/s) Air Void content (%) Fitted Model Data from literature Study experiment data =7.561+(−0.8608)/( −0.0557) R2= 0.84 R2= 0.99 Fig. 9 Fitting of Eq. (18) to the experimental data represented in Fig. 6 and literature data although the dispersion is higher. The reason for this is that the hydraulic conductivity and air void content of each dataset were measured with a range of different techniques. To have an idea of the differences between various measuring methods the authors simply suggest to see the air void content measured with the foil method in Table 2 and observe the differences with the macroporosity, obtained using CT-Scans. The asymptote of the hyperbole when the air void content tends to a very high value is lim x!1 y ¼ A; where A [ 0: ð20Þ ð20Þ Finally, the asymptote of the hyperbola when the hydraulic conductivity tends to 0 is lim y!0 x ¼ C B A ; where C\0: ð21Þ 4.3 Validation of the model lim y!0 x ¼ C B A ; where C\0: ð21Þ ð21Þ Finally, based on all the information presented in this paper, the authors have concluded that (1) hydraulic conductivity of asphalt mixture is mainly related to the air void content and (2) there is a universal relationship between the air void content and hydraulic conductivity given by Eq. (18) and the parameters at the beginning of this section. where A, B, C, D, E, R, S, a and b are constants. For this reason, the authors suggest that future studies on the hydraulic conductivity of asphalt mixture should be focused on quantifying the differences between different testing methods and uniformizing them, to be able to predict hydraulic conductivity accurately based only on the air void content. 4.3 Validation of the model To validate the results, the data have been ﬁtted to Eq. (18) using the least square method. The parameters obtained are A = 7.56, B = 0.86 and C = 0.056. Fig- ure 9 shows the ﬁtting of Eq. (18) to the experimental data in Fig. 6. Moreover, the R square values are 0.99. The high R square value for the experimental data conﬁrms that the Weibull-based statistical model is appropriatetoﬁttheexperimentalresults.Inaddition,the hydraulic conductivity of asphalt mixture in the conﬁg- urations studied is simply related to the air void content, and the topological properties of asphalt mixture, i.e. air voids diameter, aspect ratio, tortuosity, Euler number, etc.haveastronginﬂuence ontheairvoidcontentbutnot on the hydraulic conductivity of asphalt mixture. To validate the results, the data have been ﬁtted to Eq. (18) using the least square method. The parameters obtained are A = 7.56, B = 0.86 and C = 0.056. Fig- ure 9 shows the ﬁtting of Eq. (18) to the experimental data in Fig. 6. Moreover, the R square values are 0.99. where A, B, C, D, E, R, S, a and b are constants. Any of the families (15), (16) and (17) could be used to ﬁt hydraulic conductivity versus air voids data but the zero percentile curves of model (17) degen- erate in the asymptotes. This implies that in model (17) specimens with lowest hydraulic conductivity are not affected by the air void content. For this reason, model (17) will be discarded. f X; A vci ¼ f Ki; Y ð Þ: ð12Þ ð12Þ Since Y B Ki for all i, from the assumption (9) it can be inferred that X B A vci for all i. Since X = A vci then, X = min (A vci). In models (15) and (17), the hydraulic conductivity increases with the air void content. On the other hand, model (15) has the shape parameter variable with hydraulic conductivity, which as it can be seen in Fig. 7a is not correct, because all the points seem to align with a single model independently of the type of mixture used. For this reason, we will select the model (16) to ﬁt hydraulic conductivity data. Additionally, since X given Y and Y given X can be expressed as the minimum of identically distributed random variables, the extreme value theory can be applied [46]. The Fisher–Tippett–Gnedenko theorem [47, 48] establishes that there are only three types of extreme variable distributions (i) the Weibull, (ii) the Gumbel and (iii) the Fre´chet. From these distributions, only the Weibull has a ﬁnite lower limit. This applies to the hydraulic conductivity of asphalt mixture, which is lower-bounded by the molecular diffusion of water through the binder. Finally, forcing the percentile at some point and rearranging the constants again, f(x, y) has the form y ¼ A þ B x C ð18Þ ð18Þ Finally, considering Eq. (12), it can be concluded that where A, B and C are constants, different than in (15), (16) and (17). Conﬂict of interest The authors declare that they have no conﬂict of interest. Conﬂict of interest The authors declare that they have no conﬂict of interest. • When the air void content increases, (1) the average void diameter increases; (2) Euler num- ber decreases; (3) enclosed cavities decrease, and (4) tortuosity decreases. Furthermore, it has been found that the air voids percolate when their content is between 10 and 15.1% and that the Euler number is a good indication of the percolation threshold when it goes from positive to negative. 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 unre- stricted 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 Com- mons license, and indicate if changes were made. • Air voids perimeter, aspect ratio, circularity and roundness are all affected by the air void content, aggregate gradation and compaction mode. For example, compacting the aggregates using higher force reduces the air void content, increases the aspect ratio of the air voids and reduces their circularity. In addition, reducing the air void content below the percolation threshold reduces the aspect ratio and increases the circularity of air voids. Finally, when the air void content is above 23.8%, the perimeter of air voids seems to increase substantially, which is an indication that all the air voids are connected. 5 Conclusions From the results, the following conclusions have been obtained: Besides, Fig. 9 shows that the model proposed has a good ﬁtting with the literature data, R squared 0.84, 116 Page 14 of 16 Materials and Structures (2017) 50:116 • Air void content has been calculated using the foil method and X-ray tomography scans. Both meth- ods produce approximately the same results, with an error of approximately 1.3%. Acknowledgements The authors would like to acknowledge the University of Nottingham which has funded this research through the Dean of Engineering Research Scholarship for International Excellence. • Topological parameters of air voids such as the average void diameter, Euler number, enclosed cavities, percolation number, and tortuosity are all related among them and to the air void content. Funding This study was funded internally by the University of Nottingham. References doi:10.2136/vzj2015.01.0002 Materials and Structures (2017) 50:116 Page 15 of 16 116 116 11. Luo L, Lin H, Halleck P (2008) Quantifying soil structure and preferential ﬂow in intact soil using X-ray computed tomography. Soil Sci Soc Am J 72(4):1058–1069 28. Putman BJ (2012) Evaluation of open-graded friction courses: construction, maintenance, and performance. Rep No FHWA-SC-12-04, Clemson Univ, South Carolina Dept of Transportation 12. 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https://openalex.org/W2948270187	https://www.frontiersin.org/articles/10.3389/fimmu.2019.01307/pdf	English	null	Vitamin D Controls Tumor Growth and CD8+ T Cell Infiltration in Breast Cancer	Frontiers in immunology	2,019	cc-by	8,800	Edited by: Edited by: Zorica D. Juranic, Institute of Oncology and Radiology of Serbia, Serbia Women with low levels of vitamin D have a higher risk of developing breast cancer. Numerous studies associated the presence of a CD8+ T cell inﬁltration with a good prognosis. As vitamin D may play a key role in the modulation of the immune system, the objective of this work was to evaluate the impact of vitamin D on the breast cancer progression and mammary tumor microenvironment. We show that vitamin D decreases breast cancer tumor growth. Immunomonitoring of the different immune subsets in dissociated tumors revealed an increase in tumor inﬁltrating CD8+ T cells in the vitamin D-treated group. Interestingly, these CD8+ T cells exhibited a more active T cell (TEM/CM) phenotype. However, in high-fat diet conditions, we observed an opposite effect of vitamin D on breast cancer tumor growth, associated with a reduction of CD8+ T cell inﬁltration. Our data show that vitamin D is able to modulate breast cancer tumor growth and inﬂammation in the tumor microenvironment in vivo. Unexpectedly, this effect is reversed in high-fat diet conditions, revealing the importance of diet on tumor growth. We believe that supplementation with vitamin D can in certain conditions represent a new adjuvant in the treatment of breast cancers. Reviewed by: Anastasia N. Vlasova, The Ohio State University, United States Vijaya Iragavarapu-Charyulu, Florida Atlantic University, United States Reviewed by: Anastasia N. Vlasova, The Ohio State University, United States Vijaya Iragavarapu-Charyulu, Florida Atlantic University, United States Correspondence: Jacques A. Nunès jacques.nunes@inserm.fr †Present Address: Stéphanie O. Morin, UMR 7268 ADÉS, Aix-Marseille-Université - EFS, CNRS “Biologie des Groupes Sanguins”, Faculté de Médecine, Marseille, France Specialty section: This article was submitted to Nutritional Immunology, a section of the journal Frontiers in Immunology Specialty section: This article was submitted to Nutritional Immunology, a section of the journal Frontiers in Immunology Vitamin D Controls Tumor Growth and CD8+ T Cell Inﬁltration in Breast Cancer Esma Karkeni 1, Stéphanie O. Morin 1†, Berna Bou Tayeh 1, Armelle Goubard 2, Emmanuelle Josselin 2, Rémy Castellano 2, Cyril Fauriat 1, Geoffrey Guittard 1, Daniel Olive 1 and Jacques A. Nunès 1 Esma Karkeni 1, Stéphanie O. Morin 1†, Berna Bou Tayeh 1, Armelle Goubard 2, Emmanuelle Josselin 2, Rémy Castellano 2, Cyril Fauriat 1, Geoffrey Guittard 1, Daniel Olive 1 and Jacques A. Nunès 1* 1 Immunity and Cancer Team, Centre de Recherche en Cancérologie de Marseille, Equipe Labellisée Fondation pour la Recherche Médicale, Institut Paoli-Calmettes, Inserm, CNRS, Aix Marseille Université, Marseille, France, 2 Centre de Recherche en Cancérologie de Marseille, Plateforme d’essai préclinique TrGET, Institut Paoli-Calmettes, Inserm, CNRS, Aix Marseille Université, Marseille, France ORIGINAL RESEARCH published: 06 June 2019 doi: 10.3389/ﬁmmu.2019.01307 INTRODUCTION Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer-related death in women worldwide (1). It is now well established that the immune system plays an important role in disease outcome in patients aﬀected with cancer. However, the assessment of immune response to the tumor in individual patients is diﬃcult (2). Malignant tumors are associated with changes of peripheral blood lymphocyte phenotype and function (3, 4). However, it is obvious that changes detected in the peripheral blood may not necessarily reﬂect the situation in the tumor microenvironment. A large body of evidence suggests that the main events determining the outcome of the host-tumor relationship occur at the tumor site (2). A number of parameters have been assessed as biomarkers of the host immune response in the tumor microenvironment (5–7). The presence of tumor-inﬁltrating lymphocytes (TILs) has been recognized as a biomarker of anti-tumor immune response across a wide range of tumors. The presence of TILs has been Received: 15 March 2019 Accepted: 22 May 2019 Published: 06 June 2019 Keywords: immunonutrition, vitamin D, breast cancer, CD8+ T cells, inﬂammation EO771 Tumor Cell Implantation EO771 cell line, a spontaneously developing breast adenocarcinoma from C57BL/6 mice, was purchased from CH3 BioSystems LLC (Amherst, NY, USA). EO771-GFP+-Luciferase were generated by transduction of GFP-Luciferase lentiviral vector (pRRL-GFP/Luc2 vector), cells were maintained in RPMI-1640 media supplemented with 10% FCS. To establish orthotopic implantation of breast tumors, EO771-Luc/GFP cells were suspended in 100 µL of a mixture of PBS/Matrigel (v/v) (Corning). 2 × 105 EO771-Luc/GFP cells were injected into the 4th inguinal mammary of the two fat pads of 7-week-old female C57BL/6 mice. Tumor growth was monitored by caliper measurements. To avoid tumor necrosis, and in compliance with regulations for use of vertebrate animals in research, animals were euthanized before the tumors reached 1,500 mm3. VD Supplementation in Standard Diet Condition Some studies have indicated that diet may have an inﬂuence in approximately 35% of breast cancer cases (25). Weight gain since early adulthood and obesity have been associated with increased post-menopausal breast cancer risk in several prospective studies (26–29). Indeed, obesity favors an increase in circulating estrogens and entails increased risk of hormone- dependent breast cancer (30–32). The pathway of chronic inﬂammation (pro-inﬂammatory cytokines) induced by adipose tissue also contributes to both tumor transformation and resistance to therapy (33). It is known that the balance between pro-inﬂammatory and anti-inﬂammatory T cells is modiﬁed during obesity-induced inﬂammation in visceral adipose tissue, the pool of pro-inﬂammatory T cells such as CD4+ and CD8+ T cells is increased, and anti-inﬂammatory T cells such as Tregs are decreased in diet-induced obesity (34). Moreover, macrophages in obese tissue are mostly M1 macrophages (also known as classically activated macrophages), which secrete pro- inﬂammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α (35–37). To assess the impact of VD on breast cancer progression in basal conditions, the mice (n = 8–10) were fed ad libitum with standard diet (AIN-93M, 10% energy from lipids) (Safe, Augy, France) and received by gavage the native form of VD (cholecalciferol) (40 International Units (IU)/day per mouse; Sigma Aldrich) (Vitamin D group) or vehicle alone (olive oil) (control group) seven times in 2 weeks. As a fat-soluble vitamin, VD was diluted in olive oil to ensure better absorption, as previously described (38–40). VD supplementation was performed seven days after cell injection. Citation: Karkeni E, Morin SO, Bou Tayeh B, Goubard A, Josselin E, Castellano R, Fauriat C, Guittard G, Olive D and Nunès JA (2019) Vitamin D Controls Tumor Growth and CD8+ T Cell Inﬁltration in Breast Cancer. Front. Immunol. 10:1307. doi: 10.3389/ﬁmmu.2019.01307 June 2019 \| Volume 10 \| Article 1307 Frontiers in Immunology \| www.frontiersin.org Vitamin D Controls Mammary Tumor Growth Karkeni et al. associated with improved prognosis in epithelial ovarian carcinoma (8), endometrial cancer (9, 10), and also breast cancer (11–13). It was demonstrated that TILs obtained from patients with breast cancer, exhibited cytolytic activity against tumor cells (14, 15). An Immunoscore based on the combined analysis of CD8+ plus CD45RO+ T cells in speciﬁc tumor regions is a useful criteria for the prediction of tumor recurrence and survival in patients with early stage colorectal cancer (16). Moreover, a correlation between tumor inﬁltrating CD8+ T lymphocytes in invasive breast cancer and better prognosis has been demonstrated. and its consequences on CD8+ T cell inﬁltration into the tumor. Our data provide evidence that, depending of diet conditions, the VD supplementation has opposite eﬀects on both in CD8+ T cell inﬁltration and tumor growth. Frontiers in Immunology \| www.frontiersin.org Animal Experiments In accordance with European Directive 2010/63/EU on the use of animals for scientiﬁc purposes, the experimental protocol was approved by an Institutional Animal Care and Use Committee. The corresponding Project Authorization (agreement No. 11868- 2017101916238361) was delivered by the French Ministry of Research and Higher Education. 6-week-old female C57BL/6J mice were obtained from Janvier Labs (France). The animals were maintained at 21◦C under a 12 h light, 12 h dark cycle with a 55% humidity level. Behaviors such as reduction in alcohol intake and consumption of fats and red meat, together with an increase in the amount of ﬁbers and vitamin D in the diet, may be direct or indirect protective factors against breast cancer. Vitamin D (VD; cholecalciferol) is mostly known for its major role in bone metabolism and calcium homeostasis (17), but accumulating evidence also suggests that VD is recognized as an immune modulator and has positive eﬀects in the prevention and treatment of cancer (18). Several epidemiologic studies suggest inverse correlations between serum 25-hydroxyvitamin D (25(OH)D) levels and breast cancer development, risk for breast cancer recurrence, and mortality in women with early-stage breast cancer (19–21). Mouse model studies have shown that VD or its biologically active metabolite, calcitriol (1,25(OH)2D3) can inhibit cellular proliferation and angiogenesis and induce diﬀerentiation and apoptosis (22, 23). VD modulates its biological eﬀects by directly regulating target gene expression through the vitamin D receptor (VDR), a ligand-regulated transcription factor and a member of the nuclear receptor superfamily. A recent study has shown that VD, through its nuclear binding to VDR, regulates cell death by modulating autophagy in luminal-like breast cancer-cell models, and also in normal mammary gland in mice (24). The indirect biological eﬀects induced by VD in the tumor microenvironment are poorly investigated. RNA Isolation and Real-Time PCR RNA Isolation and Real-Time PCR Total cellular RNA was extracted using TRIzol reagent (Life Technologies, Villebon-sur-Yvette, France) according to the manufacturer’s instructions. cDNAs were synthesized from 1 µg of total RNA using random primers and Moloney murine leukemia virus reverse transcriptase (Life Technologies, Villebon-sur-Yvette, France). Real-time quantitative RT-PCR analyses were performed using the Power SYBR Green PCR Master Mix (Life Technologies, Villebon-sur-Yvette, France) and detected with a CFX96 Real-Time PCR Detection System (Bio-Rad). For each condition, the expression was quantiﬁed in duplicates and the ribosomal protein 18S mRNA was used as the endogenous control in the comparative cycle threshold (CT) method (2 −(11CT)). The respective sets of primers sequences are 18S-F_cgccgctagaggtgaaattct and 18S-R_cattcttggcaaatgctttcg (for 18S mRNA); Il6-F_acaagtcggaggcttaattacacat and Il6- R_ttgccattgcacaactcttttc (for Il6 gene); Ccl2-F_catccacgtgttggctca and Ccl2-R_gatcatcttgctggtgaatgagt (for Ccl2 gene) Ccl5- F_tgcagaggactctgagacagc and Ccl5-R_gagtggtgtccgagccata (for Ccl5 gene); Ccl11-F_agagctccacagcgcttct and Ccl11-R_gcaggaagttgggatgga (for Ccl11 gene); Cx3cl1- F_catccgctatcagctaaacca and Cx3cl1-R_cagaagcgtctgtgctgtgt (for Cx3cl1 gene), Adiponectin- F_tcctggagagaagggagagaaag and Adiponectin-R_tcagctcctgtcattccaaca (for Adiponectin gene); Pparg-F_caagaataccaaagtgcgatcaa and Pparg- R_gagctgggtcttttcagaataataag (for Pparg gene); Cebpa- F_agcaacgagtaccgggtacg and Cebpa-R_tgtttggctttatctcggctc (for Cebpa gene); Pgc1a-F_ ttccaaaaagaagtcccatacaca; and PGC1a-R_gataaagttgttggtttggcttga (for Pgc1a gene). CD8+ T Cell Depletion To study the impact of the depletion of CD8+ T cells on EO771 tumor growth, CD8+ T cells were depleted by 11 successive injections of the 53–5.8 mAb (BioXCell) reacting with mouse CD8β in female C57BL/6 mice. Then, 2 × 105 EO771-Luc/GFP cells were injected in the fat pad of the mammary glands (n = 12). Tumor growth was monitored by caliper measurements. Tumor Dissociation Mammary tumors were dissociated mechanically and enzymatically using collagenase/hyaluronidase (StemCell Technologies, Grenoble, France) digestion in a water bath under slight agitation at 37◦C for 30 min, to generate single-cell suspensions for ﬂow cytometry staining. To study Granzyme B production (intracellular staining after ﬁxation and permeabilization), samples were placed in the presence of Golgi transport inhibitors (Golgi stop/plug) (BD Biosciences, Le Pont-De-Claix, France) during the entire process of cell extraction. VD Supplementation in High-Fat Diet Conditions To assess the impact of VD on breast cancer progression on inﬂammatory conditions associated with overweight, the mice (n = 8) were fed ad libitum with high-fat diet (245-HF, 45% energy from lipids) (Safe, Augy, France). After 8 weeks of high-fat diet, mice were injected with EO771 cells. Seven days after cell injection, mice received VD (cholecalciferol) (40 IU)/day per mouse; Sigma Aldrich) (Vitamin D group) or Our work aimed to explore the eﬀects of VD on the breast cancer progression in mice from basal to overweight conditions June 2019 \| Volume 10 \| Article 1307 Frontiers in Immunology \| www.frontiersin.org 2 Vitamin D Controls Mammary Tumor Growth Karkeni et al. analysis and the upper phase (adipocytes) was used to quantify inﬂammatory markers. vehicle alone (olive oil) (control group) seven times in 2 weeks. This VD concentration (40 IU of VD per mouse) has been reported as being non-toxic to rodents, without major risk of hypercalcemia (38, 41). Vitamin D Limits Breast Cancer Tumor Progression To control the eﬃciency of VD supplementation, 25(OH)D was measured in the serum of mice at the end of the experiment (Supplemental Figure 1). VD decreased tumor growth starting at day 13 post injection and this diﬀerence was conserved until mouse sacriﬁce (day 20) [VD gavage (284 ± 38.9 mm3) compared with control mice (512 ± 31.2 mm3, P < 0.001)] (Figure 1A). In parallel, we detected a decrease in tumor weight (1.6-fold compared with control group) (Figure 1B). Altogether, these data show that VD supplementation is suﬃcient to reduce tumor growth. Analysis of Plasma Samples Plasma 25(OH)D concentrations were measured using an ELISA kit (Promokine, Promocell). IL-6 cytokine was quantiﬁed with Ready-SET-Go! ELISA (Life Technologies, Villebon-sur-Yvette, France). CCL5 chemokine was quantiﬁed with Instant ELISA kit (Life Technologies, Villebon-sur-Yvette, France). Plasma 25(OH)D concentrations were measured using an ELISA kit (Promokine, Promocell). IL-6 cytokine was quantiﬁed with Ready-SET-Go! ELISA (Life Technologies, Villebon-sur-Yvette, France). CCL5 chemokine was quantiﬁed with Instant ELISA kit (Life Technologies, Villebon-sur-Yvette, France). Isolation of Stromal Vascular Fraction Stromal-vascular cells were isolated from visceral adipose tissue using Clostridium histolyticum collagenase (Sigma Aldrich, Saint- Quentin Fallavier, France) as previously described (38). The samples were incubated at 37◦C under shaking conditions until complete digestion. After ﬁltration on a 70 µm sieve (Corning), samples were centrifuged for 10 min at 400 g. The pellet (stromal vascular fraction) was used for ﬂow cytometry FIGURE 1 \| Vitamin D limits EO771 tumor progression in mice. EO771 cells were injected in the fat pad of the mammary glands (n = 8–10) of female mice fed with standard diet. (A) Tumor size was measured with a caliper. The values of tumor volume (mm3) are reported (Mean ± SEM). (B) Tumors were weighed at the end of the experiment (g). Statistical signiﬁcance between control and VD groups was determined by two-tailed Student’s t-test. *P < 0.001. FIGURE 1 \| Vitamin D limits EO771 tumor progression in mice. EO771 cells were injected in the fat pad of the mammary glands (n = 8–10) of female mice fed with standard diet. (A) Tumor size was measured with a caliper. The values of tumor volume (mm3) are reported (Mean ± SEM). (B) Tumors were weighed at the end of the experiment (g). Statistical signiﬁcance between control and VD groups was determined by two-tailed Student’s t-test. P < 0.001. June 2019 \| Volume 10 \| Article 1307 3 Vitamin D Controls Mammary Tumor Growth Karkeni et al. Statistical Analysis Tissues, but Not Inside the Tumor Since VD is known to regulate inﬂammation (38, 42), we next decided to investigate this in our tumor model. A decrease in IL-6 and CCL5 protein secretion was observed after VD supplementation in mice (2.1 and 1.6-fold, respectively) (Figure 3A). To further characterize this, we studied the eﬀect of VD on cytokine/chemokine gene expression in isolated adipocytes from the visceral adipose tissue. As shown in Figure 3B, the levels of Il6, Ccl5, and Cx3cl1 transcripts were signiﬁcantly decreased (11, 4.5, and 2-fold, respectively) after VD supplementation. In addition, ﬂow cytometry was used to detect pro-inﬂammatory macrophages in the stromal vascular fraction of adipose tissue. We observed a reduction in M1 macrophages (F4/80+CD11b+) population in mice supplemented with VD (1.42-fold compared with control mice) (Figure 3C) whereas no modiﬁcation was observed in macrophage inﬁltration into the tumor (Figure 4A). We also studied these markers in lymphoid organs like tumor-draining lymph nodes and spleen. In lymph nodes, we observed a reduction in F4/80+CD11b+ macrophage inﬁltration (1.4- fold compared with control mice) (Figure 4B). Finally, VD reduced F4/80+CD11b+ and F4/80+CD11c+ macrophage levels in spleen of mice supplemented with VD (1.3 and 1.5-fold, respectively) (Figure 4C). We next evaluated the eﬀect of VD supplementation on adipogenesis. For this purpose, the mRNA levels of adiponectin, peroxisome proliferator-activated receptor gamma (Pparg), PPARγ-coactivator 1α (Pgc1a) and CCAAT enhancer binding protein-α (Cebpa) were quantiﬁed by RT-qPCR. We have shown that VD supplementation in standard diet condition signiﬁcantly limits adiponectin expression in adipocytes with a tendency for a decrease in Pparg, Pgc1a and Cebpa (Supplemental Figure 2). The data were expressed as the mean ± SEM. Prism 5.03 software (GraphPad, San Diego, CA, USA) was used for all statistical analysis. Statistical signiﬁcance between control and VD groups was determined by two-tailed Student’s t-test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. The data were expressed as the mean ± SEM. Prism 5.03 software (GraphPad, San Diego, CA, USA) was used for all statistical analysis. Statistical signiﬁcance between control and VD groups was determined by two-tailed Student’s t-test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Flow Cytometry Analysis CD8+ cells when VD was present. This observation is correlated with an increase of CD44+ T cell subsets (central and eﬀector memory cells; Figure 2B). To further investigate the activation status of the CD8+ T cells that inﬁltrated the tumor, we assessed the cytotoxic T cell phenotype by using a speciﬁc marker, Granzyme B, and for activated T cell phenotype, PD- 1. Indeed, VD-supplemented mice exhibited higher Granzyme B and PD-1 expression levels in tumor inﬁltrating CD8+ T cells (1.3 and 1.4-fold, respectively) as compared with control mice (Figure 2C). Our results argue for an increased T cell recruitment to the tumor site and T cell activation in VD context compared to control. Tumor, stromal vascular fraction, spleen and tumor-draining lymph nodes were used for ﬂow cytometry analysis. Before labeling, cells were incubated in Fc-blocking (CD16/32 antibody). Cells were labeled with ﬂuorescent antibodies against CD3, CD4, CD8, CD44, CD62L, PD-1, CD11b, CD11c, F4/80, Granzyme B (intracellular staining after ﬁxation and permeabilization). Antibodies were purchased from (Life Technologies, Villebon-sur-Yvette, France), Biolegend (San Diego, CA, USA) and BD Biosciences (Le Pont-De-Claix, France). Tumor, stromal vascular fraction, spleen and tumor-draining lymph nodes were used for ﬂow cytometry analysis. Before labeling, cells were incubated in Fc-blocking (CD16/32 antibody). Cells were labeled with ﬂuorescent antibodies against CD3, CD4, CD8, CD44, CD62L, PD-1, CD11b, CD11c, F4/80, Granzyme B (intracellular staining after ﬁxation and permeabilization). Antibodies were purchased from (Life Technologies, Villebon-sur-Yvette, France), Biolegend (San Diego, CA, USA) and BD Biosciences (Le Pont-De-Claix, France). All data were acquired on a FACS FORTESSA 3 lasers ﬂow cytometer (Becton–Dickinson, Le Pont-De-Claix, France) and analyzed with FlowJo software (Tree Star, Ashland, OR, USA). All data were acquired on a FACS FORTESSA 3 lasers ﬂow cytometer (Becton–Dickinson, Le Pont-De-Claix, France) and analyzed with FlowJo software (Tree Star, Ashland, OR, USA). Finally, to further characterize the EO771 model and its sensitivity to CD8+ T cells, these cells were depleted by the injection of CD8β mAb. CD8+ T cell depletion exacerbates tumor growth (680 ± 75 mm3) compared with control mice (413 ± 34 mm3, P < 0.01) (Figure 2D), emphasizing the importance of CD8+ T cells in controlling tumor growth. Frontiers in Immunology \| www.frontiersin.org Vitamin D Promotes CD8+ T Cell Tumor Inﬁltration Numerous studies and more recent pre-clinical or clinical data have highlighted the important role of cytotoxic T cells in breast cancer, especially with respect to immune-checkpoint immunotherapies. Therefore, we wondered whether VD had an impact on CD8+ T cells in our model. After tumor dissociation, we performed immunomonitoring to study the diﬀerent immune populations. We observed a signiﬁcant increase of tumor inﬁltrating CD8+ T cell frequency (Figure 2A). We next studied T cells subsets. We clearly saw a decrease in the naïve T cells (CD62L+) population in TIL Our data show a reduction of pro-inﬂammatory cytokines and macrophages (M1) in accordance with previously published data. June 2019 \| Volume 10 \| Article 1307 Frontiers in Immunology \| www.frontiersin.org 4 Vitamin D Controls Mammary Tumor Growth Karkeni et al. FIGURE 2 \| Vitamin D modulates the CD8+ T cell phenotype in the tumor in basal conditions. Flow cytometry analysis of the tumor from Control and Vitamin D-treated mice. (A) Quantiﬁcation of the CD8+ T cells. (B) Quantiﬁcation of the CD8+ T cell markers (CD44, CD62L) (C) and Granzyme B and PD-1. (D) CD8+ T cells were depleted by the injection of CD8β mAb in C57BL/6J (WT) mice. Then, EO771 cells were injected in the fat pad of the mammary glands (n = 12). Tumor size is measured with a caliper. The values of tumor volume (mm3) are reported (Mean ± SEM). Statistical signiﬁcance between control and VD groups was determined by two-tailed Student’s t-test. P < 0.05, P < 0.01 and P < 0.001. FIGURE 2 \| Vitamin D modulates the CD8+ T cell phenotype in the tumor in basal conditions. Flow cytometry analysis of the tumor from Control and Vitamin D-treated mice. (A) Quantiﬁcation of the CD8+ T cells. (B) Quantiﬁcation of the CD8+ T cell markers (CD44, CD62L) (C) and Granzyme B and PD-1. (D) CD8+ T cells were depleted by the injection of CD8β mAb in C57BL/6J (WT) mice. Then, EO771 cells were injected in the fat pad of the mammary glands (n = 12). Tumor size is measured with a caliper. The values of tumor volume (mm3) are reported (Mean ± SEM). Statistical signiﬁcance between control and VD groups was determined by two-tailed Student’s t-test. P < 0.05, P < 0.01 and P < 0.001. Frontiers in Immunology \| www.frontiersin.org Vitamin D Increases EO771 Tumor Progression in Mice Subjected to a High-Fat Diet reduced Il6, Ccl2, Ccl5, Cx3cl1, and Ccl11 proinﬂammatory cytokine mRNA expression in visceral adipocytes (Supplemental Figure 4B) and no modiﬁcation of adipogenic markers (Supplemental Figure 4C). Surprisingly, in contrast to basal diet conditions, VD supplementation of overweight- mice resulted in a faster tumor progression. Hence VD signiﬁcantly increased tumor growth starting at day 15 post injection. Prior to sacriﬁce, a higher tumor volume was observed in obese mice that received VD gavage (642 ± 45 mm3) compared with control mice (486 ± 53 mm3, P < 0.05) (Figure 5A). The increase of tumor volume correlated with the increase in tumor weight (1.4-fold compared with control group) (Figure 5B). We next evaluated the inﬁltration of CD8 T cells in the tumor. Unexpectedly, we observed in VD-supplemented overweight mice a reduction of inﬁltrating CD8 T cells compared with control mice (Figure 5C) but no modiﬁcation in the CD62L/CD44 T Since obesity increases tumor progression, we next wondered whether VD would reverse overweight-related inﬂammation and reduce tumor progression, similarly to basal diet conditions. Mice were ﬁrst fed for 8 weeks with a high-fat diet (45% energy from lipids) leading to a signiﬁcant gain of weight (Supplemental Figure 3A) and a reduction of 25(OH)D compared with control mice (Supplemental Figure 3B). EO771 were then injected as in previous experiments, and VD was administrated 1 week after tumor cell transplant. Similarly to standard fat diet, VD administration resulted in increase of plasmatic 25(OH)D concentration (Supplemental Figure 4A). VD administration also resulted in a lower systemic inﬂammation with reduced IL-6 and CCL5 plasmatic concentrations (Supplemental Figure 4A), June 2019 \| Volume 10 \| Article 1307 Frontiers in Immunology \| www.frontiersin.org 5 Vitamin D Controls Mammary Tumor Growth Karkeni et al. FIGURE 3 \| Vitamin D limits inﬂammation in plasma, adipocytes and stromal vascular fraction of mice. (A) In EO771 breast cancer model (Standard diet), inﬂammatory cytokines (IL-6 and CCL5) levels were measured in plasma by ELISA (n = 8–10). (B) mRNA levels of Il6, Ccl5 and Cx3cl1 were quantiﬁed through qPCR in isolated adipocytes from visceral adipose tissue (n = 10) and expressed relative to 18S ribosomal RNA. The data are expressed as relative expression ratios. (C) Inﬂammatory macrophages (F4/80+CD11b+) were quantiﬁed in stromal vascular fraction of adipose tissue (n = 8–10) by ﬂow cytometry. The values are presented as the mean ± SEM. Vitamin D Increases EO771 Tumor Progression in Mice Subjected to a High-Fat Diet P < 0.05, P < 0.01, P < 0.001 compared with control group. FIGURE 3 \| Vitamin D limits inﬂammation in plasma, adipocytes and stromal vascular fraction of mice. (A) In EO771 breast cancer model (Standard diet), inﬂammatory cytokines (IL-6 and CCL5) levels were measured in plasma by ELISA (n = 8–10). (B) mRNA levels of Il6, Ccl5 and Cx3cl1 were quantiﬁed through qPCR in isolated adipocytes from visceral adipose tissue (n = 10) and expressed relative to 18S ribosomal RNA. The data are expressed as relative expression ratios. (C) Inﬂammatory macrophages (F4/80+CD11b+) were quantiﬁed in stromal vascular fraction of adipose tissue (n = 8–10) by ﬂow cytometry. The values are presented as the mean ± SEM. P < 0.05, P < 0.01, P < 0.001 compared with control group. Interestingly, these CD8+ T cells exhibited a more active central/eﬀector memory T cell phenotype, which are considered to have increased anti-tumor properties (43). These data are in agreement with data that highlight TILs as an important predictive and prognostic biomarker in patients with breast cancer (44). Ono et al. observed an association between pathological response and TIL score in patients with triple- negative breast cancer, but not in patients with other tumor types (45). Therefore, a general consensus emerged on the central role played by eﬀector T cells in the anti-tumor response. Similar results were obtained in colorectal cancer in which quantiﬁcation of immune cell densities revealed the major positive role of cytotoxic and memory T cells for patient survival (46). The present study indeed also reports that CD8+ T cells inﬁltrating the tumor harbor an activated/cytotoxic phenotype eliciting PD- 1/Granzyme B expression. In line with these results, several studies on cancer patients have reported that PD-1 expression is cell subsets. When studying macrophage population, we observed an increase of F4/80+CD11b+ and F4/80+CD11c+ macrophages in tumor of obese mice supplemented with VD (Supplemental Figure 5A). We also assessed macrophage frequency in other tissues. We showed that there is an increase of M1 macrophages in the tumor-draining lymph nodes (Supplemental Figure 5B). Finally, VD reduced F4/80+CD11c+ macrophage levels in spleen as observed in basal conditions (Supplemental Figure 5C). DISCUSSION In the present study, using female mice injected with EO771 breast cancer cells and supplemented with VD, we report a beneﬁcial role of VD on tumor growth progression and inﬂammation. Indeed, VD supplementation reduced tumor weight and increased CD8+ T cell inﬁltration into the tumor. In the present study, using female mice injected with EO771 breast cancer cells and supplemented with VD, we report a beneﬁcial role of VD on tumor growth progression and inﬂammation. Indeed, VD supplementation reduced tumor weight and increased CD8+ T cell inﬁltration into the tumor. June 2019 \| Volume 10 \| Article 1307 Frontiers in Immunology \| www.frontiersin.org 6 Vitamin D Controls Mammary Tumor Growth Karkeni et al. FIGURE 4 \| Vitamin D modulates macrophage inﬁltration. Frequencies of macrophages “F4/80+CD11b+” and “F4/80+CD11c+” were analyzed by ﬂow cytometry in tumor (A), tumor-draining lymph nodes (B) and spleen (C) (n = 10). The values are presented as the mean ± SEM. P < 0.05, *P < 0.01 compared with control group fed with standard diet. FIGURE 4 \| Vitamin D modulates macrophage inﬁltration. Frequencies of macrophages “F4/80+CD11b+” and “F4/80+CD11c+” were analyzed by ﬂow cytometry in tumor (A), tumor-draining lymph nodes (B) and spleen (C) (n = 10). The values are presented as the mean ± SEM. P < 0.05, *P < 0.01 compared with control group fed with standard diet. highly expressed on tumor inﬁltrating CD8+ T cells in various immunogenic tumors (47–49). high-fat diet fed mice (38). In the present study, we report a beneﬁcial role of VD supplementation on inﬂammation both in basal or overweight conditions. We have shown that VD is able to limit the expression of inﬂammatory markers in adipocytes (Il6, Ccl5, and Cx3cl1) and their secretion (IL- 6 and CCL5) in the plasma. These results are in agreement with the previously reported anti-inﬂammatory eﬀects of VD on adipocytes (38, 52). Taken together, our results reveal the inﬂuence of diet on tumor growth control and CD8+ T cell inﬁltration by VD supplementation. The diﬀerence observed between normal and high-fat diet conditions could be explained by a modiﬁcation in the VD metabolism in adipocytes, in particular Cyp27a1 in overweight mice, which is involved in the ﬁrst hydroxylation of cholecalciferol (data not shown). Frontiers in Immunology \| www.frontiersin.org DISCUSSION This enzyme is known to be increased in adipose tissue of obese mice, which suggests the possible increase of 25-hydroxylation in adipose tissue, resulting in higher local g Surprisingly, in contrast to standard diet condition, VD supplementation of overweight-mice resulted in increased tumor progression without CD8+ T cell inﬁltration (illustrated in Figure 6). The impact of VD supplementation on inﬂammation associated with obesity has been largely described. It has been reported a beneﬁcial role of VD supplementation on weight gain limitation in mice (50, 51). Indeed, such supplementation reduced the weight gain induced by high-fat diet and improved insulin sensitivity. The reduced weight gain in VD supplemented mice is primarily due to a limitation of fat mass accumulation (50). We have also shown in a previous study that VD displays immunoregulatory eﬀects and reduces adipocyte inﬂammation in vitro and in vivo. Indeed, VD supplementation decreases the expression and secretion of a large range of inﬂammatory markers and macrophage inﬁltration in the adipose tissue of June 2019 \| Volume 10 \| Article 1307 Frontiers in Immunology \| www.frontiersin.org 7 Vitamin D Controls Mammary Tumor Growth Karkeni et al. FIGURE 5 \| Vitamin D increases EO771 tumor progression in overweight mice. EO771 cells were injected in the fat pad of the mammary glands (n = 8) of female mice fed with high-fat diet. (A) The tumor size was measured with a caliper. The values of tumor volume (mm3) are reported (Mean ± SEM). (B) Tumors were weighed at the end of the experiment (g). (C) CD8+ T cells and their markers were quantiﬁed by ﬂow cytometry. Statistical signiﬁcance between control and VD groups was determined by two-tailed Student’s t-test. P < 0.05. FIGURE 5 \| Vitamin D increases EO771 tumor progression in overweight mice. EO771 cells were injected in the fat pad of the mammary glands (n = 8) of female mice fed with high-fat diet. (A) The tumor size was measured with a caliper. The values of tumor volume (mm3) are reported (Mean ± SEM). (B) Tumors were weighed at the end of the experiment (g). (C) CD8+ T cells and their markers were quantiﬁed by ﬂow cytometry. Statistical signiﬁcance between control and VD groups was determined by two-tailed Student’s t-test. *P < 0.05. production of 25(OH)D in adipose tissue (53). Several studies about obesity have demonstrated that 25(OH)D is diluted in a higher volume in obese patients (54). Frontiers in Immunology \| www.frontiersin.org DISCUSSION VD supplementation was validated in both models by the quantiﬁcation of the 25(OH)D levels and inﬂammatory cytokines (IL-6 and CCL5) in plasma and mRNA levels of inﬂammatory markers (Il6, Ccl5, Ccl2, Ccl11, and Cx3cl1) in adipocytes. Tumor size was measured and CD8+ T cells were quantiﬁed in the tumor. FIGURE 6 \| Vitamin D controls breast cancer tumor growth and CD8+ T cells inﬁltrating the tumor. VD supplementation was validated in both models by the quantiﬁcation of the 25(OH)D levels and inﬂammatory cytokines (IL-6 and CCL5) in plasma and mRNA levels of inﬂammatory markers (Il6, Ccl5, Ccl2, Ccl11, and Cx3cl1) in adipocytes. Tumor size was measured and CD8+ T cells were quantiﬁed in the tumor. supplementation trials with breast cancer have been conducted, and these have suﬀered from major limitations (as inappropriate dose...) (65). Another recent multicentric, randomized, placebo- controlled trial, with a two-by-two factorial design, of vitamin D3 (cholecalciferol) and omega-3 fatty acids for the prevention of cancer, was conducted in the United States. Supplementation with VD did not result in a lower incidence of invasive cancer than placebo (66). Given the interest in using VD to reduce cancer risk in lean patients, further research is needed, particularly randomized controlled trials, to demonstrate in humans whether individuals with low levels of circulating 25(OH)D are at increased risk of developing cancer (VD in prevention strategy) and whether VD supplements can reduce cancer risk and progression and improve outcome (VD in therapy). In conditions where VD favors access for cytolytic T cells to the tumor site, VD supplementation (T-cell migration inducer) could be combined with an immune checkpoint immunotherapy (T-cell activation inducer) in the treatment of breast cancers. types harboring immunosuppressive properties: among immune cells but also in other cell lineages such as the cancer-associated ﬁbroblasts (CAFs). Adipose tissue is increasingly recognized as an active endocrine organ, which secretes several adipose tissue-speciﬁc hormones including adipokines. It plays a signiﬁcant role in the regulation of homeostasis. Importantly, tumor progression does not necessarily depend on fat mass, but may be inﬂuenced more signiﬁcantly by the speciﬁc metabolic state of the local adipocyte population (60). Adipose tissue is altered during inﬂammatory context related to obesity (61) and breast cancer (62). The factors inducing leukocyte and chemokine inﬁltration in adipose tissue are probably numerous. The paracrine, autocrine and endocrine signals appear to be responsible for this phenomenon (63). DISCUSSION We hypothesized that the increase of adipocytes in overweight-mice reduces 25(OH)D in plasma, which could have an impact on a decreased CD8+ T cell inﬁltration. diﬀerences in diet, treatment periods, cell types, and/or VD treatment protocols. Diﬀerences on CD8+ T cell inﬁltration into the tumor of standard diet and high-fat diet-fed mice could ﬁnally be explained by the decrease of expression levels of chemokine receptor CCR7 which is expressed on central memory T cells and involved in chemotaxis (58). Further studies are needed to investigate the molecular mechanisms involved in the regulation of CD8+ T cell migration inside the tumor under diﬀerent kinds of diets. We next wondered if the observed contrasting eﬀects could be due to a modiﬁcation of adipogenic markers under VD supplementation. Using RT-qPCR approach, we found that VD limited adiponectin expression and a tendency for a decrease in Pparg, Pgc1a, and Cebpa without modiﬁcation in high-fat condition. Our results obtained in normal diet fed-mice are in accordance with previous results reported in mouse models (55) but they cannot explain the diﬀerence between the two conditions. Indeed, the eﬀects of VD on adipogenesis have been analyzed in several animal models, and the majority of these studies suggest that VD plays an inhibitory role in adipogenesis. The few cell culture and supplementation studies that have investigated adipogenesis in human cells indicate that, in contrast to ﬁndings from rodent studies, VD is proadipogenic (56, 57). The diﬀerence in results among mouse and human studies could be due to A CD8+ T cell inﬁltrate is generally associated with a good cancer patient prognosis. However, other immune cell inﬁltrates are associated with a poor patient prognosis [inﬁltrates with immunosuppressive cell types such as regulatory T cells (Tregs), tumor-associated macrophages (TAMs) or myeloid- derived suppressor cells (MDSCs)] (59). In this study, only CD4+CD25+CD127low/- T cell subset has been considered (Supplemental Figure 6), as this subset could contain some Tregs. Flow cytometry analysis showed that VD did not aﬀect the presence of CD4+CD25+CD127low/- T cells in the tumor both in basal and high-fat conditions. It would be of interest to further analyze, in the tumor microenvironment, these cell June 2019 \| Volume 10 \| Article 1307 8 Vitamin D Controls Mammary Tumor Growth Karkeni et al. FIGURE 6 \| Vitamin D controls breast cancer tumor growth and CD8+ T cells inﬁltrating the tumor. Frontiers in Immunology \| www.frontiersin.org DISCUSSION Indeed, adipose tissue and adipocytes in particular produce and release a variety of adipokines notably chemokines which are deﬁned as “cytokines with selective chemoattractant properties,” coordinating leukocyte movement to sites of inﬂammation or injury (64). Most of the breast body is made up of adipose tissue which contains both adipocytes and immune cells. In following studies, it will be important to combine immunomonitoring to adipocyte behavior studies. In conclusion, our results show that VD modulates tumor growth progression and the inﬁltration of the cytotoxic CD8+ T cells in the tumor of mice depending on the type of diet. If our data can be conﬁrmed in human in randomized clinical trials, VD supplementation could represent a new adjuvant in the treatment of breast cancers. The clinical data related to the eﬀect of VD supplementation on breast cancer are still unclear. 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FUNDING This work was supported by institutional grants from the Institut National de la Santé et de la Recherche Médicale (Inserm), Centre National de la Recherche Scientiﬁque (CNRS) and Aix-Marseille Université to CRCM; by the Fondation pour la Recherche Médicale (Equipe FRM DEQ20180339209). EK was supported by a post-doctoral fellowship from the Fondation pour ETHICS STATEMENT la Recherche Médicale (SPF20160936267). GG was supported by a post-doctoral fellowship from the Fondation ARC pour la recherche sur le cancer, then the Janssen Horizon Fund. SOM and BBT were supported by a fellowship from Aix-Marseille Université. DO is senior scholar of the Institut Universitaire de France. In accordance with European Directive 2010/63/EU on the use of animals for scientiﬁc purposes, the experimental protocol was approved by an Institutional Animal Care and Use Committee. The corresponding Project Authorization (agreement No. 11868-2017101916238361) was delivered by the French Ministry of Research and Higher Education. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/ﬁmmu. 2019.01307/full#supplementary-material AUTHOR CONTRIBUTIONS The authors wish to thank Hervé Luche (Ciphe, Marseille), Jean-François Landrier (C2VN, Marseille), Magali Irla (CIML, Marseille) for advice and Valérie Ferrier-Depraetere (Institut Paoli-Calmettes, CRCM) for critical reading of the manuscript. EK, SOM, and JAN (équipe NACRe #39) thank the NACRe (Réseau National Alimentation Cancer Recherche) Network for helpful discussions. We thank Canceropôle PACA for continued support the development of the TrGET preclinical assay platform. We are grateful to the CRCM animal facility for taking care of the mouse strain colonies and the CRCM cytometry platform for FACS analysis. 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https://openalex.org/W3003882727	https://europepmc.org/articles/pmc7038146?pdf=render	English	null	Effects of Gintonin-Enriched Fraction on Methylmercury-Induced Neurotoxicity and Organ Methylmercury Elimination	International journal of environmental research and public health/International journal of environmental research and public health	2,020	cc-by	8,916	Received: 16 December 2019; Accepted: 24 January 2020; Published: 29 January 2020 Abstract: Gintonin is a newly discovered ingredient of ginseng and plays an exogenous ligand for G protein-coupled lysophosphatidic acid receptors. We previously showed that gintonin exhibits diverse eﬀects from neurotransmitter release to improvement of Alzheimer’s disease-related cognitive dysfunctions. However, previous studies did not show whether gintonin has protective eﬀects against environmental heavy metal. We investigated the eﬀects of gintonin-enriched fraction (GEF) on methylmercury (MeHg)-induced neurotoxicity and learning and memory dysfunction and on organ MeHg elimination. Using hippocampal neural progenitor cells (hNPCs) and mice we examined the eﬀects of GEF on MeHg-induced hippocampal NPC neurotoxicity, on formation of reactive oxygen species (ROS), and on in vivo learning and memory functions after acute MeHg exposure. Treatment of GEF to hNPCs attenuated MeHg-induced neurotoxicity with concentration- and time-dependent manner. GEF treatment inhibited MeHg- and ROS inducer-induced ROS formations. Long-term treatment of GEF also improved MeHg-induced learning and memory dysfunctions. Oral administration of GEF decreased the concentrations of MeHg in blood, brain, liver, and kidney. This is the ﬁrst report that GEF attenuated MeHg-induced in vitro and in vivo neurotoxicities through LPA (lysophosphatidic acids) receptor-independent manner and increased organ MeHg elimination. GEF-mediated neuroprotection might achieve via inhibition of ROS formation and facilitation of MeHg elimination from body. Keywords: Ginseng; gintonin; methylmercury; ROS; mercury elimination; neuroprotection Hyeon-Joong Kim 1,2, Sun-Hye Choi 1, Na-Eun Lee 1, Hee-Jung Cho 1 , Hyewhon Rhim 3, Hyoung-Chun Kim 4, Sung-Hee Hwang 5 and Seung-Yeol Nah 1,* 1 Ginsentology Research Laboratory and Department of Physiology, College of Veterinary Medicine, Konkuk University, Seoul 05029, Korea; aidenkim@email.unc.edu (H.-J.K.); vettman@hanmail.net (S.-H.C.); dhkdnrl@naver.com (N.-E.L.); hyeonjoongk@gmail.com (H.-J.C.) 1 Ginsentology Research Laboratory and Department of Physiology, College of Veterinary Medicine, Konkuk University, Seoul 05029, Korea; aidenkim@email.unc.edu (H.-J.K.); vettman@hanmail.net (S.-H.C.); dhkdnrl@naver.com (N.-E.L.); hyeonjoongk@gmail.com (H.-J.C.) y j g g 2 Biomedical Research Imaging Center, University of North Caroline at Chapel Hill, Chapel Hill, NC 27599, USA 3 Center for Neuroscience, Korea Institute of Science and Technology, Seoul 02792, Korea; hrhim@kist.re.kr 4 Neuropsychopharmacology and Toxicology program, College of Pharmacy, Kangwon National University, Chunchon 24341, Korea; kimhc@kangwon.ac.kr Center for Neuroscience, Korea Institute of Science and Technology, Seoul 02792, Korea; hrhim@kist.re.kr 4 Neuropsychopharmacology and Toxicology program, College of Pharmacy, Kangwon National University, Chunchon 24341, Korea; kimhc@kangwon.ac.kr 5 Department of Pharmaceutical Engineering, College of Health Sciences, Sangji University, Wonju 26339, Korea; d639sunghee@hotmail.com Korea; d639sunghee@hotmail.com * Correspondence: synah@konkuk.ac.kr; Tel.: + 82-2-450-4154 Received: 16 December 2019; Accepted: 24 January 2020; Published: 29 January 2020 International Journal of Environmental Research and Public Health International Journal of Environmental Research and Public Health www.mdpi.com/journal/ijerph Eﬀects of Gintonin-Enriched Fraction on Methylmercury-Induced Neurotoxicity and Organ Methylmercury Elimination Hyeon-Joong Kim 1,2, Sun-Hye Choi 1, Na-Eun Lee 1, Hee-Jung Cho 1 , Hyewhon Rhim 3, Hyoung-Chun Kim 4, Sung-Hee Hwang 5 and Seung-Yeol Nah 1,* 1. Introduction Mercury (Hg) is one of heavy metal elements but is the most poisonous environmental pollutants and is highly toxic to multiple organs including brain. The primary mercury in atmosphere derives from natural sources such as volcanic eruptions and decay of mercury-containing sediments. The second largest source of mercury is from fossil fuel combustion for diverse purposes [1]. In nature, mercury exists as three forms: i.e., elementary mercury or mercury vapor, inorganic mercury and organic Int. J. Environ. Res. Public Health 2020, 17, 838; doi:10.3390/ijerph17030838 2 of 14 Int. J. Environ. Res. Public Health 2020, 17, 838 mercury. Methylmercury (MeHg) is one of organic forms of mercury [2]. MeHg exhibits strong neurotoxic eﬀects to brain including neurological and developmental damages in experimental animals and human [3–6]. Organic mercury such as MeHg is also found in ﬁshes, shellﬁshes and livestock that were fed with ﬁshmeal, pesticides, fungicides, and insecticides [7–9]. One of most severe problems in human MeHg exposure is that MeHg is almost completely absorbed in gastrointestinal systems and easily penetrates the blood–brain barrier and accumulate in brain [10]. In addition, MeHg is absorbed into the placenta and stored in fetal brain in concentrations over maternal blood level [10,11]. Thus, long-term exposure to MeHg through foods such as ﬁshes with high amount of MeHg induces various symptoms and threats to human health. MeHg toxicity is a second-most common cause of acute heavy metal poisoning derived from foods and other environmental sources [1]. Ginseng, the root of Panax ginseng Meyer, has been used for keeping human healthy from a long time ago [12]. Recent studies show that ginseng contains a newly discovered bioactive ingredient named gintonin [13]. Further analysis revealed that gintonin is a complex of carbohydrates, lipid, and ginseng proteins and the main functional component of gintonin is lysophosphatidic acids (LPA, 1-acyl-2-hydroxy-sn-glycero-3-phosphate) [14]. The previous reports demonstrated that gintonin exhibits diverse in vitro and in vivo physiological and pharmacological eﬀects through LPA receptor signaling pathways in peripheral and central nervous systems. [15] Although gintonin is a kind of complex compound and exhibits a high aﬃnity with LPA receptors on neuronal cells, the previous reports did not show other functions of gintonin besides LPA receptor-mediated actions. Recently, we developed a method for gintonin-enriched fraction (GEF) preparation to mass-produce gintonin from ginseng. GEF contains various bioactive phospholipids including LPAs, phosphatidic acids, and free fatty acids such as linoleic acid [14]. 1. Introduction On the other hand, one of methods for the treatment of MeHg poisoning is to facilitate the elimination of MeHg from body or anti-oxidant agents for the inhibition of reactive oxygen species (ROS) formation by MeHg [16–18], since Hg is known to be accumulated in body, especially brain and Hg induces oxidative stresses by producing ROS to cause brain mitochondrial dysfunctions [16,18]. However, it was not directly demonstrated whether GEF could attenuate MeHg-induced in vitro and in vivo neurotoxicities and could further facilitate MeHg elimination from body. In the present study, we investigated whether GEF could attenuate MeHg-induced neurotoxicity, reduce accumulations of MeHg in main organs, and improve MeHg-induced learning and memory dysfunctions. Here, we found that GEF attenuated MeHg-induced neurotoxicity in hippocampal neural progenitor cells (hNPCs) and reduced concentrations of MeHg in various organs and improved learning and memory damaged by MeHg. We further discuss the possible molecular mechanisms on in vitro and in vivo GEF-induced neuroprotection and facilitation of MeHg excretion and pharmacological roles of GEF in MeHg neurotoxicity. 2.1. Materials Gintonin-enriched fraction (GEF) was prepared according to a previously described method [19]. Brieﬂy, 1 kg of 4-year-old ginseng was ground into small pieces (>3 mm) and reﬂuxed with 70% fermentation ethanol 8 times for 8 h at 80 ◦C each. The ethanol extracts (350 g) were concentrated. Ethanol extract was dissolved in distilled cold water at a ratio of 1 to 10 and stored in a cold chamber at 4 ◦C for 24 h. The supernatant and precipitate produced by water fractionation, after the ethanol extraction of ginseng, was separated by centrifuge (3,000 rpm, 20 min). The precipitate was lyophilized after being centrifuged. This fraction was designated GEF and had a yield of 1.3%. Other agents were purchased from Sigma-Aldrich (Plymouth Meeting, PA, USA). In vivo study, GEF was treated dissolved in saline. Control groups were treated saline only. Int. J. Environ. Res. Public Health 2020, 17, 838 3 of 14 2.4. Cell Viability As a general protocol, cells were plated in 96-well plates (20,000 cells/well) overnight, wells were divided into indicated treatment groups, and then were subjected to MeHg treatment for the indicated agents and time, and then the viability from each treatment was determined by WST-1 (water-soluble tetrazolium salt-1). WST-1 cell proliferation assay (catalog no. ab155902, Abcam plc.) was performed and analyzed on a plate reader (Synergy 2, BioTek). The stable tetrazolium salt WST-1 is cleaved to a soluble formazan by a complex cellular mechanism that occurs primarily at the cell surface. This bio-reduction is largely dependent on the glycolytic production of NAD(P)H in viable cells. Therefore, the amount of formazan dye formed directly correlates to the number of metabolically active cells in the culture. Cells grown in a 96-well tissue culture plate are incubated with the WST-1 reagent for 0.5–4 h. After this incubation period, the formazan dye formed is quantitated with a scanning multi-well spectrophotometer (Spectra Max 190, Molecular Devices, Sunnyvale, CA). The OD of each well was measured by a plate reader with a ﬁlter setting at 570 nm. Cell survival rate was obtained as follows: Survival rate of vehicle control without MeHg was set as 100% and calculate survival rate in the presence of 350 nM MeHg with or without various concentrations of GEF. 2.3. Primary Culture of Hippocampal Neural Precursor Cells (NPCs) Hippocampal NPC cultures were prepared as follows. Brieﬂy, embryos at embryonic day 14.5 (E14.5) were dissected out of C57BL/6 adult pregnant female mice. The hippocampal region of the embryonic brain was isolated in calcium/magnesium free Hanks’ Balanced Salt Solution (HBSS; Gibco), seeded at 2 × 105 cells in 10 cm culture dishes (Corning Life Sciences), which were precoated with 15 µg/mL poly-L-ornithine (Sigma) and 1 µg/mL ﬁbronectin (Invitrogen) and then cultured for 5–6 days in serum-free N2 medium supplemented with 20 ng/mL bFGF (R&D Systems). Cell clusters generated by precursor cell proliferation were dissociated in HBSS and plated at 2 × 104 cells per well on coated 24-well plates, 2 × 105 cells per well on coated 6-well plates, and 8 × 105 cells per dish on coated 6-cm culture dishes. The plated cells were allowed to proliferate further in N2 + bFGF up to 70–80% cell conﬂuence before induction of diﬀerentiation. All experiments were carried out using the passage 1 (P1) neural precursor cells. 2.2. Animals All experimental animal procedures (C57Bl/6, 2-month old, female black mouse) were approved by the Institutional Animal Care and Use Committee (IACUC) at Konkuk university under approval number 2015–0009. C57Bl/6 mice were housed under controlled conditions (12h light/dark cycles, 24 ◦C temperature, and fed ad libitum with automatic watering). Experiments were performed in accordance with the NIH guidelines. Further, the experiments were conducted in accordance with the principles of laboratory animal care (National Research Council US Committee for the Update of the Guide for the Care and Use of Laboratory Animals 2011). 2.7. Behavioral Test Mice were divided into ﬁve groups such as control, MeHg (2 mg/kg, 3 weeks, p.o.) alone, MeHg + GEF (50 mg/kg, 3 weeks, p.o.), MeHg + GEF (100 mg/kg, 3 weeks, p.o.) and GEF (100 mg/kg, p.o.) alone. Two diﬀerent dosages of GEF was administered before 30 min MeHg. After 21 days Morris water maze test was performed during consecutive 6 days. The Morris water maze was used to assess spatial reference learning and memory as previously described [22]. The maze protocol is similar to that described by Morris (1984) with modiﬁcations for mice. The maze system included a white circular pool, 1.4 m in diameter and 0.6 m in height, ﬁlled up to a depth of 30 cm with water maintained at a temperature of 21 ± 0.5 ◦C [23]. The transparency of the pool water was abolished by addition of 150 mL of non-toxic white tempera paint. An escape platform (10 cm in diameter) made of Plexiglas was positioned 1 cm below the water surface and placed in the center of one quadrant of the pool, 15 cm from the pool’s edge. The platform remained in the same position throughout the training day and was removed from the pool during the probe test. Extra maze cues surrounding the maze were placed at ﬁxed locations in the surrounding curtains, and visible to the mice while these were in the maze. Maze performance was recorded by a video camera suspended above the maze that was interfaced with a video tracking system (HVS Imaging, Hampton, UK). On the ﬁrst day (visible platform), mice were placed into the water facing the wall. If a mouse reached the platform before a 60 s cutoﬀ, it was permitted to stay on the platform for 5 s then returned to the home cage. If the mouse did not reach the platform in a 60 s, it was smoothly led onto the platform and allowed to stay for 20 s before returning to the home cage. This procedure was repeated for three more days, each starting in a diﬀerent quadrant. Repeating this four-trail training procedure is for all mice. On the next 4 days (hidden platform), the mice were trained as on the ﬁrst day but with the platform submerged. After 5 consecutive days of pre-training, the mice were tested with the platform removed. 2.7. Behavioral Test During the test, mice were placed into the water from the opposite quadrant where the platform used to be, and were tested for 60 s. Each training session consisted of three trials separated by a 10 min intertrial interval (ITI). To assess the performance in the water maze, mean escape latencies and swim distance were analyzed. The statistical analysis of behavioral data was described below. During experiments, we could not observe the morbidity or mortality in all treatment groups. 2.5. Measurement of Reactive Oxygen Species Cells were plated in 96-well plates (20,000 cells/well) overnight, wells were divided into indicated treatment groups, and then the assay was performed as described in reference paper [20]. The medium from each well was removed and each culture was treated with 10 AM H2DCF-DA (100 Al/well prepared in HBSS) for 20 min at 37 ◦C. After removal of H2DCF-DA, cells were treated with MeHg (or pyocianin, 100 Al/well prepared in HBSS) for 20 min at 37 ◦C. The ﬂuorescence of each well was detected by a ﬂuorescence plate reader (Spectra Max Gemini XS, Molecular Devices, Sunnyvale, CA) with the following settings: excitation 485 nm, emission 535 nm, and cutoﬀ530 nm. A set of cells without H2DCF-DA treatment was used as “blank” in each experiment. The reading of blank was subtracted before the results were plotted, expressed as net ﬂuorescence units (FU). Int. J. Environ. Res. Public Health 2020, 17, 838 4 of 14 2.6. Determination of Hg Concentration from Various Organs after MeHg Treatment 2.6. Determination of Hg Concentration from Various Organs after MeHg Treatment Mice were divided into ﬁve groups such as control, MeHg (2 mg/kg, p.o.) alone, MeHg + GEF (50 mg/kg, p.o.), MeHg + GEF (100 mg/kg, p.o.), and GEF (100 mg/kg, p.o.) alone. At each time points (0, 7, 14, and 21 days) plasma, brain, kidney and liver were collected after sacriﬁce. 0 time means 15 min after MeHg administration. Two diﬀerent dosages of GEF was administered before 30 min MeHg. Concentrations of Hg in plasma, brain, kidney or liver were determined in Korea Institute of Science and Technology (KIST) using a ﬂameless atomic absorption spectrophotometer equipped with a model GF-AAS Zeeman graphite furnace (Thermo Inc., Cambridge, UK). Brieﬂy, samples were diluted to 0.2% in Triton X-100 with 1% nitric acid and 0.2% diammonium hydrogen phosphate. 15 ml aliquots were injected into a graphite boat after mixing roughly. Determination of total Hg in each samples was performed by using a direct mercury analyzer (DMA-80; Milestone Inc., Shelton, CT, USA) with a gold-amalgam method [21]. Hg analyses were validated using standards and a standard reference material (SRM, 955c level 2; National Institute of Standards and Technology, Gaithersburg, MD, USA). 2.8. Data Analysis Statistical analyses were performed using one-way analysis of variance (ANOVA) or one-way repeated measures ANOVA followed by post-hoc Fisher’s least signiﬁcant diﬀerence (LSD) test. Unpaired 2-tailed t-test and Pearson’s correlation coeﬃcient were used for in vitro and in vivo statistical comparisons using IBM SPSS ver. 21.0 (IBM, Chicago, IL, U.S.A.) Values were expressed as 5 of 14 Int. J. Environ. Res. Public Health 2020, 17, 838 mean ± standard error of the mean (SEM). Statistical signiﬁcance was set at P < 0.05. The calibration curves were evaluated via weighted linear (1/x) and quadratic (1/x2) regression with their correlation coeﬃcients (R ≥0.95). The linearity of each calibration curve was determined by peak area analyte/peak area internal standard at six concentration levels of individual analytes. All values are presented as mean ± SEM (%) from the sample of GEF. In behavioral tests, a two-way ANOVA followed by the Bonferroni posttest was used to analyze escape latency and travel distance. The results are displayed as mean ± standard error of the mean (SEM). Multiple t test followed by the Sidak–Bonferroni method was used to analyze the time in quadrant. A one-way ANOVA followed by the Bonferroni posttest was used to analyze and obtain statistics of the entries to target quadrant. 3.1. Determinations of ED50 and Treatment Time in MeHg-Induced hNPC Cytotoxicity We determined the ED50 and treatment time for MeHg-induced cytotoxicity. For this, we ﬁrst treated hPNCs for 24 h with various concentrations of MeHg with 20,000 cells/96 well and the cell viability on MeHg treatment was determined. Figure 1A shows that MeHg induced a dose-dependent hNPC death. Thus, various concentrations of 100, 150, 250, 500, and 1000 nM MeHg induced cell death by 8.2 ± 2.6%, 23.8 ± 6.0%, 41.2 ± 6.3%, 67.7 ± 6.7%, and 89.0 ± 9.8%, respectively. (Control group’s natural cell death is 3.2 ± 1.2%), The concentration of MeHg to induce 50% cell death was about 350 nM. Next, we did experiments to determine the “commitment time point” of MeHg-induced cell death. We ﬁrst treated cells with 350 nM MeHg for various indicated time, changed with fresh medium without MeHg, and then determined the cell viability 24 h after the experiment was initiated. We found that treatment of MeHg with 350 nM for 0.2, 0.5, 1, 2, 4, 8, 16, 24, or 48 h induced cell death by 1.5 ± 1.3%, 9.4 ± 2.3%, 13.5 ± 4.6%, 18.4 ± 6.3%, 24.3 ± 4.6%, 55.3 ± 5.7%, 65.6 ± 7.3%, 71.2 ± 8.7%, and 83.4 ± 11.2%, respectively. These results indicated that MeHg cytotoxicity is also dependent on its concentration and incubation time in cultures (Figure 1B). Based on Figure 1A,B, we performed subsequent experiments to conﬁrm whether GEF attenuates in vitro 350 nM MeHg-induced neurotoxicity after 24 h treatment. Figure 1. Eﬀects of MeHg on cell death and eﬀects of gintonin-enriched fraction (GEF) with MeHg in hippocampal neural progenitor cell (hNPC) survival. (A) MeHg induces cell death in a concentration-dependent manner. The indicated concentration of MeHg was treated for 24 h. (B) MeHg-mediated cell death was time-dependent. hNPCs were treated with the indicated time and Figure 1. Eﬀects of MeHg on cell death and eﬀects of gintonin-enriched fraction (GEF) with MeHg in hippocampal neural progenitor cell (hNPC) survival. (A) MeHg induces cell death in a concentration-dependent manner. The indicated concentration of MeHg was treated for 24 h. (B) MeHg-mediated cell death was time-dependent. hNPCs were treated with the indicated time and 6 of 14 Int. J. Environ. Res. Public Health 2020, 17, 838 subjected to WST-1 assay as described in the Materials and Methods section. (C) Eﬀects of GEF on the decrease of MeHg-induced cell survival rate. 3.1. Determinations of ED50 and Treatment Time in MeHg-Induced hNPC Cytotoxicity hNPCs were incubated MeHg with the control vehicle (Control) or various concentrations of GEF with WST-1 assay. GEF increased cell survival rate over 30 µg/ml. The indicated concentration of GEF was pretreated 24 h before MeHg (350 nM) and the degree of cell survival rate was measured as described in Methods. LPA1/3 receptor antagonist (Ki) (Ki16425, 10 µM) or phospholipase C inhibitor (U) (U73122, 5 µM) was used. (D) GEF-mediated attenuation of cell death was observed after 24 h. Cells were treated with 30 µg/ml GEF. Data are presented as the mean ± SEM (n = 9; p < 0.01, compared with untreated control). 3.2. GEF-Mediated Attenuation on MeHg-induced Cell Death is Independent on LPA Receptor Signaling Pathways As shown in Figure 1C,D, GEF pretreatment to hNPCs attenuated MeHg-induced cell death with concentration-manner. Thus, pretreatment of cells with 30 and 100 µg/ml GEF for 24 h signiﬁcantly increased cell viability from control 57.0 ± 3.5 and 58.6 ± 3.0% to 73.3 ± 5.8 and 71.7 ± 8.1%, respectively. GEF pretreatment to hNPCs also attenuated MeHg-induced cell death with time-manner. Thus, pretreatment of cells with 30 and 100 µg/ml GEF for 24 and 48 h signiﬁcantly increased the viability from control 57.8 ± 4.5 and 55.6 ± 4.9% to 74.5 ± 6.6 and 77.8 ± 11%, respectively. When we also examined co-treatment eﬀect of GEF with MeHg, co-treatment eﬀect of GEF with MeHg showed almost same degree of protective eﬀects against MeHg cytotoxicity (data not shown). Next, we examined whether the GEF eﬀect against MeHg-induced cytotoxicity can be mediated by LPA1/3 receptor signaling pathway, since the previous reports show that most of GEF-mediated cellular eﬀects achieved via LPA receptor activation [24]. We found that LPA receptor1/3 antagonist, Ki16425, did not prevent GEF-mediated attenuations on MeHg-induced cytotoxicity. In addition, LPA C18:1, phospholipase C inhibitor, U73122, and LPA itself also did not attenuate MeHg-induced cytotoxicity (Figure 1C), indicating that GEF-mediated attenuation against MeHg-induced cytotoxicity is not related with LPA and LPA receptor-mediated signaling pathways. 3.3. Eﬀects of Phosphatidic Acids on MeHg-induced Cytoxicity In previous report, we showed that GEF consists of various lipid-derived active ingredients [14]. Since phosphatidic acids (PAs) such as PA 18:2–18:2 and PA 18:2–16:0 are major phospholipids in GEF with negative charges and MeHg exhibits positive charges, we next examined whether these PAs could represent GEF-mediated attenuation of MeHg cytotoxicity. As shown Figure 2A and B, pretreatment of PA 18:2–18:2 or PA 18:2–16:0 did not attenuate MeHg-induced cytotoxicity, indicating that PA components in GEF do not participate in attenuation of MeHg-induced cytotoxicity. In addition, we also found that ginseng total extract (GTE) and crude ginseng total saponin (cGTS) fraction had no eﬀects on MeHg-induced cytotoxicity, also indicating that GEF is an active and unique fraction to prevent MeHg-induced cytotoxicity from ginseng (Figure 4). Figure 2. Eﬀects of GEF, PA 16:0–18:2, PA 18:2–18:2 on MeHg-induced cytoxicity. (A,B) The indicated concentration of PA 16:0–18:2 and PA 18:2–18:2 have no eﬀects on MeHg-mediated cell death. MeHg (350 nM) was treated for 24 h. Data are presented as the mean ± SEM (n = 9; p < 0.01, compared with untreated controls). Figure 2. Eﬀects of GEF, PA 16:0–18:2, PA 18:2–18:2 on MeHg-induced cytoxicity. (A,B) The indicated concentration of PA 16:0–18:2 and PA 18:2–18:2 have no eﬀects on MeHg-mediated cell death. MeHg (350 nM) was treated for 24 h. Data are presented as the mean ± SEM (n = 9; p < 0.01, compared with untreated controls). Int. J. Environ. Res. Public Health 2020, 17, 838 7 of 14 7 of 14 3.4. Eﬀects of GEF on MeHg-induced Reactive Oxygen Species (ROS) Generation in hNPCs 3.4. Eﬀects of GEF on MeHg-induced Reactive Oxygen Species (ROS) Generation in hNPCs On the other hand, MeHg is well-known to cause intracellular ROS generation for induction of neuronal cytotoxicity [16,18]. However, it is unknown whether MeHg-induced cytotoxicity in hNPCs is due to ROS generation and whether GEF could attenuate MeHg-induced ROS generation. Next, we ﬁrst examined whether MeHg can produce ROS in hNPCs and GEF can decrease MeHg-induced ROS production in hNPCs. For this, hNPCs were treated with MeHg for 24 h, and then the ROS generation was determined. Results showed that MeHg caused an increase of ROS in a concentration- and time-dependent manner in hNPCs (Figure 3A,B). However, the presence of GEF greatly attenuated MeHg-induced ROS formation and the inhibitory eﬀects of GEF on MeHg-induced ROS formation were also concentration- and time-dependent manner (Figure 3C,D). 3.3. Eﬀects of Phosphatidic Acids on MeHg-induced Cytoxicity In addition, to conﬁrm whether GEF-mediated attenuation of MeHg-induced cytoxicity achieves via the inhibitions of ROS formation, we also tested the eﬀects of GEF on pyocyanin, a ROS inducer. As shown in Figure 4B,C, GEF treatment decreased pyocyanin-induced ROS formation with concentration- and time-dependent manners. These results indicate that GEF-mediated attenuation against MeHg-induced cytotoxicity achieved through the inhibition of MeHg-induced ROS formation. In addition, LPA itself, LPA receptor1/3 antagonist, Ki16425 and PLC inhibitor U73122 did not prevent GEF-mediated attenuations on MeHg- and pyocyanin-ROS formation (Figures 3C and 4B,C). Ginseng total extract (GTE), crude ginseng total saponin (cGTS) fraction and ginsenoside Rb1 and Rg1 also had no eﬀects on MeHg-induced ROS formation (Figure 4A), indicating again that GEF-mediated attenuation against MeHg- and pyocyanin-induced ROS formation is not related with ginseng saponin and LPA receptor-mediated signaling pathways Figure 3. Eﬀects of MeHg in intracellular reactive oxygen species (ROS) formation and eﬀects of GEF on MeHg-induced ROS generation. (A) MeHg induces ROS in a concentration-dependent manner. MeHg was treated for 24 h. (B) MeHg-mediated ROS generation was time-dependent. (C) Eﬀects of GEF on the increase of MeHg-induced ROS generation. hNPCs were incubated MeHg with the control vehicle (Control) or various concentrations of GEF. GEF decreased ROS generation in a concentration-dependent manner. The indicated concentration of GEF was pretreated 24 h before MeHg (350 nM). LPA1/3 receptor antagonist (Ki16425, 10 µM) and phospholipase C inhibitor (U73122, 5 µM) was used. (D) GEF-mediated ROS decrease was time-dependent. Cells were treated with 30 µg/ml GEF and subjected to WST-1 assay as described in the Materials and Methods section. Data are presented as the mean ± SEM (n = 9; p < 0.01, compared with untreated controls). Figure 3. Eﬀects of MeHg in intracellular reactive oxygen species (ROS) formation and eﬀects of GEF on MeHg-induced ROS generation. (A) MeHg induces ROS in a concentration-dependent manner. MeHg was treated for 24 h. (B) MeHg-mediated ROS generation was time-dependent. (C) Eﬀects of GEF on the increase of MeHg-induced ROS generation. hNPCs were incubated MeHg with the control vehicle (Control) or various concentrations of GEF. GEF decreased ROS generation in a concentration-dependent manner. The indicated concentration of GEF was pretreated 24 h before MeHg (350 nM). LPA1/3 receptor antagonist (Ki16425, 10 µM) and phospholipase C inhibitor (U73122, 5 µM) was used. (D) GEF-mediated ROS decrease was time-dependent. 3.3. Eﬀects of Phosphatidic Acids on MeHg-induced Cytoxicity Cells were treated with 30 µg/ml GEF and subjected to WST-1 assay as described in the Materials and Methods section. Data are presented as the mean ± SEM (n = 9; p < 0.01, compared with untreated controls). 8 of 14 Int. J. Environ. Res. Public Health 2020, 17, 838 Figure 4. Eﬀects of GEF or other ginseng components on MeHg- and pyocyanin-induced ROS generation. (A) Ginseng total extract (GTE), crude ginseng total saponin (cGTS) fraction, and ginsenoside Rb1 and Rg1 (30 µM each) had no eﬀect on MeHg-induced ROS formation. (B) Eﬀects of GEF on the pyocyanin-induced ROS generation. Hippocampal NPCs were incubated pyocyanin (30 µM) with the control vehicle (Control) or various concentrations of GEF. GEF decreased ROS generation in a concentration-dependent manner. The indicated concentration of GEF was pretreated 16 h before pyocyanin and the degree of cell survival rate was measured as described in Methods. LPA 1/3 receptor antagonist (Ki16425, 10 µM) or phospholipase C inhibitor (U73122, 5 µM) was used. (C) GEF-mediated ROS decrease was time-dependent. Cells were treated with 30 µg/ml GEF and subjected to WST-1 assay as described in the Materials and Methods section. Data are presented as the mean ± SEM (n = 6; p < 0.01, compared with untreated controls). Cells were treated with indicated concentrations and subjected to WST-1 assay as described in the Materials and Methods section. Data are presented as the mean ± SEM (n = 6; p < 0.01, compared with untreated controls). Figure 4. Eﬀects of GEF or other ginseng components on MeHg- and pyocyanin-induced ROS generation. (A) Ginseng total extract (GTE), crude ginseng total saponin (cGTS) fraction, and ginsenoside Rb1 and Rg1 (30 µM each) had no eﬀect on MeHg-induced ROS formation. (B) Eﬀects of GEF on the pyocyanin-induced ROS generation. Hippocampal NPCs were incubated pyocyanin (30 µM) with the control vehicle (Control) or various concentrations of GEF. GEF decreased ROS generation in a concentration-dependent manner. The indicated concentration of GEF was pretreated 16 h before pyocyanin and the degree of cell survival rate was measured as described in Methods. LPA 1/3 receptor antagonist (Ki16425, 10 µM) or phospholipase C inhibitor (U73122, 5 µM) was used. (C) GEF-mediated ROS decrease was time-dependent. Cells were treated with 30 µg/ml GEF and subjected to WST-1 assay as described in the Materials and Methods section. 3.3. Eﬀects of Phosphatidic Acids on MeHg-induced Cytoxicity Data are presented as the mean ± SEM (n = 6; p < 0.01, compared with untreated controls). Cells were treated with indicated concentrations and subjected to WST-1 assay as described in the Materials and Methods section. Data are presented as the mean ± SEM (n = 6; p < 0.01, compared with untreated controls). Figure 4. Eﬀects of GEF or other ginseng components on MeHg- and pyocyanin-induced ROS generation. 3.5. Eﬀects of GEF for Organ MeHg Elimination Data are presented as the mean ± SEM (n = 6; p < 0.05, compared with MeHg treatment group, #p < 0.01, compared with MeHg treatment group). Figure 5. Eﬀects of GEF on MeHg elimination after oral administration of MeHg. (A) Long-term treatment of GEF (50 and 100 mg/kg, 3 weeks, p.o.) after oral administration of MeHg (2 mg/kg, 3 weeks, p.o.) decreased plasma MeHg concentration with dose- and time-dependent manners. Each group are obtained plasma in every 7 day. (B) GEF eliminated brain MeHg concentration with dose-dependent manners. (C) GEF eliminated kidney MeHg concentration with dose-dependent manners. (D) GEF also eliminated liver MeHg concentration with dose-dependent manners. Hg concentration of brain, kidney, and liver were determined after 3 weeks of Hg administration. Data are presented as the mean ± SEM (n = 6; p < 0.05, compared with MeHg treatment group, #p < 0.01, compared with MeHg treatment group). 3.5. Eﬀects of GEF for Organ MeHg Elimination Since gintonin attenuated MeHg-induced cytotoxicity, we next examined long-term eﬀects of GEF on organ MeHg elimination after co-oral administration of MeHg with GEF. As shown in Figure 5A, long-term treatment of GEF (50 and 100 mg/kg, 3 weeks, p.o.) after acute oral administration of MeHg (2 mg/kg, p.o.) signiﬁcantly decreased plasma Hg with dose- and time-dependent manners compared to MeHg alone. In addition, we also determined the amount of Hg in brain (Figure 5B), kidney (Figure 5C) and liver (Figure 5D). Compared to MeHg alone administration, long-term administration of GEF (50 and 100 mg/kg, 3 weeks, p.o.) after oral administration of MeHg also signiﬁcantly decreased Hg of brain, liver and kidney with a dose-dependent manner, respectively. Control vehicle and GEF alone administration only show a basal Hg amount. These results show that oral long-term administration of GEF facilitates tissue MeHg elimination. 9 of 14 Int. J. Environ. Res. Public Health 2020, 17, 838 Figure 5. Eﬀects of GEF on MeHg elimination after oral administration of MeHg. (A) Long-term treatment of GEF (50 and 100 mg/kg, 3 weeks, p.o.) after oral administration of MeHg (2 mg/kg, 3 weeks, p.o.) decreased plasma MeHg concentration with dose- and time-dependent manners. Each group are obtained plasma in every 7 day. (B) GEF eliminated brain MeHg concentration with dose-dependent manners. (C) GEF eliminated kidney MeHg concentration with dose-dependent manners. (D) GEF also eliminated liver MeHg concentration with dose-dependent manners. Hg concentration of brain, kidney, and liver were determined after 3 weeks of Hg administration. Data are presented as the mean ± SEM (n = 6; p < 0.05, compared with MeHg treatment group, #p < 0.01, compared with MeHg treatment group). Figure 5. Eﬀects of GEF on MeHg elimination after oral administration of MeHg. (A) Long-term Figure 5. Eﬀects of GEF on MeHg elimination after oral administration of MeHg. (A) Long-term treatment of GEF (50 and 100 mg/kg, 3 weeks, p.o.) after oral administration of MeHg (2 mg/kg, 3 weeks, p.o.) decreased plasma MeHg concentration with dose- and time-dependent manners. Each group are obtained plasma in every 7 day. (B) GEF eliminated brain MeHg concentration with dose-dependent manners. (C) GEF eliminated kidney MeHg concentration with dose-dependent manners. (D) GEF also eliminated liver MeHg concentration with dose-dependent manners. Hg concentration of brain, kidney, and liver were determined after 3 weeks of Hg administration. 3.6. Eﬀects of GEF on MeHg-Induced Memory Deﬁcits Long-term treatment of GEF (50 and 100 mg/kg, 3 weeks, p.o) group has tendency to ﬁnd target early. (B) Velocity (cm/s) for each group. (C) Increase of time spent in target quadrant means mice remember the target space. Time spent in target quadrant in the Morris water maze during a 60 s spatial memory trial 24 h after the last training session. It shows that GEF 100 mg/kg group spent more time in the target quadrant. (D) Percentage of distance spent in the target quadrant on the seventh day to assess memory strength in mice. Increase of distance traveled in target quadrant means mice remember the target space. GEF 100 mg/kg groups have more distance traveled in target quadrant. For C–D, data are representative of two independent experiments analyzed using unpaired 2-tailed t-tests. n = 10; p < 0.01, compared with MeHg treatment. 3.6. Eﬀects of GEF on MeHg-Induced Memory Deﬁcits Since MeHg is also known as causing memory-deﬁcit [25,26] and GEF attenuated MeHg-induced cytotoxicity and increased MeHg elimination from body organs including brain, we next examined the eﬀects of GEF on MeHg-induced memory deﬁcits. As shown in Figure 6A, long-term treatment of GEF after co-oral administration of MeHg with GEF (100 mg/kg, 3 weeks, p.o.) signiﬁcantly lowers escape latency in consecutive days. Time speed to ﬁnd out target is not diﬀerent signiﬁcantly in each group (Figure 6B). Figure 6C,D show that mice co-treated group with GEF and MeHg signiﬁcantly spent more time and traveled more distances in target quadrant than MeHg alone. These results also raise a possibility that GEF-mediated attenuation of in vitro cytotoxicity and in vivo enhancement of MeHg elimination from brain might contribute to GEF-mediated improvement from learning and memory dysfunctions damaged by MeHg treatment. 10 of 14 10 of 14 Int. J. Environ. Res. Public Health 2020, 17, 838 Figure 6. Morris water maze test of control, MeHg alone, MeHg + GEF (50 or 100 mg/kg, 3 weeks, p.o.) and GEF (100 mg/kg) alone groups. (A) Long-term oral treatment of MeHg (2 mg/kg, p.o) group need more time to ﬁnd target. Long-term treatment of GEF (50 and 100 mg/kg, 3 weeks, p.o) group has tendency to ﬁnd target early. (B) Velocity (cm/s) for each group. (C) Increase of time spent in target quadrant means mice remember the target space. Time spent in target quadrant in the Morris water maze during a 60 s spatial memory trial 24 h after the last training session. It shows that GEF 100 mg/kg group spent more time in the target quadrant. (D) Percentage of distance spent in the target quadrant on the seventh day to assess memory strength in mice. Increase of distance traveled in target quadrant means mice remember the target space. GEF 100 mg/kg groups have more distance traveled in target quadrant. For C–D, data are representative of two independent experiments analyzed using unpaired 2-tailed t-tests. n = 10; p < 0.01, compared with MeHg treatment. Figure 6. Morris water maze test of control, MeHg alone, MeHg + GEF (50 or 100 mg/kg, 3 weeks, p.o.) Figure 6. Morris water maze test of control, MeHg alone, MeHg + GEF (50 or 100 mg/kg, 3 weeks, p.o.) and GEF (100 mg/kg) alone groups. (A) Long-term oral treatment of MeHg (2 mg/kg, p.o) group need more time to ﬁnd target. 4. Discussion Mercury is currently used for various purposes from pharmaceutical companies to manufacturing industrial companies [1]. However, mercury is regarded as an industrial hazard as a neurotoxicant. MeHg is a toxic form of mercury and is mainly formed by bacteria through methylation of mercury and is biomagniﬁed in marine animals through the aquatic food chains [2]. The long-term exposure to MeHg through environmental contaminations or dietary consumption of aquatic foods results in adverse neurological problems [27,28]. Although ginseng extract exhibits diverse beneﬁcial eﬀects for human health, relatively little is known on the eﬀects of ginseng component on MeHg-induced neurotoxicity. In the present study, we investigated the eﬀects of GEF on MeHg-induced neurotoxicity. We found that GEF attenuated MeHg-induced in vitro neurotoxicities and in vivo memory impairments. Interestingly, LPA alone did not show the protective eﬀect on MeHg-induced in vitro neurotoxicity. In addition, the protective eﬀects of GEF on MeHg-induced neurotoxicity were not also attenuated by LPA receptor antagonist and PLC inhibitor (Figure 1), although co-administration of GEF with MeHg facilitates MeHg elimination and improved learning and memory damaged by MeHg (Figures 5 and 6). Thus, the present study shows that GEF-mediated anti-MeHg eﬀects might not be achieved through LPA receptors signaling pathways. Although it is well known that prenatal exposure to MeHg induces abnormal brain development, recent study shows that MeHg vulnerability declines with age, and that early exposure impairs later neurogenesis in older juveniles [29]. Interestingly, in vitro study also showed that co-culture of astrocytes with neurons was less vulnerable to MeHg assaults than neuron alone. Thus, astrocytes might help to increase neuronal resistance, raising a possibility that astrocytes might show a protective 11 of 14 Int. J. Environ. Res. Public Health 2020, 17, 838 role in MeHg neurotoxicity [30]. Furthermore, accumulating evidences show that MeHg neurotoxicity is not induced by a simple cause and showed that MeHg neurotoxicity includes multiple mechanisms such as disturbance of intracellular calcium homeostasis, alteration of glutamate homeostasis resulting in excitotoxicity and oxidative stress through ROS formations [31]. Thus, multifactorial mechanisms might be involved in MeHg-induced in vitro and in vivo neurotoxicity. Based on GEF-induced attenuations of MeHg neurotoxicity without involvement of LPA receptor regulations, it will be interesting to consider what are the molecular mechanisms of GEF-mediated attenuation on in vitro MeHg-induced neurotoxicity and in vivo MeHg-induced memory impairments. MeHg has positive charges and GEF contains negative charged components such as phosphatidic acids [32–34]. 4. Discussion Thus, the ﬁrst possibility is that the negative charged components of GEF might bind positive MeHg and interrupt MeHg-induced neurotoxicity. However, pre-treatment or co-treatment of PAs, a major negative charged component of GEF, did not attenuate MeHg-induced neurotoxicity (Figure 2). Thus, it is unlikely that the negative charged components of GEF are involved in attenuation of MeHg-induced neurotoxicity. On the other hand, the excess productions of ROS initiate peroxidative cell damage [35–37]. It is well-known that MeHg is a strong ROS producer and MeHg-induced ROS induces mitochondrial dysfunctions of neurons in nervous systems [10,38]. The central nervous system is very sensitive to peroxidative damages, since brain is rich in oxidizable lipids and catecholamine neurotransmitters [37]. Thus, the ﬁrst possibility is that GEF might attenuate MeHg-induced neurotoxicity through the inhibition of ROS formation by MeHg (Figure 3). As shown in Figure 3, GEF signiﬁcantly inhibited MeHg-induced ROS formation. To further prove that GEF attenuates MeHg-induced neurotoxicity through inhibition of ROS formation, we also used another speciﬁc ROS inducing agent, pyocyanin. As shown Figure 4, GEF inhibited pyocyanin-induced ROS formation with concentration-dependent manner, showing a possibility that GEF-mediated neuroprotection against MeHg-induced cytotoxicity achieved via the inhibition of MeHg-induced ROS formation. When we compared the eﬀects of GEF with other ginseng components such as whole ginseng total extract, crude ginseng total saponin fraction, and representative ginsenosides, we found that other ginseng components except GEF showed had no eﬀects on MeHg-induced neurotoxicity and MeHg-induced ROS formations (Figure 4). GEF might be a main component of ginseng and exhibit better anti-oxidative activity than other ginseng components. The second possibility is that GEF-mediated rapid elimination of MeHg from body might contribute to attenuation of MeHg-induced neurotoxicity. As shown in Figure 5, oral administration of GEF after MeHg administration facilitated MeHg elimination from major organs, although we could currently not explain how GEF helps rapid MeHg excretion compared to MeHg alone. The last possibility is that the combinational eﬀects of GEF on inhibition of MeHg-induced ROS formation and on rapid elimination of MeHg might contribute to GEF-induced attenuations of MeHg neurotoxity (Figures 4 and 5). Taken together, these results raise a possibility that GEF utilizes multiple ways to exert its anti-MeHg activity. However, it is unknown exactly which component(s) of GEF play an active role for anti-oxidant eﬀects through the inhibition of MeHg-induced ROS formation and MeHg elimination. 4. Discussion Further studies in future will be required to identify the active component in GEF. In previous reports, we showed that gintonin elicits [Ca2+] i transient via LPA receptor signaling pathways and modulates Ca2+-dependent various cellular eﬀects from ion channel regulations to hormone and neurotransmitter release. The gintonin actions via LPA receptor-Ca2+-dependent regulations are further associated with pharmacologically beneﬁcial eﬀects such as anti-Alzheimer’s disease through activation of non-amyloidogenic pathway [24,39]. In the present study, GEF attenuated MeHg-induced neurotoxicity with LPA receptor-independent manner but through multiple ways. Supporting this notion is that GEF mitigated in vitro and in vivo anti-Parkinson’s disease activity through the inhibition of major signaling pathways of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced ROS formation [40,41]. Co-administration of GEF with MeHg also facilitates MeHg elimination from organs and improved learning and memory damaged by MeHg have other biological activity (Figures 5 and 6). Thus, the present study further expanded that GEF possesses biological 12 of 14 Int. J. Environ. Res. Public Health 2020, 17, 838 functions such as anti-oxidant activity and rapid elimination of MeHg from body besides the role of exogenous LPA receptor ligand. Reactive oxygenspeciessuch as superoxideanion, hydro-gen peroxide, ferry! ion, and hydroxyl radical, if present in excess, are thought to be initiators of peroxidative cell damage (Freeman and Crapo, 1982; Halliwell and Gutteridge, 1984, 1986). The nervous system is exquisitely sensitive to per-oxidative damage since it is rich in oxidizable substrates such as lipids and catecholamines (Halliwell and Gutteridge, 1985). Reactive oxygen species such as superoxide anion, hydro-gen peroxide, ferry! ion, and hydroxyl radical, if present in excess, are thought to be initiators of peroxidative cell damage (Freeman and Crapo, 1982; Halliwell and Gutteridge, 1984, 1986). The nervous system is exquisitely sensitive to peroxidative damage since it is rich in oxidizable substrates such as lipids and catecholamines (Halliwell and Gutteridge, 1985). Reactive oxygen species such as superoxide anion, hydro-gen peroxide, ferry! ion, and hydroxyl radical, if present in excess, are thought to be initiators of peroxidative cell damage (Freeman and Crapo, 1982; Halliwell and Gutteridge, 1984, 1986). The nervous system is exquisitely sensitive to per-oxidative damage since it is rich in oxidizable substrates such as lipids and catecholamines (Halliwell and Gutteridge, 1985). In conclusion, GEF-mediated in vitro and in vivo anti-MeHg eﬀects might achieve via the inhibition of MeHg-induced ROS formation and rapid MeHg eliminations from organs. 4. Discussion Finally, the present study shows that GEF can be also used as a preventive and/or therapeutic agent for attenuation of MeHg-induced neurotoxicity and for inhibition of MeHg accumulation from body. Author Contributions: K.H.J., C.S.H., L.N.E. and C.H.J. collected the data. H.S.H. and K.H.C. elaborated the study design. R.H. and K.H.J. contributed to data analysis and interpretation. K.H.J., N.S.Y. drafted the article. All authors critically reviewed the content and approved the ﬁnal version for publication. Acknowledgments: This work was supported by the Basic Science Research Program (NRF-2017R1D1A1A09000520) and NRF2016M3C7A1913894 to S.Y. Nah from the Ministry of Science, ICT, and Future Planning. Acknowledgments: This work was supported by the Basic Science Research Program (NRF-2017R1D1A1A09000520) and NRF2016M3C7A1913894 to S.Y. Nah from the Ministry of Science, ICT, and Future Planning. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Doney, S.C. The growing human footprint on coastal and open-ocean biogeochemistry. Science 2010, 328, 1512–1516. [CrossRef] [PubMed] 1. Doney, S.C. 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https://openalex.org/W3013012320	https://europepmc.org/articles/pmc7230927?pdf=render	English	null	Segmentation of Polish Households Taking into Account Food Waste	Foods	2,020	cc-by	10,147	Received: 21 February 2020; Accepted: 20 March 2020; Published: 25 March 2020 Abstract: Currently, food waste is estimated at more than one-third of all food produced, and the primary responsibility for this phenomenon is attributed to households. Therefore, it seems reasonable to take action to limit food waste and to raise awareness about this link in the chain. To develop and implement educational programs addressed at consumers it is necessary to understand the factors determining food waste in households. Segmentation is a tool that can help eﬀectively reach consumers who are to the greatest extent wasting food which identiﬁes homogeneous clusters of consumers. The aim of this study was to perform segmentation to identify consumer groups with similar behaviors in relation to food, with particular emphasis on food wastage. We carried out segmentation on a representative sample of Polish people over 18 years of age and to identiﬁed three clusters of consumers. The three consumer segments diagnosed diﬀered in sociodemographic terms, i.e., number of adults, number of children, subjective assessment of the ﬁnancial situation, and percentage of spending on food. The segment exhibiting a high frequency of discarding food due to too large package size included single and double households. Keywords: food waste; food waste segmentation; households; consumers; cluster analysis; behavior; food practices foods foods Foods 2020, 9, 379; doi:10.3390/foods9040379 Segmentation of Polish Households Taking into Account Food Waste Beata Bilska 1,* , Marzena Tomaszewska 1 , Danuta Koło˙zyn-Krajewska 1 and Małgorzata Piecek 2 Beata Bilska 1,* , Marzena Tomaszewska 1 , Danuta Koło˙zyn-Krajewska 1 and Małgorzata Piecek 2 foods foods Małgorzata Piecek 2 1 Department of Food Gastronomy and Food Hygiene, Institute of Human Nutrition Sciences, Warsaw University of Life Sciences—SGGW, Nowoursynowska 159C St., 02−776 Warsaw, Poland; marzena_tomaszewska@sggw.pl (M.T.); danuta_kolozyn_krajewska@sggw.pl (D.K.-K.) 2 Polish Food Technologists’ Society, Nowoursynowska 166C St., 02−787 Warsaw, Poland; wrzosekmalgorzata@wp.pl 1 Department of Food Gastronomy and Food Hygiene, Institute of Human Nutrition Sciences, Warsaw University of Life Sciences—SGGW, Nowoursynowska 159C St., 02−776 Warsaw, Poland; marzena_tomaszewska@sggw.pl (M.T.); danuta_kolozyn_krajewska@sggw.pl (D.K.-K.) 2 Polish Food Technologists’ Society, Nowoursynowska 166C St., 02−787 Warsaw, Poland; wrzosekmalgorzata@wp.pl 1 Department of Food Gastronomy and Food Hygiene, Institute of Human Nutrition Sciences, Warsaw University of Life Sciences—SGGW, Nowoursynowska 159C St., 02−776 Warsaw, Poland; marzena_tomaszewska@sggw.pl (M.T.); danuta_kolozyn_krajewska@sggw.pl (D.K.-K.) 2 Polish Food Technologists’ Society, Nowoursynowska 166C St., 02−787 Warsaw, Poland; wrzosekmalgorzata@wp.pl * Correspondence: beata_bilska@sggw.pl; Tel.: +48-2259-70-75 * Correspondence: beata_bilska@sggw.pl; Tel.: +48-2259-70-75 1. Introduction Waste, which is estimated at more than one-third of all food produced, is becoming a serious global problem that threatens the sustainable system, generating negative social, ecological, economic, and ethical consequences [1,2]. It is estimated that the level of food waste in the European Union, in 2012, was approximately 88 million tons [3]. With respect to the quantity of food produced, this value is approximately 20%. [3]. Food waste is produced along the various stages of the food supply chain [4]. The sectors contributing the most to food waste are households (47 million tons) and processing (17 million tons). These two sectors account for 72% of the EU’s food waste. Of the remaining 28% of food waste, 11 million tons (12%) comes from food service, nine million tons (11%) comes from primary production, and ﬁve million tons (5%) comes from wholesale and retail [3]. Despite the fact that there are multiple ways of decreasing the generation of waste, the common pillar is the prioritization of food waste prevention [5]. The second most attractive option involves distributing food surplus to groups aﬀected by food poverty [6]. The need to ensure food security to people in food poverty has led to the development of new technologies in the food business that Foods 2020, 9, 379; doi:10.3390/foods9040379 www.mdpi.com/journal/foods www.mdpi.com/journal/foods 2 of 19 Foods 2020, 9, 379 are aimed at preventing food losses and waste and at improving the quality of products recovered by charities [7,8]. A smart portable device associated with good prevention practices could potentially be useful to reduce food waste in the agrifood supply chain and in the food recovery ﬁeld for charitable purposes [9]. The next option is converting food waste to animal feed [6]. According to Castrica et al. [10] feed product derived from food waste is compliant with the EU safety requirements, is nutritionally valuable, and has great potential. g p It should be noted that within the structure of costs incurred in the food chain losses in the European Union, the highest level of wastage is generated by the last link, namely households (more than 50%, the cost of approximately 98 billion euros) [3]. Using data received from the EU-27, the BIO Intelligence, Paris, France, Service calculated that, at the household level, one household member discards approximately 76 kg of food per year. 1. Introduction An analysis of food waste across the EU shows that there are substantial diﬀerences in the quantity per one household member. For example, the level recorded in the Czech Republic was 25 kg/person/year, whereas in the United Kingdom it was 133 kg per person per year. In Poland, the waste level was 54 kg of waste food per person per year [11]. Estimates show that approximately 60% of food discarded at this stage was still suitable for human consumption. Therefore, it seems reasonable to take action to limit the phenomenon of food waste and to raise awareness of the problem among consumers [3], especially because consumers are concerned about the scale of this phenomenon and believe that discarding food is wrong and evokes negative emotions (often shame and resentment) and ethical objections. It should be noted, however, that in the subjective assessment they do not see themselves as the main source of discarded food imputing responsibility to other links in the food chain [12]. In addition to knowledge and awareness, food waste is aﬀected by many factors which cannot be interpreted in isolation. The tendency to discard is rather the result of how a household copes with all activities related to food. This process consists of activities such as planning and doing shopping, food storage, meal preparation, and consumption [3]. One of the main factors that motivates action to limit food waste more strongly than environmental and prosocial awareness is a desire to minimize one’s own expenses. In the context of loss, consumers often mention losing time for buying products, bringing them home, and preparing a meal that eventually is not consumed or is partially consumed. The link between ecological impacts such as global warming or excessive consumption of natural resources is not clearly identiﬁed with the quantity of food that is wasted [12]. The research by Neﬀet al. [13] and Qi and Roe [14] has shown that only 40% and 58.4% of the respondents are aware of the consequences and the negative inﬂuence of food waste on the environment, respectively. Therefore, the key is to build awareness and knowledge that discarding food unduly wastes personal ﬁnancial resources at the same time exerting a negative impact on the environment [12]. Food waste is the result of many complex factors and consumer behavior [15,16]. 1. Introduction Among the edible fraction, food is more likely to be thrown in the bin resulting from excess left on the plate after eating, excess prepared but not served, and products opened but not ﬁnished [17]. According to Bilska et al. [18], the most common causes of food waste include food being spoiled and missing the expiry date. Zieli´nska et al. [19] noticed that the majority of respondents have diﬃculty distinguishing and properly understanding the terms “use by” and “best before” on the label. As pointed by Ramírez et al. [20], an electronic nose could be useful for consumers, as well as meat industries and related food services, that would allow real-time characterization of these products. The results have shown that pork meat trays should not be labelled with the same expiration date, because of observed wide variation in the typical hygiene and sanitary quality parameters [20]. According to many researchers [16,21–23], the quantity of wasted food is correlated with demographic factors, notably household size. Single households waste more per person, which implies an economic scale associated with the size of commercially available packages. The Waste and Resources Action Programme Report [15] indicated that young people, both single, young families, young people, and children, tend to waste more food. Similarly, Hanssen et al. [24] pointed out that Foods 2020, 9, 379 3 of 19 young people tend to waste more food, which is why educational programs are addressed largely to these groups. According to Aschemann-Witzel et al. [25], demographic data does not play as important a role in explaining food waste at the consumer level as do psychographics. The Fusions Report draws attention to three factors, namely social factors (type of household and family stage), individual behavior, as well as lack of awareness, knowledge, and skills [26]. A good understanding of the factors determining food waste in a household [27] facilitates the development and implementation of consumer education programs, A tool that can eﬀectively help reach consumers who waste the most food is segmentation, which is considered one of the most accepted and important concepts, both in academic research and real world practice [28]. A one-size-ﬁts-all approach cannot meet all the varied requirements; therefore, companies need to consider diﬀerentiated approaches to customers, products, and supply [29]. 1. Introduction As pointed by French [30], segmentation adds real value to most social programs and is a common technique that has been applied as part of social marketing programs. According to Johnson et al. [31], market segmentation is a valuable tool for identifying consumers with similar needs who are likely to respond in a similar way to marketing communication. Each segment requires individual products or marketing mixes [32,33]. These rules also apply to educational campaigns. Demographic and socioeconomic variables are the most popular basic elements for segmentation, because the information is relatively easy to collect and measure and is the best example of a priori descriptive methods. These variables include age, sex, size of household, household income, profession, and education [31]. The aim of the study was to perform segmentation to identify consumer groups with similar behavior in relation to food, with particular emphasis on its wastage. The question was the following: (1) Whether food waste in households is associated with sociodemographic features such as household size, personal composition, subjective assessment of the ﬁnancial situation, and level of spending on food as well as (2) the impact these factors have on food waste. 2.1. Sample Collection The survey was conducted in February and March 2019, among a group of 1115 adult consumers using computer-assisted personal interviews (CAPIs). The selection of the sampling from the address survey of the Central Statistical Oﬃce in Poland, Warsaw fulﬁlled the condition of representativeness of the general population for the Polish people over 18 years in terms of gender, age, and size of the place of residence. The survey was conducted in each of the sixteen voivodeships in Poland. After drawing the starting addresses, the so-called method of random route was used in the selection of the sample. The factors taken into account in the segmentation of the respondents included characteristics such as the number of adults in the household (over 18 years), number of children, subjective assessment of the ﬁnancial situation of the household, and percentage of spending on food. The detailed sociodemographic proﬁle of the respondents is presented in Table 1. The surveyed households were dominated by families consisting of two adults. Much lower percentage values concerned households which consisted of one (and three or more adults). The majority of the respondents declared that there were no minors in their households. As for families with children (under 18 years), the survey was dominated by families with one child (respondents were asked about their ﬁnancial situation). For this purpose, were used subjective ﬁnancial assessments as a measure of ﬁnancial well-being. A majority of the respondents declared an average ﬁnancial situation. The actual survey was preceded by a pilot study conducted on 30 people. 4 of 19 Foods 2020, 9, 379 Table 1. Sociodemographic proﬁle of consumer groups (n = 1115). Table 1. Sociodemographic proﬁle of consumer groups (n = 1115). Table 1. Sociodemographic proﬁle of consumer groups (n = 1115). Feature Characteristics % No. of people over 18 years in household 1 2 3 or more 17.2 61.2 21.6 No. of children in household 0 1 2 or more 72.7 17.3 10.0 Subjective assessment of the ﬁnancial situation Good Average Refusal to provide an answer 41.6 57.6 0.8 Portion of the household budget for food expenditure Large (100–61%) Average (60–40%) Small (39–0%) Hard to say 12.3 50.2 27.7 9.8 2.2. Questionnaire Schematic diagram of the issues used for segmentation of households. 2.3. Statistical Analysis Figure 1. Schematic diagram of the issues used for segmentation of households. 2.2. Questionnaire The study was performed using a speciﬁcally designed questionnaire which consisted of two parts. The ﬁrst part contained questions regarding consumer behavior and knowledge in relation to food was divided into seven areas These topics were selected on the basis of the literature [34–36], which showed that these aspects aﬀect food waste in households. The ﬁrst group of questions concerned preparation for shopping and behavior during shopping (using a 5-point scale “always, usually, sometimes, rarely, never”) (Figure 1Q1,Q2). Food products were divided into 32 groups, and then the respondents were asked about the place of purchase (market, small private shop, small chain store, discount store, hypermarket, specialized shop, and via the internet) and frequency of doing shopping (daily, on average every other day, on average 1−2 times a week, on average 1−2 times per month, less than once a month, never) (Figure 1Q3). In addition, the respondents were asked to identify food products purchased in bulk (possibility of indicating several responses) (Figure 1Q3). The survey was also aimed at examining the degree to which information on the product package was important to the respondents (using a 5-point scale from “strongly agree” to “strongly disagree”) (Figure 1Q4). A signiﬁcant issue is the proper storage of food at home (Figure 1Q5). This question used a 5-point scale ranging from “always” to “never”. In addition, the respondents were asked to select 20 products kept in the refrigerator from among the products listed (Figure 1Q5). The key issue was 32 food groups waste, i.e., the reasons and frequency (Figure 1Q6). In the questions concerning the reasons for discarding food, the respondents could indicate several responses. The respondents were also asked questions regarding the minimum “best before” date (Figure 1Q7). The scale was deliberately diﬀerentiated and adapted to the type of question. The second section contained questions concerning the demographic and social aﬃliation of the respondents as follows: number of people in household, number of children in household, subjective assessment of the ﬁnancial situation, and portion of the household budget spent on food. (Figure 1). Foods 2020, 9, 379 5 of 19 Figure 1. Schematic diagram of the issues used for segmentation of households. 2.3. Statistical Analysis A taxonomic analysis was performed using Statistica 12.1. PL (StatSoft, Cracow, Poland). Its aim was to select several groups of respondents diﬀering in behavior related to food waste. A 5 of 19 Foods 2020, 9, 379 Figure 1. 2.3. Statistical Analysis A taxonomic analysis was performed using Statistica 12.1. PL (StatSoft, Cracow, Poland). Its aim was to select several groups of respondents diﬀering in behavior related to food waste. A multidimensional cluster analysis was used for this purpose. A non-hierarchical k-means method was used to create clusters because it allows establishment of a priori, of the number of clusters. This algorithm uses the Euclidean distance. Homogeneous clusters of respondents were created 6 of 19 Foods 2020, 9, 379 based on the average frequency rate. The starting point of the analysis was the identiﬁcation of a speciﬁed number of segments of respondents on the basis of their descriptive (“sociodemographic”) characteristics. In this way, 35 diﬀerent segments were created as follows: number of adults in the household, number of children, subjective assessment of the ﬁnancial situation, and percentage of spending on food. Subsequently, based on the average frequency rate (for each segment) illustrating discarding 32 groups of non-consumed food products, the respondent segments (35) were grouped into three uniform clusters. Cluster analysis was complemented by the examination of the signiﬁcance of diﬀerences between the mean levels for each element (consisting of a multidimensional criterion for the creation of clusters) in the identiﬁed clusters. The null hypothesis of equality of the mean value (calculated for each cluster) was veriﬁed using the Fisher–Snedecor F test, and a post-hoc analysis was performed using the LSD (least signiﬁcant diﬀerence). test. This allowed identiﬁcation of homogeneous groups of arithmetic means. This veriﬁcation was carried out with a signiﬁcance level of p = 0.05. The basis of inferences was the test probability values “p”. This enabled indication of homogeneous arithmetic mean groups. The null hypothesis was rejected when p < 0.05. A preliminary analysis was performed assuming 2, 3, 4, and 5 expected clusters. This analysis showed that the clearest proﬁle of the groupings of elements (segments) in the individual clusters was obtained when assuming 3 clusters. 3.2. Characteristics of the Deﬁned Segments Taking into Account Common Features aracteristics of the Deﬁned Segments Taking into Account Common Features The analysis of variance (F test) showed that the three segments diagnosed were similar in terms of preparation for shopping. Almost a quarter of the consumers declared that the did not check their supplies before going shopping (22.6%) or prepare a shopping list (27%) (answers “never” and “rarely”). It was also found that the people classiﬁed in the three segments were, to the same extent, willing to purchase unplanned products (18.9% response “always” or “usually”). A relatively small percentage of the respondents declared that they “always” or “usually” buy food in bulk (16.6%). It was also found that the people classiﬁed in the three segments generally did not arrange food based on the expiry date in the fridge and cupboard (41.8% of answers “rarely” or “never”). The analysis of variance (F-test) showed that in the case of products such as bread, cereals, and legumes, the frequency of discarding was similar for all three clusters. Particularly noteworthy is that bread was one of the products most commonly discarded by the Polish respondents. Almost a quarter (23.8%) declared that they “often” and “sometimes” discard this product. There was also no diﬀerence among the three segments in terms of the frequency of buying 23 groups of food products, including bread, which was purchased by the vast majority (82.3%) of the respondents daily or on average every other day. Generally, there were no diﬀerences in the reasons for discarding food, with the exception of package size. The vast majority of the respondents declared that they discarded food because of its deterioration (65.2%) and missed expiry date (42%). Nearly one-quarter of the respondents wasted food because they prepared (26.5%) or bought (22.2%) too much. The diagnosed clusters similarly interpreted the meaning of the “best before” and “use by” dates. Almost half of the respondents (42.8%) believed that these dates mean the same thing, more than one third of the respondents (36%) were of the opposite view, and every ﬁfth respondent (21.2%) answered “I do not know”. The respondents in the three segments failed to correctly indicate the products which had a “best before” date. Forty per cent of the respondents correctly indicated groats, and nearly 2% more (41.7%) incorrectly indicated yoghurt and buttermilk. 3.2. Characteristics of the Deﬁned Segments Taking into Account Common Features A similar percentage of the respondents believed that the term “best before” meant the date after which a product becomes dangerous for the consumer (39.8%) or loses its quality (37.4%). 3.1. Sociodemographic Proﬁle of Segments Segmentation was carried out on a representative sample of Polish people over 18 years of age and we identiﬁed three clusters with varying percentage share as follows: Cluster 1 “saving food” 41.8%, Cluster 2 “wasting vegetables and fruit” 46.3%, and Cluster 3 “wasting food” 11.9%. The three clusters diﬀer signiﬁcantly in sociodemographic terms (Table 2). More than half of households in Cluster 1 are formed by two adults and one in ﬁve households consists of a single person, or three or more adults. In Cluster 2, there are no single households and it is dominated by families consisting of two adults with one child. Segment 3 is dominated by single households. All three segments are dominated by households without children, which deﬁnitively prevail in Cluster 3. Nearly half of households forming Cluster 1 declared a good ﬁnancial situation and a small proportion of spending on food. Most households classiﬁed in Segments 2 and 3 are characterized by average ﬁnancial situation. In Cluster 3 almost half of the people declared a large proportion of spending on food. Table 2. Sociodemographic proﬁle of clusters. Feature % Share in the Segment - Saving Food Wasting Vegetables and Fruit Wasting Food Number of people 1 adult 22.4 0.0 68.2 2 adults 58.2 72.6 31.7 3 and more adults 19.4 27.4 8.5 At least 3 persons 37.4 51.7 15.5 Number of children No children 73.4 73.2 93.8 One child 16.0 18.3 0.0 At least two children 10.6 8.5 6.2 Financial situation Good * 50.1 33.6 33.3 Average * 47.2 64.8 66.7 Table 2. Sociodemographic proﬁle of clusters. Table 2. Sociodemographic proﬁle of clusters. 7 of 19 Foods 2020, 9, 379 Table 2. Cont. Feature % Share in the Segment - Saving Food Wasting Vegetables and Fruit Wasting Food Proportion of spending on food Low (0−39%) * 33.6 27.8 0.0 Average (40−60%) * 47.9 66.2 53.5 High (61−100%) * 12.0 4.4 46.5 * In the case of Segments 1 and 2, the numbers do not add up to 100% due to no response to a question. 3.3. Description of the Deﬁned Segments in Terms of Handling Food The “saving food” cluster, more rarely than the other segments, declare buying fresh red meat (Table 3). People in Cluster 1 least frequently refrigerate fresh herbs and carrots as compared with the other clusters. This segment stood out with a lower share of persons declaring discarding 13 groups of products, both of animal origin (e.g., milk and dairy products, eggs, fresh poultry meat, sausages) and of plant origin (e.g., potatoes, fruit and vegetable products) as compared with the other segments (Table 4). People in the “saving food” cluster do not discard any food product more often than the other two segments. 8 of 19 Foods 2020, 9, 379 Table 3. Selected aspects of food handling for the identiﬁed segments, average level of frequency rate, arithmetic mean and the results of the variance analysis and the LSD test. 3.3. Description of the Deﬁned Segments in Terms of Handling Food - Saving Food Wasting Vegetables and Fruit Wasting Food p-Value - Saving Food Wasting Vegetables and Fruit Wasting Food p-Value - Shopping Frequency (Arithmetic Mean *) - - Refrigeration (Average Level of Frequency Rate ) - Milk 3.05 a 3.09 a 3.42 b 0.038 Fresh poultry meat 0.80 ab 0.86 b 0.75 a 0.042 Fresh red meat 3.79 b 3.54 a 3.47 a 0.002 Fresh ﬁsh 0.76 ab 0.83 b 0.68 a 0.010 Fresh ﬁsh 4.40 a 4.34 a 3.86 b 0.001 Yogurt, buttermilk 0.91 ab 0.94 b 0.83 a 0.031 Canned meat and ﬁsh 4.85 b 4.69 b 4.09 a 0.000 Fresh milk 0.84 b 0.89 b 0.67 a 0.000 Fresh fruit 3.02 a 3.01 a 3.45 b 0.002 Carrot 0.21 a 0.36 b 0.32 b 0.040 Fruit and vegetable preserves (including dried, canned) 4.48 b 4.39 ab 4.13 a 0.010 Butter 0.84 b 0.87 b 0.75 a 0.049 Legume seeds 4.69b 4.74 b 4.11 a 0.000 Tomatoes 0.32 a 0.42 b 0.34 a 0.040 Chilled ready meals 4.72 b 4.76 b 4.35 a 0.005 Onion 0.14 a 0.17 a 0.30 b 0.006 Frozen food 4.57 b 4.59 b 4.27 a 0.029 Juices after opening 0.63 ab 0.71 b 0.54 a 0.035 - Fresh herbs 0.05 a 0.14 b 0.11 b 0.008 Frequency of action (Arithmetic mean ) - Purchase frequency (Arithmetic mean ) - Observance of the storage conditions speciﬁed by the manufacturer 2.17 a 2.10 a 2.45 b 0.012 Deformed, small fruit and vegetables 4.17 b 4.27 b 3.71 a 0.002 Refrigeration of food immediately after bringing home 1.67 a 1.60 a 2.05 b 0.002 Food with short expiry dates 3.69 b 3.74 b 3.42 a 0.005 Signiﬁcance of the information on the label (Average value *) - Food with short expiry dates at a discounted price 3.73 b 3.76 b 3.39 a 0.016 Expiry date 1.43 a 1.35 a 1.76 b 0.001 Food products in a deformed package 4.09 b 4.23 b 3.85 a 0.027 Price 1.56 a 1.47 a 1.83 b 0.009 Buying in bulk (Average level of frequency rate ) - Reason for discarding food (Average level of frequency rate ) - Cereal products 0.56 b 0.61 b 0.37 a 0.018 Too big package size 0.16 a 0.15 a 0.37 b 0.001 Other fats 0.32 b 0.33 b 0.15 a 0.041 - Coﬀee, tea, cocoa 0.60 ab 0.65 b 0.47 a 0.043 The term “best before” means (Average level of frequency rate ) - Products on which the phrase “best before” is placed (Average level of frequency rate ) - Date after which the product can be consumed 0.08 a 0.14 b 0.06 a 0.048 Do not know 0.27 b 0.22 b 0.11 a 0.015 An identical letter at the arithmetic mean value or frequency rate means that there are no signiﬁcant diﬀerences between the clusters. 3.3. Description of the Deﬁned Segments in Terms of Handling Food The highest frequency rate means the highest frequency. Rank order for reported behavior “1” being the highest reported frequency and “5” being the lowest reported frequency. Frequency of action (Arithmetic mean ) 9 of 19 Foods 2020, 9, 379 Table 4. Frequency of discarding 32 groups of food products and place of purchase, average level of frequency rate for the designated clusters, and the results of the variance analysis and the LSD test. 3.3. Description of the Deﬁned Segments in Terms of Handling Food - Frequency of Discarding Food Products p-Value Place of Shopping p-Value - - Average Level of Frequency Rate * Average Level of Frequency Rate * - Cluster 1 Cluster 2 Cluster 3 Cluster 1 Cluster 2 Cluster 3 Place of Shopping Bread 0.58 0.69 0.60 ns 0.45 b 0.54 c 0.35 a 0.007 Small private shop Cereal products 0.42 0.45 0.49 ns 0.08 a 0.16 b 0.05 a 0.045 Large-format shop Cakes 0.42 a 0.48 a 0.60 b 0.009 0.08 a 0.05 a 0.18 b 0.041 Small chain store Sugar and substitutes 0.21 a 0.24 a 0.40 b 0.01 0.03 a 0.02 a 0.11 b 0.009 Bazaar 0.67 b 0.62 b 0.49 a 0.021 Discount store Sweets and snacks 0.20 a 0.28 a 0.49 b 0.000 ns ns ns ns ns Milk 0.38 a 0.55 b 0.54 b 0.001 ns ns ns ns ns Diary drinks 0.36 a 0.59 b 0.55 b 0.000 ns ns ns ns ns Cream 0.33 a 0.55 b 0.61 b 0.000 ns ns ns ns ns Curd cheese 0.34 a 0.50 b 0.47 b 0.000 ns ns ns ns ns Rennet cheeses 0.30 a 0.46 b 0.53 b 0.004 0.04 a 0.02 a 0.17 b 0.001 Small chain store Eggs 0.18 a 0.29 b 0.44 c 0.000 ns ns ns ns ns Fresh poultry meat 0.19 a 0.28 b 0.39 c 0.001 0.06 a 0.04 a 0.16 b 0.005 Small chain store Fresh red meat 0.21 a 0.26 a 0.39 b 0.000 0.07 a 0.15 b 0.07 a 0.023 Large-format shop Fresh ﬁsh 0.20 a 0.29 a 0.48 b 0.000 0.20 b 0.34 c 0.08 a 0.001 Specialized store Sausage (packed and packaged) 0.43 a 0.60 b 0.56 b 0.000 ns ns ns ns ns Smoked ﬁsh 0.26 a 0.34 b 0.43 b 0.008 0.22 b 0.22 b 0.10 a 0.041 Small private shop 0.07 ab 0.04 a 0.12 b 0.045 Small chain store Canned meat and ﬁsh 0.24 a 0.20 a 0.40 b 0.007 0.06 ab 0.02 a 0.11 b 0.036 Small chain store Butter, Margarines, mixes 0.16 a 0.22 ab 0.28 b 0.030 ns ns ns ns ns Other fats 0.15 a 0.19 a 0.37 b 0.000 ns ns ns ns ns Potatoes 0.39 a 0.59 c 0.51 b 0.000 ns ns ns ns ns Table 4. ns, not signiﬁcant; an identical letter at the frequency rate means that there are no signiﬁcant diﬀerences between the c 3.3. Description of the Deﬁned Segments in Terms of Handling Food Frequency of discarding 32 groups of food products and place of purchase, average level of frequency rate for the designated clusters, and the results of the variance analysis and the LSD test. 10 of 19 Foods 2020, 9, 379 Table 4. Cont. Table 4. Cont. - Frequency of Discarding Food Products p-Value Place of Shopping p-Value - - Average Level of Frequency Rate * Average Level of Frequency Rate * - Cluster 1 Cluster 2 Cluster 3 Cluster 1 Cluster 2 Cluster 3 Place of Shopping Root vegetables 0.48 a 0.60 b 0.41 a 0.008 0.06 a 0.04 a 0.15 b 0.038 Small chain store Vegetables, except root vegetables 0.55 a 0.67 b 0.55 a 0.007 ns ns ns ns ns Fresh fruit 0.50 a 0.69 b 0.54 a 0.002 ns ns ns ns ns Fruit and vegetable preserves (including dried, canned) 0.28 a 0.35 b 0.45 c 0.045 ns ns ns ns ns Carbonated drinks, still drinks 0.21 a 0.24 a 0.37 b 0.038 ns ns ns ns ns Coﬀee, tea, cocoa 0.15 a 0.14 a 0.32 b 0.000 0.05 a 0.07 a 0.18 b 0.014 Small chain store Food concentrates 0.19 a 0.22 a 0.44 b 0.000 ns ns ns ns ns Ketchup, mayonnaise, mustard and other sauces 0.23 a 0.29 a 0.48 b 0.000 0.07 a 0.06 a 0.19 b 0.006 Small chain store Legume seeds 0.21 a 0.25 a 0.44 b 0.000 ns ns ns ns ns Preparations from pulses 0.28 0.27 0.40 ns ns ns ns ns ns Chilled ready meals 0.23 a 0.39 b 0.51 b 0.000 ns ns ns ns ns Frozen 0.23 a 0.34 b 0.41 b 0.004 ns ns ns ns ns ns, not signiﬁcant; an identical letter at the frequency rate means that there are no signiﬁcant diﬀerences between the clusters; * the highest frequency rate means the highest frequency. Foods 2020, 9, 379 11 of 19 People in the “wasting vegetables and fruit” cluster most frequently refrigerate tomatoes as compared with the other clusters (Table 3). People in this cluster most frequently buy bread in small private shops, grain products and fresh red meat in supermarkets, and fresh ﬁsh in specialized shops (Table 4). Cluster 2 was characterized by people who declare most frequently discarding fresh fruit, potatoes, root vegetables and other vegetables as compared with the other two clusters (Table 4, Figure 2). 3.3. Description of the Deﬁned Segments in Terms of Handling Food More people classiﬁed in this segment believe that the term “best before” means the date after which a product can be consumed (Table 3). Figure 2. The highest frequency of discarding individual product groups declared by the three segments identiﬁed. Figure 2. The highest frequency of discarding individual product groups declared by the three segments identiﬁed. Households classiﬁed in the “wasting food” cluster purchase food products with a very short “best before” or with short “use by” dates which are oﬀered at a discounted price more often than others. Nevertheless, for the people classiﬁed in Cluster 3 the expiry date and price are less important than for those in the other clusters. It should also be noted that the respondents forming Cluster 3 more often than others buy deformed, too little fruit and vegetables, and food products in deformed packaging (Table 3). p g g Cluster 3 includes respondents buying less milk and fresh fruit. They buy fresh ﬁsh, canned meat and ﬁsh, legume seeds and preserves, ready meals and frozen foods more often as compared with Clusters 1 and 2. The factor diﬀerentiating the clusters is also the place of purchase. The LSD test indicates that people in Cluster 3 most frequently buy cakes, rennet cheese, fresh poultry meat, root vegetables, ketchup and other sauces, coﬀee, tea and cocoa in small chain stores, and sugar and substitutes-in markets as compared with the other clusters (Table 4). Foods 2020, 9, 379 12 of 19 12 of 19 People in Cluster 3 less frequently buy sugar and substitutes in discount stores, smoked ﬁsh in small private shops, and fresh ﬁsh in specialized shops as compared with the other clusters (Table 4). The factor that could inﬂuence the level of food waste in households is the model of buying food products “in bulk”. It has been shown that buying in bulk 3 groups of products substantially distinguishes between the clusters in the test sample. Cluster 3 is distinguished by people who less frequently buy in bulk cereal products and fats (Table 3). In addition, the respondents belonging to this cluster more frequently do not observe the storage conditions speciﬁed by the manufacturer on the package, or place food products requiring refrigeration temperatures in the refrigerator immediately after bringing them home as compared with the other clusters. 4. Discussion This study diagnosed three consumer segments diﬀering in sociodemographic terms, i.e., number of adults, number of children, subjective assessment of the ﬁnancial situation, and percentage of spending on food. Despite the many diﬀerences observed in the behavior of the three segments, several similarities were also found. The respondents in the three clusters generally do not check their product stocks at home before going shopping, or prepare shopping lists, and “sometimes, often” buy unplanned items. According to many researchers [16,21,23,37] careful planning of grocery shopping is an eﬀective tool for the prevention of food waste. While conscious estimation of demand for a given product and its corresponding quantity relevant to the number of members in the household can reduce buying excessive products, which consequently have no chance of being consumed [38]. It has been estimated that such preparation for shopping could reduce the quantity of food discarded by a household by approximately 20% per one family member [12]. The respondents from all three segments do not tend to arrange products by the expiry date in their cupboards or refrigerator. Sorting products from the shortest to the longest expiry dates undoubtedly helps in the proper management of food storage in the household [10,39]. Consumers can use practical advice as well as all sorts of ‘’goodies” to ensure the proper storage of food at home. A good example of a campaign directed to consumers is that conducted by the Netherlands Nutrition Centre, The Hague, the Netherlands which is in an accessible way helps not only to gain knowledge of the proper handling of food at home, but also to acquire practical accessories useful in food management [40]. All three segments discarded bread with almost the same high frequency. Many researchers have observed that the product is among the most frequently wasted foods [21,25,41]. According to [42], consumers require that bread meets high requirements for freshness, and stale bread is most often thrown away by them. A survey of 1000 Austrians aged 15 and over showed that 78% of respondents rated freshness as the most important attribute of bread [43]. In addition, the respondents belonging to the three segments have diﬃculty interpreting the “use by” or “best before” dates placed on packages. Boxstael et al. [44] also showed inadequate consumer knowledge of these two terms. 3.3. Description of the Deﬁned Segments in Terms of Handling Food Cluster 3 has less people keeping in the refrigerator fresh poultry meat, fresh ﬁsh, yogurt, buttermilk, fresh milk and butter. In contrast, a higher percentage of the respondents refrigerate onion (Table 4). In the course of studies, it was also shown that people in Cluster 3 most frequently indicated too large package size as the main reason for discarding food as compared with the other clusters. The “wasting food” cluster is characterized by the people who most frequently discard 15 groups of food products, including sweets and salty snacks, eggs, fresh poultry and red meat, fresh ﬁsh, canned meat and ﬁsh, and ready meals (Table 4, Figure 2). People in Cluster 3 least frequently indicated that they do not know which products could contain the term “best before” as compared with the other clusters (Table 3). The features diﬀerentiating the segments are shown in Figure 3. Figure 3. Characteristics diﬀerentiating three segments diagnosed. Figure 3. Characteristics diﬀerentiating three segments diagnosed. Foods 2020, 9, 379 13 of 19 13 of 19 4. Discussion According to many researchers [13,15,22,45–48], various diﬀerent languages on date labels and misunderstanding of the meaning of expiry dates is closely associated with a higher frequency of discarding food. As assessed in the UK, approximately one ﬁfth of avoidable food waste is wasted due to the lack of understanding, by 40% to 49% of consumers, of the information concerning the ‘’best before” dates on the packaging. Therefore, it is also necessary to educate consumers, in terms of distinguishing between the “use by” and “best before” dates on food packaging. Thanks to the campaign ‘’Love Food Hate Waste” conducted in the UK in 2008, avoidable food waste was reduced in households by 3% [11]. The most distinctive segment were single and double households without children (Segment 3), exhibiting the highest frequency of discarding 15 food product groups. Mallinson et al. [49] made a similar observation that high wastage of food occurs in the segment consisting of single households. Many available studies indicate a correlation between the quantity of wasted food and household size. Single households waste much food per person [21,22,50–52]. Cluster 2 consisting primarily of families with children is characterized by the highest frequency of discarding fruit and vegetables. The available research indicates that the number of children can impinge disproportionately on the level of food waste due to the unpredictability of behavior and food preferences of children, and parents desire to serve fresh and the highest quality products [53–55]. Most people in Segment 1 declared a good ﬁnancial situation, the lowest level of spending on food, as well as the lowest frequency of discarding 13 product groups. This observation is conﬁrmed by the study by Mallinson et al. [49], conducted in the UK, which showed that the cluster with the lowest percentage of consumers discarding food were people with the highest income levels as compared with the other clusters. Pearson et al. [56] arrived at an opposite conclusion ﬁnding that higher income is associated with higher food waste, which is relatively cheaper than other goods. Foods 2020, 9, 379 14 of 19 The reason for discarding food by small households, which was observed in our study in Poland, was too large package size. This observation was conﬁrmed in studies [21,52] which showed that it is more proﬁtable for small households to purchase goods in larger packages. According to Williams et al. 4. Discussion [57], approximately 20% of food is wasted because of a package which is too large or diﬃcult to empty. Products in smaller packages are disproportionately expensive, so despite being aware of the inability to use a product, consumers buy larger packages. A factor stronger than the awareness of wastage which determines consumers decisions is a signiﬁcant diﬀerence in price based on product weight. These decisions result in an increase in food waste in households in total by approximately 20% to 25% [58]. The quantity of food wasted in households is determined by the frequency and place of purchasing. The publication by Williams et al. [57] and Glanz [58] indicated that most food is discarded when consumers shop exclusively in large supermarkets. This quantity decreases when food products are purchased in a variety of commercial facilities, including in small shops and local markets. In the case of self-supply, the quantity of wasted food is the lowest because people are more aware of the amount of work, time and eﬀort related to its production, breeding and growing. More frequent shopping also minimizes the quantity of discarded products because the planning perspective and current demand for food are not far away [12]. These observations have not been conﬁrmed in our study. The “wasting food” segment prefers small chain shops. In addition, the segment buys and discards fresh and canned ﬁsh more often than the other two segments. The respondents assigned to the “wasting food” cluster most frequently buy products with short expiry dates and at a discounted price, while noting the importance of low prices when buying food. As has been demonstrated by Koivupuro et al. [22] and Schmidt and Matthies [59], the factor exerting excessive inﬂuence on purchasing are marketing activities (discounts) that stimulate impulsive purchases. Nevertheless, researchers have shown that families who often buy food at a reduced price or consider low prices as an important factor when shopping, waste less food. Studies show, on the one hand, that households in which the price of food is an important determinant of purchase are more willing to use promotional oﬀers, including “buy 2 and get 1 free”, and purchase products at a discount, waste less food [22,59]. On the other hand, households that spend more money on groceries per person tend to generate more food waste [12]. 4. Discussion The factor motivating some consumer groups to buy food with short expiry dates is an eco-friendly attitude and counteracting food waste rather than a price reduction ensuring only a ﬁnancial advantage. Increasing consumer awareness of the problem of food waste has a positive eﬀect on a greater desire to prevent this phenomenon, and sometimes it is a stronger incentive than the economic aspect [59]. A factor that can prevent food waste at the stage of selecting food products by consumers in retail facilities is the willingness to buy “imperfect” food, i.e., undersized, with uneven staining, slightly distorted, in torn bulk package, including short expiry dates [12]. It should be noted that people in the “wasting food” segment are more likely than other clusters to buy deformed food in crooked packaging, as well as with short expiry dates, but their motivation is unknown. This segment more often than other segments buys ready frozen and chilled meals, which are also often discarded. According to Mallinson et al. [49], the use of convenience food restricts the purchase of products and semi-ﬁnished products required for conventional cooking, and therefore it can be deduced that it has a positive eﬀect on the level of food waste in households. In the Swedish study by [60] it was demonstrated that such a correlation is not present. The respondents, in Segment 3, that wasted the most food are characterized by improper food storage and disregard for storage conditions speciﬁed by the manufacturer on the packaging. The fact that the persons belonging to this cluster less frequently keep unstable products under refrigeration is disturbing. According to Terpstra et al. [61], consumers have some knowledge of food storage, but do not always use it, for example, improperly store vegetables and set too high temperatures in the refrigerator. While Visschers et al. [62] found no direct relationship between knowledge about storage and the quantity of wasted food. According to Papargyropoulou et al. [6], households that improperly store food contribute to its waste Foods 2020, 9, 379 15 of 19 Many studies have pointed to the insuﬃcient knowledge of consumers concerning issues related to temperature during the technological process [63–66]. Meanwhile, temperature control during the manufacturing process is one of the primary tools in controlling the growth of microorganisms in food. Limitations and Further Research One limitation of this study is that food waste behavior included self-reported measures by respondents, which could be biased. Future studies should consider other sociodemographic factors not included in this segmentation, i.e., gender, age and other factors related to lifestyle, such as meals in the restaurant and ordering meals home. In our study, the segmentation used seven groups of issues and, in the future, studies should be extended to include other factors related to food preparation, planning portions, and use of leftovers. It is also necessary to further analyze the issues raised in the study to learn the motives that guide persons purchasing deformed fruit and vegetables, and food products in deformed packages. To do this, qualitative research could be used. In our study, the respondents answered to the question “how often do you discard food products?”, but other study methods should still be used to specify the quantity of wasted food. 4. Discussion Failure to observe the recommended values is the main cause of the proliferation of microbial cells and, consequently, a number of risks, including food poisoning [67]. According to Ishangulyyev et al. [68], consumers should be educated, among others, in ﬁnancial management, interpretation of expiry dates on packaging, and proper storage of food. Conﬂicts of Interest: The authors declare no conﬂict of interest. Author Contributions: Conceptualization, B.B.; methodology, B.B. and M.T.; validation, D.K.-K., B.B. and M.T.; formal analysis, B.B.; data curation, B.B. and M.T.; writing—original draft preparation, B.B. and M.P.; writing—review and editing, B.B. and M.T.; visualization, B.B.; supervision, D.K.-K.; project administration, B.B. All authors have read and agreed to the published version of the manuscript. Funding: This publication has been developed under the contract with the National Center for Research and Development no. Gospostrateg1/385753/1/NCBR/2018 for carrying out and funding of a project implemented as part of the “The social and economic development of Poland in the conditions of globalizing markets—GOSPOSTRATEG” program called “Developing a system for monitoring wasted food and an eﬀective program to rationalize losses and reduce food wastage” (acronym PROM). References 1. Gustavsson, J.; Cederberg, C.; Sonesson, U.; Van Otterdijk, R.; Meybeck, A. Global Food Losses and Food Waste. Extent, Causes and Prevention; FAO: Rome, Italy, 2011. 2. Martin-Rios, C.; Demen-Meier, C.; Gössling, S.; Cornuz, C. Food waste management innovations in the foodservice industry. Waste Manag. 2018, 79, 196–206. [CrossRef] [PubMed] 3. Stenmarck, A.; Jensen, C.; Quested, T.; Moates, G. Estimates of European Food Waste Levels; Swedish Environmental Research Institute: Stockholm, Sweden, 2016. . Parﬁtt, J.; Barthel, M.; Macnaughton, S. Food waste within food supply chains: Quantiﬁcation and poten for change to 2050. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 2010, 365, 3065–3081. [CrossRef] [PubMed] 5. Gustavsson, J.; Cederberg, C.; Sonesson, U.; Emanuelsson, A. The Methodology of the FAO Study: Global Food Losses and Food Waste-Extent, Causes and Prevention-FAO, 2011; SIK Institutet för Livsmedel och Bioteknik: Göteborg, Sweden, 2013. 6. Papargyropoulou, E.; Lozano, R.; Steinberger, J.K.; Wright, N.; Bin Ujang, Z. The food waste hierarchy as a framework for the management of food surplus and food waste. J. Clean. Prod. 2014, 76, 106–115. [CrossRef] 7. Vittuari, M.; De Menna, F.; Gaiani, S.; Falasconi, L.; Politano, A.; Dietershagen, J.; Segrè, A. The second life of food: An assessment of the social impact of food redistribution activities in Emilia Romagna, Italy. Sustainability 2017, 9, 1817. [CrossRef] 8. Cavaliere, A.; Ventura, V. Mismatch between food sustainability and consumer acceptance toward innovation technologies among millennial students: The case of shelf life extension. J. Clean. Prod. 2018, 175, 641–650. [CrossRef] 9. Castrica, M.; Panseri, S.; Siletti, E.; Borgonovo, F.; Chiesa, L.; Balzaretti, C.M. Evaluation of smart portable device for food diagnostics: A preliminary study on cape hake ﬁllets (M. capensis and M. paradoxus). J. Chem. 2019. [CrossRef] 10. Castrica, M.; Tedesco, D.E.; Panseri, S.; Ferrazzi, G.; Ventura, V.; Frisio, D.G.; Balzaretti, C.M. Pet food as the most concrete strategy for using food waste as feedstuﬀwithin the European context: A feasibility study. Sustainability 2018, 10, 2035. [CrossRef] 11. Monier, V.; Mudgal, S.; Escalon, V.; O’Connor, C.; Gibon, T.; Anderson, G.; Montoux, H. Final Report—Preparatory Study on Food Waste across EU 27; European Commission BIO Intelligence Service: Brussels, Belgium, 2010; Available online: https://ec.europa.eu/environment/eussd/pdf/bio_foodwaste_report.pdf (accessed on 25 July 2017). 12. Schanes, K.; Dobernig, K.; Gözet, B. Food waste matters—A systematic review of household food waste practices and their policy implications. J. Clean. Prod. 2018, 182, 978–991. [CrossRef] 13. 5. Conclusions Three segments diﬀering in size, composition, assessment of the economic situation, and the level of spending on food were identiﬁed. The segment dominated by single and double households without children exhibited a high frequency of discarding food due to too large package. At the same time, this segment did not store food in accordance with the manufacturer’s instructions, which could also contribute to its waste. Segment 2 formed mostly by couples with one child and preferring large-surface stores was distinguished by the highest frequency of discarding fruit and vegetables. g y g q y g g People in Segment 1 declared the lowest frequency of discarding 13 product groups due to a good ﬁnancial situation and the lowest level of spending on food. The identiﬁed segments were similar in terms of the way of preparing for shopping and knowledge of the term “best before”. Knowledge of the factors inﬂuencing consumer behavior related to food waste is essential for the development and implementation of eﬀective education programs in order to reduce this negative phenomenon. It should be borne in mind that consumers are a heterogeneous population and it is necessary to divide them into clusters. Our study showed that segmentation is an eﬀective tool to identify consumers with similar sociodemographic characteristics and behaving similarly in terms of purchase, storage, as well as the causes and frequency of discarding food. Such separate segments require the development of educational programs that most eﬀectively respond to their needs and bring the desired eﬀect which is to reduce food waste. Author Contributions: Conceptualization, B.B.; methodology, B.B. and M.T.; validation, D.K.-K., B.B. and M.T.; formal analysis, B.B.; data curation, B.B. and M.T.; writing—original draft preparation, B.B. and M.P.; writing—review and editing, B.B. and M.T.; visualization, B.B.; supervision, D.K.-K.; project administration, B.B. All authors have read and agreed to the published version of the manuscript. Funding: This publication has been developed under the contract with the National Center for Research and Development no. 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https://openalex.org/W3092569123	https://publicatio.bibl.u-szeged.hu/19763/1/31629691Kovacs-2020-The-sensitivity-and-specificity-of-.pdf	English	null	The sensitivity and specificity of chest CT in the diagnosis of COVID-19	European radiology	2,020	cc-by	4,427	Abstract Multidetector computed tomography . Real-time polymerase chain reaction . Sensitivity . Specificity European Radiology https://doi.org/10.1007/s00330-020-07347-x European Radiology https://doi.org/10.1007/s00330-020-07347-x CHEST CHEST CHEST Anita Kovács1 & Péter Palásti1 & Dániel Veréb1 & Bence Bozsik1 & András Palkó1 & Zsigmond Tamás Kincses1 Received: 5 July 2020 /Revised: 1 September 2020 /Accepted: 24 September 2020 # The Author(s) 2020 * Zsigmond Tamás Kincses kincses.zsigmond.tamas@med.u-szeged.hu; https://www.u- szeged.hu/szakk/radiology/kezdolap 1 Department of Radiology, Albert Szent-Györgyi Clinical Center, University of Szeged, Semmelweis u. 6, Szeged 6725, Hungary The sensitivity and specificity of chest CT in the diagnosis of COVID-19 Anita Kovács1 & Péter Palásti1 & Dániel Veréb1 & Bence Bozsik1 & András Palkó1 & Zsigmond Tamás Kincses1 Abstract Purpose The identification of patients infected by SARS-CoV-2 is highly important to control the disease; however, the clinical presentation is often unspecific and a large portion of the patients develop mild or no symptoms at all. For this reason, there is an emphasis on evaluating diagnostic tools for screening. Chest CT scans are emerging as a useful tool in the diagnostic process of viral pneumonia cases associated with COVID-19. This review examines the sensitivity, specificity, and feasibility of chest CT in detecting COVID-19 compared with real-time polymerase chain reaction (RT-PCR). Methods Sensitivity and specificity of chest CT in detecting COVID-19 in its various phases was compared using RT-PCR as a gold standard. A “reverse calculation approach” was applied and treated chest CT as a hypothetical gold standard and compared RT-PCR to it point out the flaw of the standard approach. Results High sensitivity (67–100%) and relatively low specificity (25–80%) was reported for the CT scans. However, the sensitivity of RT-PCR was reported to be modest (53–88%), hence cannot serve as an appropriate ground truth. The “reverse calculation approach” showed that CT could have a higher specificity (83–100%) if we consider the modest sensitivity of the RT- PCR. Conclusions The sensitivity and specificity of the chest CT in diagnosing COVID-19 and the radiation exposure have to be judged together. Arguments are presented that chest CT scans have added value in diagnosing COVID-19 especially in patients, who exhibit typical clinical symptoms and have negative RT-PCR results in highly infected regions. i CT scans have higher specificity if we take into account the low sensitivity of the RT-PCR. • CT scans have higher specificity if we take into account the low sensitivity of the RT-PCR. • CT scans have higher specificity if we take into account the low sensitivity of the RT-PCR. A id h t CT l di ti h f COVID 19 i f ti • Avoid chest CT as a sole diagnostic approach for COVID-19 infection. • Patients who had negative RT-PCR result with typical clinical symptoms in highly infected regions or with close contact of COVID-19-infected patients; the use of chest CT is warranted. • Patients who had negative RT-PCR result with typical clinical symptoms in highly infected regions or with close contact of COVID-19-infected patients; the use of chest CT is warranted. Keywords COVID-19 . Multidetector computed tomography . Real-time polymerase chain reaction . Se Keywords COVID-19 . Appearance of the disease on the chest CT The SARS-CoV-2 is a member of the family of Coronaviridae and causes systemic and/or respiratory tract infections, rarely acute respiratory distress syndrome, or multi-organ failure. Its cellular entry is the angiotensin- converting enzyme 2 receptor which is expressed partly on the alveolar cells of the lung epithelium [1]. In another set of patients, also from Wuhan, 80 patients with clinical symptoms and positive RT-PCR were investigat- ed. Chest CT was positive in 76 (sensitivity of 95%) patients [5]. The CT manifestation of this viral pneumonia is non- specific but it has characteristic features based on which experienced radiologists can diagnose the disease. Findings are similar to other viral pneumonias but the lo- calization of the signs is rather typical (see Fig. 1). The most frequent and earliest pattern is ground-glass opacity (GGO) that at first may be unifocal, but usually multifocal, bilateral, and peripheral distribution with a posterior pre- dominance especially in inferior lobes. In the area of GGO, common findings are the widening of vessels and traction bronchiectasis [2]. In a study from Shanghai, China, on 38 suspicious COVID-19 patients (presumably all symptomatic), chest CT showed a sensitivity of 100%, specificity of 25%, and accu- racy 47% [7]. A study from a non-high-epidemic area of Japan analyzed the results from 21 patients suspected to have COVID-19 at CT scans at least 3 days after symptom onset [6]. Six patients out of 21 had positive RT-PCR and 15 were proven to have other causes of the symptoms (Moraxella, legionella, pneumocystis, etc.). The sensitivity of the two raters was 67% and 83%, and the specificity 93% and 80%. A study from Rome, Italy, also reported high sensitivity (97%) but moderate specificity (56%) of chest CT in compar- ison with RT-PCR in symptomatic patients [8]. Strengthening their results, RT-PCR was repeated within 24 h if negative on the first occasion. The CT can be positive in the early phase, several days after the onset of the opening symptom (0–4 days). Over time, the CT findings change characteristically. In the progressive stage (5–8 days), the affected areas usually grow and some- times thickened interlobular and intralobular lines appear in- side the GGO. This combination pattern is called crazy pav- ing. It is not characteristic for other viral pneumonias; hence, it can help the differential diagnosis [3]. Long presented data was from the city Yichang, China. Introduction On the other hand, of all 1014 patients, only 601 (59%) had positive RT-PCR results. In these patients, the chest CT was positive in almost all cases (97%). In those patients who had negative RT-PCR result, chest CT was positive in 75%. If the authors considered the RT-PCR as a gold standard, the sensi- tivity, specificity, and accuracy of chest CT indicating COVID-19 infection were 97%, 25%, and 68%, respectively. Appearance of the disease on the chest CT In the study out of 87 symptomatic patients, who had both RT- PCR and chest CT performed, RT-PCR test was positive only in 36 cases. Out of the 36 patients, chest CT was normal only in one (sensitivity 97%) [9]. The peak stage (9–13 days) is at about the 10th day. Consolidation often appears mixed with or after GGO. It can be seen as an early sign in elderly patients. The most severe clinical status is acute respiratory distress syndrome, which is radiologically equivalent to diffuse alveolar damage. A recent meta-analysis, based on sixteen studies, calculated a pooled sensitivity of 92%. In their review, the authors iden- tified only two studies [4, 10] reporting specificity (25–33%). Another meta-analysis found a pooled sensitivity of 94% and specificity of 37% [11]. After that, in the absorption phase, organizing pneumonia pattern appears, and fibrous stripes can be seen with reverse halo sign and mild architectural distortion [3]. The abnormal- ities resolve in about 1 month. Introduction Abbreviations COVID-19 Coronavirus disease 2019 GGO Ground-glass opacity RT-PCR Real-time polymerase chain reaction SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2 A novel coronavirus, named the severe acute respiratory syn- drome coronavirus 2 (SARS-CoV-2) was identified in 2019 in China. The disease caused by the highly contagious virus is called the coronavirus disease 2019 (COVID-19). It spread over the world in a couple of months, causing high fatality and enormous burden on the health care providers. The iden- tification of the infected patients has a high importance to control the disease. However the clinical picture might not be helpful, since a large majority of patients are asymptomatic or having only mild symptoms. Even if the patient had symp- toms, those are rather unspecific (fever, cough, dyspnea). * Zsigmond Tamás Kincses kincses.zsigmond.tamas@med.u-szeged.hu; https://www.u- szeged.hu/szakk/radiology/kezdolap Eur Radiol Eur Radiol the outbreak in Wuhan [4]. The 1014 patients were assigned into three groups: The first two groups consisted of patients having typical clinical symptoms and positive chest CT with or without typical dynamic changes (81%). In the third group (19%), the patients had only one positive CT scan and pre- sumably clinical symptoms (Table 1). Consequently, the performance of various diagnostic tools such as molecular biological tests and imaging are in the focus of the current scientific interest. To date, one of the biggest questions is where chest CT stands in the screening and diag- nostic process in comparison with real-time polymerase chain reaction (RT-PCR) test. In this paper, we try to summarize the results available so far in this topic. We evaluate the available data on the sensitivity, specificity, and accuracy of chest CT. Eighty-eight of all patients had a positive initial chest CT. On the other hand, of all 1014 patients, only 601 (59%) had positive RT-PCR results. In these patients, the chest CT was positive in almost all cases (97%). In those patients who had negative RT-PCR result, chest CT was positive in 75%. If the authors considered the RT-PCR as a gold standard, the sensi- tivity, specificity, and accuracy of chest CT indicating COVID-19 infection were 97%, 25%, and 68%, respectively. In another set of patients, also from Wuhan, 80 patients with clinical symptoms and positive RT-PCR were investigat- ed. Chest CT was positive in 76 (sensitivity of 95%) patients [5]. Eighty-eight of all patients had a positive initial chest CT. High sensitivity, but poor specificity of chest CT in the diagnosis of symptomatic patients It was shown in a recent manuscript (submitted, not accepted at the time of the drafting of this manuscript) that the perfor- mance of the RT-PCR from various respiratory specimens is modest. During the first 2 weeks after symptom onset, 74– 88% of sputum samples are positive and only 53–73% of the One of the largest case series on the correlation of chest CT and RT-PCR in COVID-19 is available from the epicenter of Eur Radiol Fig. 1 Appearance of COVID-19 on CT. a, b A 35-year-old male presented 13 days after the symptom onset with unproductive cough, fatigue, and anosmia. Mild CT signs: GGOs in only one lobe. c, d A 60-year-old male having symptoms for 7 days: muscle pain, weakness, fever, and effort dyspnea. Bilateral, multilobar GGOs and halo sign (small con- solidation surrounded by GGO) on the lower section. e, f A 73- year-old woman experiencing weakness, muscle pain, inappe- tence, and mild effort dyspnea. e Bilateral GGOs, thickened ves- sels, and traction bronchiectasis on the right. f 4 days later, the abnormalities are more extensive and crazy paving appeared within the GGO. g, h An 82-year-old man having symptoms for a week: dry cough, fever, weak- ness, inappetence, and low oxy- gen saturation at presentation. Several features are visible on the CT scans: GGOs, consolidation, organizing pneumonia with re- verse halo sign Starting with the largest study from Wuhan, if one con- siders the chest CT as a gold standard, the sensitivity of RT- PCR is 65%, the specificity is 83%, and the accuracy is 67%. Considering Cheng’s study, if the chest CT was the refer- ence, the sensitivity of PCR was only modest (47%) and ob- viously specificity is high (100%). Similarly, on the Italian sample, RT-PCR had a modest sensitivity and high specificity [8]. nasal swabs. Consequently, one might ask if RT-PCR can be considered as a gold standard. In the following section, we will re-evaluate the abovementioned studies as if the chest CT with typical clinical presentation was the reference (Table 2). This approach certainly underestimates the sensitivity of RT- PCR and overestimates the specificity of chest CT because of not considering other diseases having very similar clinical and CT presentation. However, the traditional estimation consid- ering the RT-PCR as a gold standard (see previous chapter) might suffer from not considering the modest sensitivity of the RT-PCR. High sensitivity, but poor specificity of chest CT in the diagnosis of symptomatic patients Starting with the largest study from Wuhan, if one con- siders the chest CT as a gold standard, the sensitivity of RT- PCR is 65%, the specificity is 83%, and the accuracy is 67%. Considering Cheng’s study, if the chest CT was the refer- ence, the sensitivity of PCR was only modest (47%) and ob- viously specificity is high (100%). Similarly, on the Italian sample, RT-PCR had a modest sensitivity and high specificity [8]. The study of Himoto offers an in depth insight into the performance of chest CT [6]. In this study, in the negative RT-PCR patients, alternative diagnosis was established. Eur Radiol Eur Radiol Table 1 Sensitivity, specificity, and accuracy of chest CT. RT-PCR gold standard Publication Country Confirmed cases/1M* Sensitivity Specificity Accuracy Sample size Days from symptom onset [4] Ai T et al China 19.49 97% 25% 68% 1014 N.R. [5] Wu, J. et al China 31.07 95% N.A. 80 7 ± 4 [6] Himoto, Y. et al Japan 6.9 67–83% 93–80% 86–81% 21 4-26 [7] Cheng, Z. et al China 19.49 100% 25% 47% 38 1–9 [8] Caruso, D. et al Italy 18.66 97% 56% 72% 158 N.R. N.R. not reported, N.A. not applicable *Prevalence data from https://ourworldindata.org/coronavirus at the time of data collection reported in the referenced papers Table 1 Sensitivity, specificity, and accuracy of chest CT. RT-PCR gold standard p , pp valence data from https://ourworldindata.org/coronavirus at the time of data collection reported in the referenced papers Unfortunately, not all patients had RT-PCR test for COVID- 19 and only six of the 15 non-COVID patients had proven infectious agent other than SARS-CoV-2. importance of multiple RT-PCR tests were emphasized by Xie too [14]. It also has to be noted that the evolution of signs of the disease on chest CT is dynamic and peaks at around the 10th day after the first symptoms appeared [3]. Furthermore, it is also an important question when one should judge a chest CT positive. The experience of the reading radiologist may also influence the result diagnosing COVID-19. It is probably not a binary decision, but more of a spectrum to which a cutoff could be determined [15]; therefore, the already reported sen- sitivity and specificity values can be fairly over or underestimated. Similarly, the definition of positive chest CT can also influence the provided performance of RT-PCR. High sensitivity, but poor specificity of chest CT in the diagnosis of symptomatic patients The collaboration of radiologists from Changsha (Hunan province of China) and Province, RI, USA, is providing a picture probably closer to the truth [12]. The authors collected chest CTs from 219 COVID-19 patients from China and 205 patients with positive Respiratory Pathogen Panel for viral pneumonia from Province, RI, from a time period between 2017 and 2019 when no COVID-19 was reported in the USA. The sensitivity for COVID-19 of the Chinese radiolo- gists ranged from 72 to 94%, the specificity 24 to 94%, and the accuracy 60 to 83%. In a smaller subsample of patients, US radiologists had sensitivity of 70 to 93%, specificity of 93 to 100%, and accuracy of 84 to 97%. Chest CT in asymptomatic patients Fang’s report from Eastern China showed that the sensitiv- ity of the RT-PCR might not be optimal at the beginning of the disease. The first RT-PCR test performed in the first 3 (± 3) days after symptom onset was positive in only 36 of the 51 patients. Further 12 patients had positive RT-PCR test on the second occasion (24–48 h after the first), 2 patients by three tests (2–5 days), and one patient by four tests (7 days). On the other hand, 98% of the patients had positive chest CT scan on the first occasion (36 patients with typical and 14 with atypical CT manifestations) [13]. Similarly, in Long’s report, there were 36 patients having positive RT-PCR out of the 87 who had been included in the study [9]. However, 6 cases were missed on the first presentation with RT-PCR. Repeated RT- PCR test 48–72 h later identified further 3 patients and retest for the third time (5–8 days after the first) identified 3 patients. Importantly, the initial CT scan was positive in all but one patient. Therefore, the sensitivity of chest CT at the initial presentation was 97.2% and the RT-PCR only 84.6%. The The modest performance of RT-PCR testing raised the ques- tion whether it could be used in the early, asymptomatic stage of the disease and if chest CT could have an added value. Up to date, only a few reports presented data on this topic. Hu and co-workers showed that 50% of the asymptomatic SARS- CoV-2-infected patients had typical ground-glass opacities and further 20% atypical CT presentation [16]. Half of the CT-positive patients never developed any symptoms. Lin and colleagues reported about a patient having multiple GGOs in the right lung and not having clinical symptoms [17]. Later on, when the patient developed mild symptoms, the appearance of the CT scan changed accordingly. In the early publication by Shi and colleagues from Wuhan, 93% of the 15 preclinical patients had GGO on the chest CT [18]. In the homogenous cohort of Princess Diamond cruise In the homogenous cohort of Princess Diamond cruise ship, 73% of the 104 infected patients were asymptomatic. Table 2 Sensitivity, specificity, and accuracy of RT-PCR accord- ing to the “reverse calculation” approach Publication Sensitivity Specificity Accuracy Sample size Days from symptom onset [4] Ai, T. et al 65% 83% 67% 1014 N.R. [7] Cheng, Z. Table 2 Sensitivity, specificity, and accuracy of RT-PCR accord- ing to the “reverse calculation” approach Chest CT in asymptomatic patients et al 47% 100% 65% 38 1–9 [8] Caruso, D. et al 58% 96% 72% 158 N.R. N.R. not reported Eur Radiol 99% efficiency. Similar time is needed for cleaning, making the throughput of a CT suit to 1–2 patients per hour. Fifty-four percent of these asymptomatic patients had lung opacities on chest CT. Asymptomatic cases showed more GGO than symptomatic patients, in whom the most frequent finding was consolidation [19]. Importantly, in regions where the prevalence of the disease is low, the introduction of chest CT into the screening protocol might not be warranted, since the relatively high specificity might only be true for regions with high occurrence of the disease. Moreover, this could certainly increase the unneces- sary radiation exposure of the population. Compliance with ethical standards Guarantor The scientific guarantor of this publication is Zsigmond Tamás Kincses MD. Conflict of interest The authors of this manuscript declare no relation- ships with any companies whose products or services may be related to the subject matter of the article. It also has to be seen that the specificity of chest CT is not as high as it is indicated by our above presented reverse cal- culation, because the ground truth is not known. But it is expected to be between the values presented earlier and the values from our reverse calculation. Statistics and biometry No complex statistical methods were necessary for this paper. Informed consent Written informed consent was not required for this study because it is a review. To judge the feasibility of chest CT in COVID-19 screening, other factors have to be considered. In the use of chest CT as a first-line screening tool in large population, the risk-benefit ratio should be considered. Medical imaging is the largest man-made source of exposure that is about 0.6 mSv/year [20]. A standard- dose chest CT is in the range of 1.8 mSv, but low-dose protocol was shown to be effective identifying COVID-19 infection that has a dose about 0.2 mSv [21]. Introducing chest CT on a large- scale in the diagnosis of COVID-19 would increase the radiation exposure of the population significantly. Ethical approval Institutional Review Board approval was not required because of the review study design. Study subjects or cohorts overlap Study subjects and cohorts have been reported previously as detailed in the methods and results as well as reference. Feasibility of chest CT for screening COVID-19 infection After a careful consideration of the sensitivity, specificity, the risk of radiation exposure, and the throughput rate of chest CT and RT-PCR: COVID-19 pandemic is spreading around the world with un- precedented speed. In this situation, it is important to analyze the available data to provide guidelines. From the studies we have reviewed above, it is clear that the chest CT has a high sensitivity to detect COVID-19. It seems, however, that while the identification of the virus RNA is the unanimous proof of the disease, the RT-PCR approach is not able to detect all the infections especially in the early phase of the disease. Even more, some studies suggest that chest CT might be more sen- sitive in this phase. & We advise not to use chest CT as a sole diagnostic ap- proach for COVID-19 infection. & However, we think the use of chest CT in patients with typical clinical symptoms in highly infected regions or with close contact of COVID-19-infected patients who had negative RT-PCR result is warranted. & The risk of radiation exposure probably outweighs the sensitivity of chest CT in asymptomatic patients. On the other hand, most of the studies so far suggested that the specificity of the chest CT is low. But it was compared with the performance of the RT-PCR, what is known to have a modest sensitivity. By selecting an inappropriate ground truth and consequently the number of true positives and negatives are being unknown, the calculation of performance will be errone- ous. This fault in the literature was indicated by those studies which showed that multiple RT-PCR tests are increasing the detection rate of the disease. The higher specificity of chest CT was also showed by studies in which COVID-19 and other viral pneumonias were judged, by expert radiologists [12]. Funding Open access funding provided by University of Szeged. References 13. Fang Y, Zhang H, Xie J et al (2020) Sensitivity of chest CT for COVID-19: comparison to RT-PCR. Radiology:200432 1. Cui J, Li F, Shi ZL (2019) Origin and evolution of pathogenic coronaviruses. Nat Rev Microbiol 17(3):181–192 14. Xie X, Zhong Z, Zhao W, Zheng C, Wang F, Liu J (2020) Chest CT for typical 2019-nCoV pneumonia: relationship to negative RT- PCR testing. Radiology 296(2):E41–E45 2. Kanne JP, Little BP, Chung JH, Elicker BM, Ketai LH (2020) Essentials for radiologists on COVID-19: an update. Radiology 296(2):E113–E114 15. Li K, Fang Y, Li W et al (2020) CT image visual quantitative evaluation and clinical classification of coronavirus disease (COVID-19). Eur Radiol 30(8):4407–4416 3. Pan F, Ye T, Sun P et al (2020) Time course of lung changes on chest CT during recovery from 2019 novel coronavirus (COVID- 19) pneumonia. Radiology 295(3):715–721 16. Hu Z, Song C, Xu C et al (2020) Clinical characteristics of 24 asymptomatic infections with COVID-19 screened among close contacts in Nanjing, China. Sci China Life Sci 63(5):706–711 4. Ai T, Yang Z, Hou H et al (2020) Correlation of chest CT and RT- PCR testing in coronavirus disease 2019 (COVID-19) in China: a report of 1014 cases. Radiology 296(2):E32–E40 17. Lin C, Ding Y, Xie B et al (2020) Asymptomatic novel coronavirus pneumonia patient outside Wuhan: the value of CT images in the course of the disease. Clin Imaging 63:7–9 5. Wu J, Wu X, Zeng W et al (2020) Chest CT findings in patients with coronavirus disease 2019 and its relationship with clinical features. Invest Radiol 55(5):257–261 18. Shi H, Han X, Jiang N et al (2020) Radiological findings from 81 patients with COVID-19 pneumonia in Wuhan, China: a descrip- tive study. Lancet Infect Dis 20(4):425–434 6. Himoto Y, Sakata A, Kirita M et al (2020) Diagnostic performance of chest CT to differentiate COVID-19 pneumonia in non-high- epidemic area in Japan. Jpn J Radiol 38(5):400–406 19. Inui S, Fujikawa A, Jitsu M et al (2020) Chest CT findings in cases from the cruise ship “Diamond Princess” with coronavirus disease 2019 (COVID-19). Radiology Cardiothoracic Imaging 2(2): e200110 7. Cheng Z, Lu Y, Cao Q et al (2020) Clinical features and chest CT manifestations of coronavirus disease 2019 (COVID-19) in a single-center study in Shanghai, China. AJR Am J Roentgenol 215(1):121–126 20. Methodology • performed at one institution Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adap- tation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, pro- vide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain It also has to be considered whether the chest CT could increase the risk of transmission of the disease. With dedicated protection devices, the execution of the examination should not carry a higher risk for the medical staff than the risk during the swab test. With careful cleaning and appropriate air exchange rate, the nosocomial transmission of the disease can be avoided. Importantly, CT suits with usual 6–8 air changes per hour require 35–45 min for airborne-contaminant removal with Eur Radiol permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 11. Kim H, Hong H, Yoon SH (2020) Diagnostic performance of CT and reverse transcriptase-polymerase chain reaction for coronavirus disease 2019: a meta-analysis. Radiology 296(3):E145–E155 12. Bai HX, Hsieh B, Xiong Z et al (2020) Performance of radiologists in differentiating COVID-19 from viral pneumonia on chest CT. Radiology 296(2):E46–E54 Publisher’s note Springer Nature remains neutral with regard to jurisdic- tional claims in published maps and institutional affiliations. References Inui S, Fujikawa A, Jitsu M et al (2020) Ionising radiation exposure from medical imaging - a review of patient’s (un) awareness. Radiography (Lond) 26(2):e25–e30 8. Caruso D, Zerunian M, Polici M et al (2020) Chest CT features of COVID-19 in Rome, Italy. Radiology 296(2):E79–E85 21. Kang Z, Li X, Zhou S (2020) Recommendation of low-dose CT in the detection and management of COVID-2019. Eur Radiol 30(8): 4356–4357 9. Long C, Xu H, Shen Q et al (2020) Diagnosis of the coronavirus disease (COVID-19): rRT-PCR or CT? Eur J Radiol 126:108961 10. Zhu W, Xie K, Lu H, Xu L, Zhou S, Fang S (2020) Initial clinical features of suspected coronavirus disease 2019 in two emergency departments outside of Hubei, China. J Med Virol 13. https://doi. org/10.1002/jmv.25763 Publisher’s note Springer Nature remains neutral with regard to jurisdic- tional claims in published maps and institutional affiliations.
https://openalex.org/W3162357096	https://link.springer.com/content/pdf/10.1007/s00526-022-02190-y.pdf	English	null	Gamma-convergence of a gradient-flow structure to a non-gradient-flow structure	Calculus of variations and partial differential equations	2,022	cc-by	25,593	Calc. Var. (2022) 61:103 https://doi.org/10.1007/s00526-022-02190-y Calc. Var. (2022) 61:103 https://doi.org/10.1007/s00526-022-02190-y Calculus of Variations B Mark A. Peletier m.a.peletier@tue.nl Gamma-convergence of a gradient-ﬂow structure to a non-gradient-ﬂow structure Mark A. Peletier1,2 · Mikola C. Schlottke1 Received: 4 May 2021 / Accepted: 28 January 2022 / Published online: 7 April 2022 © The Author(s) 2022 Abstract We study the asymptotic behaviour of a gradient system in a regime in which the driving energy becomes singular. For this system gradient-system convergence concepts are ineffec- tive. We characterize the limiting behaviour in a different way, by proving -convergence of the so-called energy-dissipation functional, which combines the gradient-system compo- nents of energy and dissipation in a single functional. The -limit of these functionals again characterizes a variational evolution, but this limit functional is not the energy-dissipation functional of any gradient system. The system in question describes the diffusion of a par- ticle in a one-dimensional double-well energy landscape, in the limit of small noise. The wells have different depth, and in the small-noise limit the process converges to a Markov process on a two-state system, in which jumps only happen from the higher to the lower well. This transmutation of a gradient system into a variational evolution of non-gradient type is a model for how many one-directional chemical reactions emerge as limit of reversible ones. The -convergence proved in this paper both identiﬁes the ‘fate’ of the gradient system for these reactions and the variational structure of the limiting irreversible reactions. Mathematics Subject Classiﬁcation 35K10 · 35K15 · 35K57 · 35R06 · 35K67 · 35B25 · 35B27 · 49S99 · 60H10 · 60F10 · 70G75 · 37L05 Mathematics Subject Classiﬁcation 35K10 · 35K15 · 35K57 · 35R06 · 35K67 · 35B25 · 35B27 · 49S99 · 60H10 · 60F10 · 70G75 · 37L05 Mathematics Subject Classiﬁcation 35K10 · 35K15 · 35K57 · 35R06 · 35K67 · 35B25 35B27 · 49S99 · 60H10 · 60F10 · 70G75 · 37L05 Communicated by A. Mondino. Mathematics Subject Classiﬁcation 35K10 · 35K15 · 35K57 · 35R06 · 35K67 · 35B25 · 35B27 · 49S99 · 60H10 · 60F10 · 70G75 · 37L05 2 Institute for Complex Molecular Systems (ICMS), Eindhoven University of Technology, Eindhoven, The Netherlands 1 Department of Mathematics and Computer Science, Eindhoven University of Technology, Eindhoven, The Netherlands 1.1 Diffusion in an asymmetric potential landscape Our interest in this paper is the limit ε →0 in the family of Fokker–Planck equations in one dimension deﬁned by Communicated by A. Mondino. 1 Department of Mathematics and Computer Science, Eindhoven University of Technology, Eindhoven, The Netherlands 2 Institute for Complex Molecular Systems (ICMS), Eindhoven University of Technology, Eindhoven, The Netherlands 123 103 Page 2 of 44 M. A. Peletier, M. C. Schlottke Fig. 1 A typical asymmetric potential V (x) Fig. 2 The time evolution of a solution ρε(t, x) to (1) whose initial distribution is supported on the left. Time increases from left to right. At the ﬁnal time, the solution is close to the equilibrium distribution, which is proportional to exp{−V (x)/ε}. The smaller the value of ε, the sharper the equilibrium distribution concentrates around the global minimum xb Fig. 2 The time evolution of a solution ρε(t, x) to (1) whose initial distribution is supported on the left. Time increases from left to right. At the ﬁnal time, the solution is close to the equilibrium distribution, which is proportional to exp{−V (x)/ε}. The smaller the value of ε, the sharper the equilibrium distribution concentrates around the global minimum xb ∂tρε = τε ε ∂xxρε + ∂x ρεV ′ , on R+ × R. (1) (1) Here we take an asymmetric double-well potential V : R →R as depicted in Fig. 1. A typical solution ρε(t, x) is displayed in Fig. 2, showing mass ﬂowing from left to right. There are two parameters, ε > 0 and τε > 0. The ﬁrst parameter ε controls how fast mass can move between the potential wells, where smaller values of ε correspond to larger transition times. The second parameter τε sets the global time scale, and is chosen such that typical transition times from the local minimum xa to the global minimum xb are of order one as ε →0 (see Eq. (3) below). The small-ε limit in the PDE (1) is known as the high activation energy limit in the context of chemical reactions. In this setting, the PDE can be derived from the stochastic evolution of a chemical system, modelled by a one-dimensional diffusion process Y ε t = Y ε(t) in R, satisfying dY ε t = −V ′(Y ε t ) dt + √ 2ε dBt, where Bt is a standard Brownian motion. 1.1 Diffusion in an asymmetric potential landscape For example, consider a particle starting in the left minimum xa and propagating from left to right. This propagation models a reaction event in which a molecule’s state changes from a low-energy state xa via a high-energy state x0 to another low-energy state xb. The assumption of asymmetry of the potential V corresponds to modelling a reaction in which the ﬁnal energy is lower than the initial energy. The energy barrier that the particle has to overcome, V (x0) −V (xa), is the activation energy of the reaction. Hendrik Antony Kramers was the ﬁrst to translate the question of determining the rate of a chemical reaction into properties of PDEs such as (1) [20, 21]. Decreasing ε reduces the noise level in comparison to the potential energy barrier, and a transition from xa to xb becomes more unlikely, and hence the average time until a transition xa ⇝xb increases. Gamma-convergence of a gradient-ﬂow structure to... Page 3 of 44 103 103 Kramers derived an asymptotic expression for this average time: Kramers derived an asymptotic expression for this average time: E inf{t > 0 : Y ε t = xb} Y ε 0 = xa = [1 + o(1)ε→0] 2π √V ′′(xa)\|V ′′(x0)\| exp{ε−1(V (x0) −V (xa))}, (2) (2) which now is known as the Kramers formula. It shows that the average transition time scales exponentially with respect to the ratio of the energy barrier V (x0) −V (xa) to the diffusion coefﬁcient ε. For further details and background on this model, we refer to the monographs of Bovier and den Hollander [3], and of Berglund and Gentz [5]. We are interested in the limit ε →0 in the Eq. (1). In this limit we expect the solution ρε to concentrate at the minima xa and xb. Furthermore, transitions from left to right face a lower energy barrier than from right to left, and because of the exponential scaling in the energy barrier in (2), we expect that in the limit ε →0 transitions occur much more often from left to right than from right to left. Since we want to follow left-to-right transitions, we choose the global time-scale param- eter τε approximately equal to the left-to-right transition time: τε := 2π √V ′′(xa)\|V ′′(x0)\| exp{ε−1(V (x0) −V (xa))}. 1.1 Diffusion in an asymmetric potential landscape (3) (3) Speeding up the process Y ε(t) by τε as Xε(t) := Y ε(τεt), the accelerated process Xε satisﬁes the SDE dXε t = −τεV ′(Xε t ) dt + 2ετε dBt, (4) (4) and the Eq. (1) is the Fokker-Planck equation for the transition probabilities ρε(t, dx) := P Xε t ∈dx . In the rescaled Eq. (1) we therefore expect the limiting dynamics to be characterized by mass being transferred at rate one from the local minimum xa to the global minimum xb, and to see no mass move in the opposite direction. In terms of the solution ρε, we expect that ρε →ρ0 = zδxa + (1 −z)δxb, (5) (5) where the density z = z(t) of particles at xa satisﬁes ∂tz = −z, corresponding to left-to-right transitions happening at rate 1. The time evolution of the limiting density is depicted in Fig. 3. The main results of this paper imply the convergence (5), but they provide more informa- tion: they describe the fate of the gradient-system, variational-evolutionary structure satisﬁed by (1). We describe this next. where the density z = z(t) of particles at xa satisﬁes ∂tz = −z, corresponding to left-to-right transitions happening at rate 1. The time evolution of the limiting density is depicted in Fig. 3. where the density z = z(t) of particles at xa satisﬁes ∂tz = −z, corresponding to left-to-right transitions happening at rate 1. The time evolution of the limiting density is depicted in Fig. 3. The main results of this paper imply the convergence (5), but they provide more informa- The main results of this paper imply the convergence (5), but they provide more informa- tion: they describe the fate of the gradient-system, variational-evolutionary structure satisﬁed by (1). We describe this next. Fig.3 The time evolution of ρ0, deﬁned as the ε →0 limit of the solution ρε(t, x) to (1). The initial distribution is supported solely on the left. Mass ﬂows only from left to right, with rate one Fig.3 The time evolution of ρ0, deﬁned as the ε →0 limit of the solution ρε(t, x) to (1). The initial distribution is supported solely on the left. Mass ﬂows only from left to right, with rate one 123 103 Page 4 of 44 M. A. Peletier, M. C. Schlottke 1.2 Gradient systems and convergence Both the convergence of stochastic processes and the convergence of PDEs are classical problems, and the particular case of the small-noise or high-activation-energy limit is very well studied; see the monographs of Berglund–Gentz and Bovier–Den Hollander that we already mentioned for much more on this topic [3, 5]. In this paper, however, our main interest in the ε →0 limit of Eq. (1) is the relation with convergence of gradient systems. One of the main points of this paper is that while the ε > 0 systems are of gradient type, there is no reasonable convergence that remains within the class of gradient systems. Instead we prove a convergence result to a more general variational evolution that is not of gradient type. In this paper we focus on gradient systems in the space of probability measures on R with a continuity-equation structure. Eq. (1) is of this form; it can be written as the triplet of equations ∂tρε + ∂x jε = 0, (continuity equation), (6a) jε = J ρ ε , (speciﬁcation of ﬂux), (6b) J ρ ε := −τε [ε ∂xρε + ρε∂xV ] , (deﬁnition of J ρ ε in terms of ρε). (6c) ∂tρε + ∂x jε = 0, (continuity equation), (6a) jε = J ρ ε , (speciﬁcation of ﬂux), (6b) J ρ ε := −τε [ε ∂xρε + ρε∂xV ] , (deﬁnition of J ρ ε in terms of ρε). (6c) (6c) For pairs (ρε, jε) satisfying (6a), the second Eq. (6b) can formally be written as For pairs (ρε, jε) satisfying (6a), the second Eq. (6b) can formally be written as Iε(ρε, jε) ≤0, (7) (7) in terms of the trivially nonnegative functional Iε, in terms of the trivially nonnegative functional Iε, Iε(ρε, jε) := 1 2 T 0 R 1 ε τε 1 ρ(t, x) jε(t, x) −J ρ ε (t, x) 2 dxdt. (8) (8) By expanding the square in Iε (see Lemma 2.2 for details) one ﬁnds the equivalent form of (7), By expanding the square in Iε (see Lemma 2.2 for details) one ﬁnds the equivalent form of (7), Iε(ρε, jε) = Eε(ρ) t=T t=0 + T 0 Rε(ρ, j) + R∗ ε ρ, −DEε ρ) dt =: DT ε (ρ, j) ≤0. 1.2 Gradient systems and convergence (9a) (9a) In (9a) the functional Eε is given as In (9a) the functional Eε is given as Eε(ρ) := H(ρ\|γε) where γε(dx) := 1 Zε e−V (x)/ε dx, (9b) (9b) and H(μ\|ν) is the relative entropy of μ with respect to ν. The dual pair (Rε, R∗ ε) of dissipation potentials is formally deﬁned as and H(μ\|ν) is the relative entropy of μ with respect to ν. The dual pair (Rε, R∗ ε) of dissipation potentials is formally deﬁned as Rε(ρ, j) := 1 2ετε R j2 ρ and R∗ ε(ρ, ξ) := ετε 2 R ξ2 ρ. (9c) (9c) The inequality (9a) is known as the EDP-formulation of the gradient system deﬁned by Eε, Rε, and the continuity equation; see e.g. [1, 28, 32] for a general discussion of gradient systems, and [35] for a speciﬁc treatment of gradient systems with continuity- equation structure. The dissipation potential R∗ ε in (9c) and its dual Rε can be interpreted as inﬁnitesimal versions of the Wasserstein metric, and for this reason system (6) or equivalently Eq. (1) is known as a Wasserstein gradient ﬂow [1, 32, 39]. 123 Gamma-convergence of a gradient-ﬂow structure to... 103 Page 5 of 44 The EDP-formulation (9) can be used not only to deﬁne gradient-system solutions, but also to deﬁne convergence of a sequence of gradient systems to a limiting gradient system. Although this method will not be directly of use to us for the proofs in this paper, since the limiting system of this paper will not be of gradient-system type, we will use a number of elements of this method. In addition, it is useful to contrast the method of this paper with this convergence concept. Deﬁnition 1.1 (EDP-convergence) A sequence (Eε, Rε) EDP-converges to a limiting gradi- ent system (E0, R0) if →E0, →DT 0 for all T , and 1. Eε −→E0, 2. DT ε −→DT 0 for all T , and 2. DT ε −→DT 0 for all T , and ε 0 3. the limit functional DT 0 can again be written in terms of the limiting functional E0 and a dissipation potential R0 as DT 0 (ρ, j) = T 0 R0(ρ, j) + R∗ 0(ρ, −DE0(ρ)) dt. 1.2 Gradient systems and convergence (10) (10) EDP-convergence implies convergence of solutions: If (ρε, jε) is a sequence of solutions of (1) or equivalently of (9) that converges to a limit (ρ0, j0), and if the initial state ρε(0) satisﬁes the well-preparedness condition Eε(ρε(t = 0)) →E0(ρ0(t = 0)), (11) (11) then the limit (ρ0, j0) is a solution of the gradient ﬂow associated with (E0, R0). See [28, 29] for an in-depth discussion of EDP-convergence. 103 Page 6 of 44 103 Page 6 of 44 M. A. Peletier, M. C. Schlottke 2. Other scalings of Eε also fail One could mitigate the blow-up of Eε by choosing a different scaling of Eε, Eε(ρ) := εEε(ρ) = ε R ρ(x) log ρ(x) dx + R ρ V + ε log Zε , which -converges to the functional ρ → ρV . With this scaling the well-preparedness condition (11) is simple to satisfy, and by general compactness arguments (e.g. [13, Ch. 10]) the correspondingly rescaled functionals DT ε := εDT ε also -converge to a limit DT 0 . How- ever, this limit functional DT 0 fails to characterize an evolution; we prove this in Sect. 1.6.4 below. Other rescaling choices suffer from similar problems. 3. EDP-convergence should fail There also is a more abstract argument why EDP- convergence should fail, and in fact why any gradient-system convergence should fail. In the limit ε →0 the ratio of forward to reverse transitions diverges, leading to a situation in which motion becomes one-directional. On the other hand, in gradient systems motion can be reversed by appropriate tilting of the driving functional. Therefore the one-directionality is incompatible with a gradient structure. Note that the limiting equation itself, ˙z = −z (see Sect. 1.5), can be given a gradient structure, even many different gradient structures; one example is E(z) := 1 2z2, R(˙z) := 1 2 ˙z2. Our claim here is the following: although the limiting equation can in fact be given a multitude of gradient structures, none of these structures can be found as the limit of the Wasserstein gradient structure of Eq. (1). The simplest proof of this statement is the -convergence theorem that we prove in this paper (Theorem 1.3), which identiﬁes the limit functional; this functional does not generate a gradient structure. Summarizing, although for each ε > 0 the Eq. (1) is a Wasserstein gradient ﬂow with components Eε and Rε, these components diverge in the limit ε →0, and only trivial gradient-system convergence is possible. On the other hand, the functional Iε combines the components Eε, Rε, and R∗ ε in such a way that their divergences compensate each other; in the case of solutions of (1), Iε even is zero for all ε. 103 Page 6 of 44 This suggests that Iε is a better candidate for a variational convergence analysis, and the rest of this paper is devoted to this. Indeed we ﬁnd below that the limit of Iε is not of gradient-ﬂow structure, conﬁrming the earlier suggestion that the sequence leaves the class of gradient systems. Remark 1.2 In [36, 37] one of us developed convergence results for this same limit ε →0 for the case of a symmetric potential V , using a functional framework based on L2-spaces that are weighted with the invariant measure γε. This approach suffers from a similar problem as the Wasserstein-based approach above. The limiting state space is the space L2, weighted by the limiting invariant measure δb, which is a one-dimensional function space; in combination with the constraint of unit mass, the effective state space is a singleton. Consequently the limiting evolution would be trivial. 1.3 (Non-)convergence as ε →0 in the Kramers problem For symmetric potentials V , EDP-convergence of the gradient systems (Eε, Rε) of (9b–9c) has been proved in [2, 24]. For non-symmetric potentials as in this paper, however, we claim that the sequence (Eε, Rε) can not converge in this sense, and we now explain this. 1. The functional Eε blows up The ﬁrst argument for non-convergence follows from the singular behaviour of the driving functional Eε. We can rewrite this functional as Eε(ρ) = H(ρ\|Z−1 ε e−V /ε) = R ρ(x) log ρ(x) dx + R ρ 1 ε V + log Zε . (12) (12) Since the normalization constant Zε is chosen such that γε has mass one, the term in paren- theses converges to +∞at all x except for the global minimizer x = xb (this follows from Lemma 4.4). Therefore Eε -converges to the singular limit functional Since the normalization constant Zε is chosen such that γε has mass one, the term in paren- theses converges to +∞at all x except for the global minimizer x = xb (this follows from Lemma 4.4). Therefore Eε -converges to the singular limit functional E0(ρ) := 0 if ρ = δxb +∞otherwise. This implies that if ρε(0) retains any mass in the higher well around xa as ε →0, then Eε(ρε(0)) →∞. The ‘well-preparedness condition’ (11) therefore can only be satisﬁed in a trivial way, with the initial mass being ‘already’ in the lower of the two wells. Indeed, a gradient system driven by E0 admits only constants as solutions, and does not allow us to follow transitions from xa to xb. 123 123 are sequentially compact in CE(0, T ); 2. Along sequences (ρε, jε) satisfying ρε(t = 0) −⇀ρ◦ 0(dx) := z◦δxa(dx) + (1 −z◦)δxb(dx) as ε →0, (14) unctional Iε -converges to a limit I0. ρε(t = 0) −⇀ρ◦ 0(dx) := z◦δxa(dx) + (1 −z◦)δxb(dx) as ε →0, (14) the functional Iε -converges to a limit I0. (14) ρε(t = 0) ρ0(dx) := z δxa(dx) + (1 z )δxb(dx) as ε →0, (14) the functional Iε -converges to a limit I0. In the next section we deﬁne the limit functional I0 and show that it characterizes the limit evolution as z′ = −z. Remark 1.4 The condition (14) can be interpreted as a well-preparedness property: it states that the initial datum converges to a measure of the same structure as the subsequent evolution (see (17a) below). The bound (13) on the initial energy provides a second type of control on the initial data. 1.4 Main result—0-convergence of Iε In the previous section we introduced the functional Iε of a pair (ρ, j) with the property that solutions of the Eq. (1) are minimizers of Iε at value zero. As for gradient structures, we can 123 123 Gamma-convergence of a gradient-ﬂow structure to... Page 7 of 44 103 103 therefore reformulate the question of convergence as ε →0 in terms of -convergence of these functionals. The main questions then are: therefore reformulate the question of convergence as ε →0 in terms of -convergence of these functionals. The main questions then are: (i) Compactness For a family of pairs (ρ′ ε, j′ ε) depending on ε, does boundedness of Iε(ρ′ ε, j′ ε) imply the existence of a subsequence of (ρ′ ε, j′ ε) that converges in a certain topology T ? (i) Compactness For a family of pairs (ρ′ ε, j′ ε) depending on ε, does boundedness of Iε(ρ′ ε, j′ ε) imply the existence of a subsequence of (ρ′ ε, j′ ε) that converges in a certain topology T ? (ii) Convergence along sequences Is there a limit functional I0 such that (ii) Convergence along sequences Is there a limit functional I0 such that −lim ε→0 Iε = I0 ? (iii) Limit equation Does the equation I0(ρ, j) = 0 characterize the evolution of (ρ, j)? (iii) Limit equation Does the equation I0(ρ, j) = 0 characterize the evolution of (ρ, j)? We answer the ﬁrst question in Theorem 4.7, which establishes that sequences (ρ′ ε, j′ ε) such that Iε(ρ′ ε, j′ ε) remains bounded are compact in a certain topology. We answer the ﬁrst question in Theorem 4.7, which establishes that sequences (ρ′ ε, j′ ε) such that Iε(ρ′ ε, j′ ε) remains bounded are compact in a certain topology. The second question is answered by Theorems4.7 (liminf bound) and Theorem 5.4 (limsup bound), which together establish a limit of Iε in the sense of -convergence. Here, we give a short version that combines these theorems into one statement. For convenience we collect pairs (ρ, j) that satisfy the continuity Eq. (6a) in a set CE(0, T ; R); convergence in this set is deﬁned in a distributional sense (see Deﬁnitions 3.1 and 3.2 ). The following theorem summarizes Theorems 4.7 and 5.4 . Theorem 1.3 (Main result) Let V satisfy Assumption 4.1. Then 1. Sequences (ρε, jε) for which there exists a constant C such that 1. Sequences (ρε, jε) for which there exists a constant C such that Iε(ρε, jε) ≤C and Eε(ρε(0)) ≤C ε (13) (13) are sequentially compact in CE(0, T ); 1.5 The limiting functional I0 Introduce the function S : R2 →[0, ∞], S(a\|b) := ⎧ ⎪⎪⎨ ⎪⎪⎩ a log a b −a + b, a, b > 0, b, a = 0, b > 0, +∞, otherwise. (15) →[0, ∞], S(a\|b) := ⎧ ⎪⎪⎨ ⎪⎪⎩ a log a b −a + b, a, b > 0, b, a = 0, b > 0, +∞, otherwise. (15) (15) otherwise. The map I0 : CE(0, T ) →[0, ∞] is deﬁned by The map I0 : CE(0, T ) →[0, ∞] is deﬁned by I0(ρ, j) := 2 T 0 S( j(t)\|z(t)) dt, (16) (16) 103 Page 8 of 44 M. A. Peletier, M. C. Schlottke 103 whenever whenever whenever ρ(t, dx) = z(t)δxa(dx) + (1 −z(t))δxb(dx) for almost all t, (17a) z(0) = z◦ (see (14)), and (17b) j is piecewise constant in x and given by j(t, x) = j(t)1(xa,xb)(x). (17c) ρ(t, dx) = z(t)δxa(dx) + (1 −z(t))δxb(dx) for almost all t, (17a) z(0) = z◦ (see (14)), and (17b) z(0) = z◦ (see (14)), and (17b) j is piecewise constant in x and given by j(t, x) = j(t)1(xa,xb)(x). (17c) j is piecewise constant in x and given by j(t, x) = j(t)1(xa,xb)(x). (17c) j is piecewise constant in x and given by j(t, x) = j(t)1(xa,xb)(x). (17c) j is piecewise constant in x and given by j(t, x) = j(t)1(xa,xb)(x). (17c) Otherwise, we set I0(ρ, j) = +∞. Otherwise, we set I0(ρ, j) = +∞. Otherwise, we set I0(ρ, j) = +∞. Lemma 1.5 (See Lemma 4.11) If I0(ρ, j) < ∞, then Lemma 1.5 (See Lemma 4.11) If I0(ρ, j) < ∞, then Lemma 1.5 (See Lemma 4.11) If I0(ρ, j) < ∞, then Lemma 1.5 (See Lemma 4.11) If I0(ρ, j) < ∞, then 1. The function z in (17a) is absolutely continuous and non-increasing, 2. The function j(t) in (17c) satisﬁes j(t) = −z′(t) for almost all t. 1. The function z in (17a) is absolutely continuous and non-increasing, 1. The function z in (17a) is absolutely continuous and non-increasing, 2. The function j(t) in (17c) satisﬁes j(t) = −z′(t) for almost all t. 1. The function z in (17a) is absolutely continuous and non-increasing, 2. The function j(t) in (17c) satisﬁes j(t) = −z′(t) for almost all t. 1.5 The limiting functional I0 F ll ( j) I ( j) ≥0 if I ( j) 0 th ti ﬁ ′(t) ( For all (ρ, j), I0(ρ, j) ≥0; if I0(ρ, j) = 0, then z satisﬁes z′(t) = −z(t) for all t. The ﬁnal part of this lemma allows us to characterize any limit of solutions (ρε, jε) of (6). Such solutions satisfy Iε(ρε, jε) = 0; therefore any limit (ρ0, j0) along a subsequence εk →0 satisﬁes 0 ≤I0(ρ0, j0) ≤lim inf εk→0 Iεk(ρε, jε) = 0, and therefore ρ0 has the structure (17a) and the corresponding function z satisﬁes z′ = −z. Since the limit is unique, in fact any sequence (ρεℓ, jεℓ) converges. The evolution of such a function ρ0 is depicted in Fig. 3. and therefore ρ0 has the structure (17a) and the corresponding function z satisﬁes z′ = −z. Since the limit is unique, in fact any sequence (ρεℓ, jεℓ) converges. The evolution of such a function ρ0 is depicted in Fig. 3. Remark 1.6 (I0 does not deﬁne a gradient structure) While the limiting equation z′ = −z has multiple gradient structures (see Sect. 1.3), the limiting functional I0 does not deﬁne any gradient structure. This example therefore is another illustration of how convergence of gradient structures is a stronger property than convergence of the equations (see [28] for more discussion on this topic). To see that I0 does not deﬁne a gradient system, at least formally, assume for the moment that there exist E and R such that 2 T 0 S(−z′\|z) dt = T 0 R(z, z′) + R∗(z, −E′(z)) dt + E(z) T 0 . (18) (18) By taking a short-time limit we deduce that 2S(−v\|z) = R(z, v) + R∗(z, −E′(z)) + E′(z) · v for all z, v, and by differentiating with respect to v we ﬁnd Dv R(z, v) = −2 log −v z −E′(z) for all z, v. Part of the deﬁnition of a gradient system is the requirement that R(z, ·) is minimal at v = 0 for each z (see the discussion in [30, p. 1296]), and the expression for the derivative DR(z, v) above shows that this can not be the case. This mathematical argument backs up the more philosophical arguments in Sect. 1.3 that that I0 does not deﬁne a gradient system. 1.6.1 Main conclusions The main mathematical question in this paper is to understand the ‘fate’ of a gradient structure in a limit in which this gradient structure itself must break down. What we ﬁnd can be summarized as follows: 1. Although the energy Eε and the dissipation potentials Rε and R∗ ε diverge, the single functional Iε that captures the Energy-Dissipation-Principle persists; 1. Although the energy Eε and the dissipation potentials Rε and R∗ ε diverge, the single functional Iε that captures the Energy-Dissipation-Principle persists; 2. This functional Iε provides sufﬁcient control for a proof of compactness and - convergence; 2. This functional Iε provides sufﬁcient control for a proof of compactness and - convergence; 3. The limiting functional I0 deﬁnes a ‘variational-evolution’ system, but not a gradient system; 3. The limiting functional I0 deﬁnes a ‘variational-evolution’ system, but not a gradient system; 4. Both the EDP functional Iε and its limit I0 have a clear connection to large deviations (see below). 4. Both the EDP functional Iε and its limit I0 have a clear connection to large deviations (see below). Although the convergence proved in Theorem 1.3 is not a gradient-system convergence and the energies Eε do not converge, we do use a small component of the typical gradient- system evolutionary-convergence proof. We need some control on the initial data; this is visible in the bound on Eε in (13), which stipulates that Eε(ρε(t = 0)) is allowed to diverge, but not too fast. In fact, the requirement in the proof of Theorem 4.7 is that Eε(ρε(t = 0)) diverges more slowly than exponentially. 1.5 The limiting functional I0 Part of the deﬁnition of a gradient system is the requirement that R(z, ·) is minimal at v = 0 for each z (see the discussion in [30, p. 1296]), and the expression for the derivative DR(z, v) above shows that this can not be the case. This mathematical argument backs up the more philosophical arguments in Sect. 1.3 that that I0 does not deﬁne a gradient system. 123 123 Page 9 of 44 103 103 Gamma-convergence of a gradient-ﬂow structure to... 1.6.3 Connections to chemical reactions There is a strong connection between the philosophy of this paper and results in the chem- ical literature on the appearance of irreversible chemical reactions as limits of reversible reactions, for instance using mass-action laws to describe the dynamics. Gorban, Mirkes, and Yablonksy [18] perform an extensive analysis of such limits and the corresponding behaviour of thermodynamic potentials. Although the gradient-system description of (1) has a clear thermodynamic interpretation (see e.g. [32, Ch. 4–5]), the current paper is different in that the starting point is a diffusion problem, not a discrete reaction system. However, the connections between these two approaches do merit deeper study. This is the top arrow in Fig. 4. The limit functional I0, on the other hand, similarly characterizes the n →∞large deviations of ﬂux-density pairs of n independent particles jumping between two points xa and xb, with jump rates given by ra→b = 1 and rb→a = 0 (see e.g. [22, 38]). This is the bottom arrow in Fig. 4. The right-hand arrow in Fig. 4 is the main result of this paper, Theorem 1.3, which establishes the -convergence of Iε to I0 in the limit ε →0. In the case at hand, in which the n particles constituting the stochastic processes on the left-hand side of the diagram are independent, the left-hand arrow also follows from the results of this paper: The zero sets of Iε and I0 are the forward Kolmogorov equations for the corresponding single-particle stochastic processes, for which the -convergence implies convergence of solutions to solutions; in turn, this implies that the stochastic processes converge. conclusion, with the results of this paper we see that the diagram of Fig. 4 commutes. close connection between Gamma convergence of rate functions and convergence of In conclusion, with the results of this paper we see that the diagram of Fig. 4 commut In conclusion, with the results of this paper we see that the diagram of Fig. 4 commutes. A close connection between Gamma-convergence of rate functions and convergence of processes is observed more broadly; see [7, 11, 27]. A close connection between Gamma-convergence of rate functions and convergence of processes is observed more broadly; see [7, 11, 27]. 1.6.2 Connection to large-deviation principles Both the pre-limit functionals Iε and the limit functional I0 have a clear interpretation as large-deviation rate functions of stochastic processes. In addition, the main result of this paper makes the diagram in Fig. 4 into a commuting diagram. We now explain this. Let Xε i (t) be independent copies of the upscaled diffusion process satisfying (4), and deﬁne formally the empirical ﬂux-density pair (ρε,n, jε,n) by ρε,n = 1 n n i=1 δXε i (t) and jε,n ≈1 n n i=1 δXε i (t)∂t Xε i (t), (19) (19) Fig. 4 The top row corresponds to the empirical ﬂux-density pairs (19) stemming from i.i.d. copies of the reversible diffusion process Xε i (t) from (4), whose Fokker-Planck equation is (1). The bottom row corresponds similarly to a jump process deﬁned on two states {a, b}, with jumps only from a to b. We prove the right arrow by Theorem 1.3, and the left arrow also follows from this result. More explanation is given in the text 03 Page 10 of 44 103 M. A. Peletier, M. C. Schlottke The functional Iε characterizes the large deviations of (ρε,n, jε,n) in the limit n →∞for ﬁxed ε [10, 16] (see also [4, (1.3) and (2.8)]) P (ρε,n, jε,n) t∈[0,T ] ≈(ρε, jε) t∈[0,T] n→∞ ∼ exp −nIε(ρε, jε) . This is the top arrow in Fig. 4. This is the top arrow in Fig. 4. 1.7 Notation CE(0, T ) set of pairs (ρ, j) satisfying the continuity equation Def. 3.1 Cn,m(X×Y) space of functions that are n times differentiable on X and m times on Y γε invariant measure normalized to one Eq. (9b) γ ℓε left-normalized invariant measure Sec. 4.2 Eε energy Eq. (9b) ˆEε, ˆIε, ˆI0 rescaled functionals Def. 5.1 E(μ\|ν, A) localized relative entropy Sec. 4.1 Iε functional for pre-limit variational formulation Def. 20 I0 functional for limit variational formulation Eq. (16) ˆjε ﬂux transformed under yε Eq. (39c)(39d) M(), P() signed Borel and probability measures Sec. 3.1 M≥0() non-negative Borel measures Sec. 3.1 QT , Q0 T QT = [0, T ] × R and Q0 T = [0, T ] × [−1/2, 1/2]. R(μ\|ν, A) localized relative Fisher-information Sec. 4.1 ˆρε, ˆγ ℓε measures transformed under yε Eq. (39a) S(a\|b) function in limit functional I0 Eq. (15) τε exponential time-scale parameter Eq. (3) ˆuℓε density transformed under yε Eq. (39b) ˆu0 limit density Eq. (47) V potential/energy landscape Ass. 4.1 yε, φε auxiliary functions Sec. 4.3 1.6.4 The renormalized gradient system (Eε, Rε) also does not converge As we remarked in Sect. 1.3, the functionals Eε diverge as ε →0, but the rescaled functionals Eε := εEε -convergetoawell-deﬁnedlimit E0(ρ) := ρV .Itisanaturalquestionwhether switching to the rescaled gradient system (Eε, Rε) might solve the singularity problems described in Sect. 1.3. Here the rescaled potentials are deﬁned by Rε(ρ, j) := εRε(ρ, j) and R∗ ε(ρ, ξ) := εR∗ ε ρ, 1 ε ξ , Rε(ρ, j) := εRε(ρ, j) and R∗ ε(ρ, ξ) := εR∗ ε ρ, 1 ε ξ , and EDP-convergence of (Eε, Rε) would follow from the -convergence of Rε(ρ, j) := εRε(ρ, j) and R∗ ε(ρ, ξ) := εR∗ ε ρ, 1 ε ξ , and EDP-convergence of (Eε, Rε) would follow from the -convergence of and EDP-convergence of (Eε, Rε) would follow from the -convergence of DT ε (ρ, j) := εDT ε (ρ, j) = T 0 Rε(ρ, j) + R∗ ε ρ, −DEε(ρ) dt. Even if (Eε, Rε) does converge in the EDP sense to (E0, R0), for some dissipation poten- tial R0, then this limiting gradient system (E0, R0) admits a very wide class of curves as ‘solutions’. This can be recognized as follows. 123 Page 11 of 44 103 Gamma-convergence of a gradient-ﬂow structure to... of 44 103 103 Let z ∈C1([0, T ]) with z′ ≤0, and deﬁne (ρ0, j0) according to (17); since z′ is bounded we have I0(ρ0, j0) < ∞. By the recovery-sequence Theorem 5.9 there exists a sequence (ρε, jε) converging to (ρ0, j0) such that Iε(ρε, jε) →I0(ρ0, j0) and lim infε→0 Eε(ρε(t = 0)) ≥E0(ρ0(t = 0)). We then calculate DT 0 (ρ0, j0) + E0(ρ0(T )) ≤lim inf ε→0 DT ε (ρε, jε) + Eε(ρε(T )) = lim inf ε→0 εIε(ρε, jε) + Eε(ρε(0)) ≤E0(ρ0(0)). It follows that (ρ0, j0) is a solution of the gradient system (E0, R0). This shows that any decreasing function z generates a solution of the gradient system (E0, R0); this explains our claim that it is too degenerate to be of any use. It follows that (ρ0, j0) is a solution of the gradient system (E0, R0). This shows that any decreasing function z generates a solution of the gradient system (E0, R0); this explains our claim that it is too degenerate to be of any use. 2 Elements of the proofs The proofs of compactness and -convergence hinge on a number of ingredients. Dual form of the functional Iε The deﬁnition of Iε given in (8) is formal, since it only makes sense for sufﬁciently smooth measures ρε and jε. The dual formulation that arises naturally from the large-deviation context (see Sect. 1.6.2) solves this deﬁnition problem: Deﬁnition 2.1 The functional Iε : CE(0, T ) →[0, ∞] is deﬁned by Iε(ρ, j) := sup T 0 R jb −ετερ ∂xb −1 ε bV ′ + 1 2b2 : 123 103 Page 12 of 44 M. A. Peletier, M. C. Schlottke b ∈C0,1 c ([0, T ] × R) . (20) (20) Note how this dual form of Iε remains singular in multiple ways: the factor ετερ is expo- nentially large in any region where ρ has O(1) mass, and it is small near the saddle x0 where ρ is expected to behave as γε. The following lemma makes the connection rigorous between Iε and the (Eε, Rε) gradient system. Lemma 2.2 Let (ρ, j) ∈CE(0, T ) satisfy Eε(ρ(0)) < ∞. Then Iε(ρ, j) = Eε(ρ(T )) −Eε(ρ(0)) + T 0 R 1 2ετε v(t, x) 2ρε(t, dx) Rε(ρε(t), jε(t)) + ετε 2 ∂x u(t, x) 2 γε(dx) “R∗ε ρε(t),−DEε(ρε(t)) ” dt. (21) ε(ρ( )) ε(ρ( )) + T 0 R 1 2ετε v(t, x) 2ρε(t, dx) Rε(ρε(t), jε(t)) + ετε 2 ∂x u(t, x) 2 γε(dx) “R∗ε ρε(t),−DEε(ρε(t)) ” dt. (21) (21) re the integral in (21) should be considered equal to +∞unless the following are satisﬁed: Here the integral in (21) should be considered equal to +∞unless the following are satisﬁed: Here the integral in (21) should be considered equal to +∞unless the following are satisﬁed: 1. j is absolutely continuous with respect to ρ on QT , with density v := d j/dρ; 2 i L b b l l i Q i h d i d /d 1. j is absolutely continuous with respect to ρ on QT , with density v := d j/dρ; 2. ρ is Lebesgue-absolutely continuous on QT , with density u := dρ/dγε; 3. ∂xu ∈L1 loc(QT ). 3. ∂xu ∈L1 loc(QT ). 3. ∂xu ∈L1 loc(QT ). The details are given in Sect. 4. The form of the limit functional I0 One can understand how the limiting functional I0 appears in at least three different ways. The ﬁrst is by observing that I0 is the rate function for the Sanov large-deviation principle of a two-point jump process; see Sect. 1.6.2 above. The second understanding of the structure of I0 follows from the proof of the lower bound. This bound follows from making a speciﬁc choice for the function b in the dual formula- tion (20), of the form b(t, x) = −2 f (t)δε x0(x), where δε x0 indicates an appropriately rescaled derivative of the classical committor function (see Sect. 4.3); in the limit δε x0 converges to a Dirac measure at the saddle x0. With this choice we ﬁnd the lower bound (Theorem 4.7) lim inf ε→0 Iε(ρε, jε) ≥2 T 0 z(t) f ′(t) from jb −(e f (t) −1) from ετερ(... ) dt + z◦f (0) lim inf ε→0 Iε(ρε, jε) ≥2 T 0 z(t) f ′(t) from jb −(e f (t) −1) from ετερ(... ) dt + z◦f (0) for any f ∈C1 b([0, T ]) with f (T ) = 0. for any f ∈C1 b([0, T ]) with f (T ) = 0. The supremum of the right-hand side over functions f equals the functional I0, expressed in terms of z. This argument is explained in detail in Sect. 4. The third way to understand the form of I0 is through the construction of the recovery sequence. This sequence is obtained by ﬁrst applying a spatial transformation x →y = yε(x), where the mapping yε is similar to the mapping ˆs used in [2, Sec. 2.1]. The choice of yε and τε leads to a desingularization of Iε, which takes the formal form ˆIε( ˆρ, ˆj) = 1 2 T 0 R 1 ˆuℓ(t, y) ˆj(t, y) + ∂y ˆuℓ(t, y) 2 dydt. (23) (23) Here ˆρ and ˆj are transformed versions of ρ and j that again satisfy the continuity equation, and ˆuℓis the density of ˆρ with respect to the ‘left-rescaled invariant measure’; see Sect. 5 for details. Here ˆρ and ˆj are transformed versions of ρ and j that again satisfy the continuity equation, and ˆuℓis the density of ˆρ with respect to the ‘left-rescaled invariant measure’; see Sect. 5 for details. 2 Elements of the proofs This does require us to assume uniform convexity of each of the two wells separately. 2. However, to prove concentration onto the two points xa and xb, we need to use the polynomial divergence of the ‘Fisher information’ integral that is guaranteed by (22). By applying Logarithmic Sobolev inequalities localized to each of the wells, this divergence is sufﬁciently slow to force concentration onto {xa, xb}. This does require us to assume uniform convexity of each of the two wells separately. 2. However, to prove concentration onto the two points xa and xb, we need to use the polynomial divergence of the ‘Fisher information’ integral that is guaranteed by (22). By applying Logarithmic Sobolev inequalities localized to each of the wells, this divergence is sufﬁciently slow to force concentration onto {xa, xb}. This does require us to assume uniform convexity of each of the two wells separately. 2 Elements of the proofs This type of reformulation is fairly standard, but we did not ﬁnd an explicit proof for this case; we provide a proof in Appendix A.4. In (21) we place R∗ ε ρε(t), −DE(ρε(t)) between quotes, since this expression is only formal; in fact, the expression above the brace could be considered a rigorous interpretation of R∗ ε ρε(t), −DE(ρε(t)) . Forcing concentration onto the two points xa and xb The starting point of the proofs of compactness and the lower bound in Theorem 4.7 is the ‘fundamental estimate’ of every -convergence and compactness proof, Iε(ρε, jε) ≤C. Restricting in (20) to functions b supported in [0, t], and taking into account the divergence of Eε(ρε(0)) as C/ε (see Sect. 1.3) and the bound on Iε, we obtain for each t ∈[0, T ] the estimate t 0 R ετε 2 ∂x uε(t, x) 2 γε(dx) dt + Eε(ρε(t)) ≤C ε Since the integral is non-negative and the constant C is independent of t, there are con- stants C1, C2 such that for every t ∈[0, T ], Since the integral is non-negative and the constant C is independent of t, there are con- stants C1, C2 such that for every t ∈[0, T ], T 0 R ετε 2 ∂x uε(t, x) 2 γε(dx) dt ≤C1 ε , Eε(ρε(t)) ≤C2 ε . Hence Hence T 0 R ετε 2 ∂x uε(t, x) 2 γε(dx) dt + sup t∈[0,T] Eε(ρε(t)) ≤C ε . (22) (22) The divergence of the right-hand side in (22) has consequences for compactness: The divergence of the right-hand side in (22) has consequences for compactness: 1. Because of the growth of V at ±∞, the divergence at rate C/ε of Eε(ρε(t)) sufﬁces to prove tightness of ρε(t); 1. Because of the growth of V at ±∞, the divergence at rate C/ε of Eε(ρε(t)) sufﬁces to prove tightness of ρε(t); 123 Gamma-convergence of a gradient-ﬂow structure to... Page 13 of 44 103 Page 13 of 44 103 2. However, to prove concentration onto the two points xa and xb, we need to use the polynomial divergence of the ‘Fisher information’ integral that is guaranteed by (22). By applying Logarithmic Sobolev inequalities localized to each of the wells, this divergence is sufﬁciently slow to force concentration onto {xa, xb}. 3.1 Preliminary remarks Throughout this paper we use the following conventions and notation. We write QT for the time-space domain [0, T ] × R. Cn,m b (QT ) is the space of functions f : QT →R that are n times differentiable in t and m times differentiable in x, and these derivatives are continuous and bounded. (In the uses below we will require no mixed derivatives). M(QT ) and M(R) are the sets of ﬁnite signed Borel measures on QT and R. We will use two topologies for measures: • The narrow topology, generated by duality with continuous and bounded functions; and The narrow topology, generated by duality with continuous and bounded functions; and • The narrow topology, generated by duality with continuous and bounded functions; and • The wide topology, generated by duality with continuous functions with compact support. • The wide topology, generated by duality with continuous functions with compact suppo The sets M≥0(R) and P(R) are the subsets of non-negative measures and probability mea- sures with the same topology. The sets M≥0(R) and P(R) are the subsets of non-negative measures and probability mea- sures with the same topology. For a measure μ ∈M(R) that is absolutely continuous with respect to the Lebesgue measure, we write μ(dx) for the measure and μ(x) for the density, so that μ(dx) = μ(x)dx. The push-forward measure of a measure μ ∈M(R) under a map T : R →R is given by (T#μ)(A) := μ(T −1(A)) for all Borel sets A ⊂, or equivalently R ϕ(y)(T#μ)(dy) = R ϕ(T (x))μ(dx) for all Borel measurable ϕ : R →R. The details are given in Sect. 4. The remarkable aspect of this rescaling is that the expression (23) no longer contains any singular parameters. The recovery sequence is constructed by solving an auxiliary PDE for ˆuℓ, based on (23), which then is transformed back to a pair (ρε, jε). After transformation to the coordinate y, the left well at xa and the right well interval [xb−, xb+] (see Fig. 1) are mapped to −1/2 and 1/2. From (23) one then ﬁnds an alternative expression for the function S of (15) in terms of functions ˆu(y) (see Lemma A.4): S( j\|z) = 1 4 inf u +1/2 −1/2 1 ˆu(y) j + ˆu′(y) 2 dy : ˆu : [−1/2, 1/2] →(0, ∞), ˆu(−1/2) = z, and ˆu(+1/2) = 0. This formula is closely related to the expression for the limiting rate functional in [2, Eq. (1.30)]; see also [24, App. A]. This formula is closely related to the expression for the limiting rate functional in [2, Eq. (1.30)]; see also [24, App. A]. 123 103 Page 14 of 44 M. A. Peletier, M. C. Schlottke 3.2 Full definition of the continuity equation The functionals Iε are deﬁned on pairs of measures (ρ, j) satisfying the continuity equation ∂tρ + ∂x j = 0 in the following sense. Deﬁnition 3.1 (Continuity Equation) We say that a pair (ρ(t, ·), j(t, ·)) of time-dependent Borel measures on R satisﬁes the continuity equation if: Deﬁnition 3.1 (Continuity Equation) We say that a pair (ρ(t, ·), j(t, ·)) of time-dependent Borel measures on R satisﬁes the continuity equation if: Deﬁnition 3.1 (Continuity Equation) We say that a pair (ρ(t, ·), j(t, ·)) of time-dependent Borel measures on R satisﬁes the continuity equation if: (i) For each t ∈[0, T ], ρ(t, ·) is a probability measure on R. The map t →ρ(t, ·) ∈P(R) is continuous with respect to the narrow topology on P(R). (i) For each t ∈[0, T ], ρ(t, ·) is a probability measure on R. The map t →ρ(t, ·) ∈P(R) is continuous with respect to the narrow topology on P(R). (i) For each t ∈[0, T ], ρ(t, ·) is a probability measure on R. The map t →ρ(t, ·) ∈P(R) is continuous with respect to the narrow topology on P(R). (ii) For each t ∈[0, T ], j(t, ·) is a locally ﬁnite Borel measure on R. The map t →j(t, ·) ∈ M(R) is measurable with respect to the wide topology on M(R), and the joint measure on QT = [0, T ] × R given by (ii) For each t ∈[0, T ], j(t, ·) is a locally ﬁnite Borel measure on R. The map t →j(t, ·) ∈ M(R) is measurable with respect to the wide topology on M(R), and the joint measure on QT = [0, T ] × R given by t∈A \| j(t, B)\| dt for A ⊂[0, T ], B ⊂R bounded, t∈A \| j(t, B)\| dt for A ⊂[0, T ], B ⊂R bounded, is locally ﬁnite on QT . (iii) The pair solves ∂tρ + ∂x j = 0 in the sense that for any test function ϕ ∈C1 c (QT ) with ϕ = 0 at t = T , we have ϕ = 0 at t = T , we have T 0 R [ρ(t, dx) ∂tϕ(t, x) + j(t, dx) ∂xϕ(t, x)] dt + R ρ(0, dx)ϕ(0, x) = 0. 3.2 Full definition of the continuity equation (24 (24) We denote by CE(0, T ) the set of all pairs (ρ, j) satisfying the continuity equation. We denote by CE(0, T ) the set of all pairs (ρ, j) satisfying the continuity equation. This deﬁnition gives rise to a corresponding concept of convergence. This deﬁnition gives rise to a corresponding concept of convergence. 123 Gamma-convergence of a gradient-ﬂow structure to... Page 15 of 44 103 Deﬁnition 3.2 (Convergence in CE) We say that (ρε, jε) converges in CE(0, T ) to (ρ0, j0) ∈ CE(0, T ) if Deﬁnition 3.2 (Convergence in CE) We say that (ρε, jε) converges in CE(0, T ) to (ρ0, j0) ∈ CE(0, T ) if 1. ρε(0, ·) converges narrowly to ρ0(0, ·) on R; 1. ρε(0, ·) converges narrowly to ρ0(0, ·) on R; 1. ρε(0, ·) converges narrowly to ρ0(0, ·) on R; ρε(0, ·) converges narrowly to ρ0(0, ·) on R; 2. ρε converges narrowly to ρ0 on QT ; 1 2. ρε converges narrowly to ρ0 on QT ; 1 3. for all ϕ ∈C1 c (QT ) with ϕ = 0 at t = T , 3. for all ϕ ∈C1 c (QT ) with ϕ = 0 at t = T , lim ε→0 T 0 R jε(t, dx) ∂xϕ(t, x) dt = T 0 R j0(t, dx) ∂xϕ(t, x) dt. (25) (25) Note that then the identity (24) for (ρε, jε) passes to the limit. Remark 3.3 (The convergence arises from a metric) The narrow convergence of ρε is gener- ated by well-known metrics such as the Lévy–Prokhorov or Bounded-Lipschitz metrics [14, Sec. 11.3]. Since Cc(R) is separable, a metric can also be constructed for the wide topology in the usual way. Remark 3.4 (Other deﬁnitions of the continuity equation) Deﬁnition 3.1 is weaker than the common continuity-equation concept for Wasserstein-continuous curves [1, Sec. 8.1], in which j is of the form j = vρ with ρ\|v\|2 < ∞. While for curves (ρε, jε) with Iε(ρε, jε) < ∞the ﬂux jε indeed has this structure (see (21)), in the limit j no longer is absolutely continuous with respect to ρ (see the characterization of ﬁnite I0 in (17c)). In addition, we choose to incorporate the initial datum in the distributional deﬁnition of the continuity Eq. (24), as is common in the theory of parabolic equations with weak time regularity (see e.g. [25, Sec. 3.2 Full definition of the continuity equation I.3]). The explicit initial datum is used below in proving that the limit of ρε connects continuously to the limiting initial datum; see steps 3 and 4 of the proof of Theorem 4.7. Remark 3.5 (Different topologies for ρ and j) It may seem odd that for ρ we require narrow continuity in Deﬁnition 3.1 and narrow convergence in Deﬁnition 3.2, but for j we require only wide convergence in Deﬁnition 3.2. This difference arises from the following considerations. For jε, convergence of the weak form (25) is what we obtain in the proof of the compactness (Theorem 4.7) and of the convergence of the recovery sequence (Theorem 5.9). In both cases it is not clear whether jε converges in a stronger manner than widely. For ρε, however, it is important that in the limit no mass is lost at inﬁnity; this requires narrow convergence. In the setup above, this narrow convergence follows from the wide convergence of jε on [0, T ] × R, which also implies wide convergence for ρε on the same space; since the limit ρ(t, ·) is again required to be a probability measure for all t, no mass escapes to inﬁnity, and the convergence of ρε in fact is narrow. The narrow continuity of t →ρ(t, ·) in Deﬁnition 3.1 follows from the the conditions on j: the local bounds on j imply wide continuity of ρ, and the requirement that ρ is a probability measure at all t upgrades this continuity to narrow. 4 Compactness The limit ε →0 is accompanied by the concentration of ρε onto the two minima of the wells, at xa and xb. This concentration is essential for the further analysis of the functionals Iε and their -limits; if ρε would maintain mass at other points in R, then the main statement and the corresponding analysis of the functionals Iε both would fail. 123 103 Page 16 of 44 103 Page 16 M. A. Peletier, M. C. Schlottke Fig. 5 Illustration of Assumption 4.1 Fig. 5 Illustration of Assumption 4.1 In the case of a potential V with wells of equal depth (as in [2, 24]), a constant bound on the initial energy Eε(ρε(t = 0)) leads to a similar bound on later energies Eε(ρe(t)), which in turn leads to concentration onto {xa, xb}. In the unequal-well case of this paper, as we discussed in the introduction, we are forced to allow for divergent Eε; consequently the concentration onto {xa, xb} has to come from different arguments. Here we choose to obtain this concentration from the ‘Fisher-information’ or ‘local-slope term’; this is the second term in DT ε in (9a), or equivalently the second half of the integral in (21). This requires imposing conditions on the convexity of the wells, which we do in part 5 of the following set of assumptions on V . Assumption 4.1 Let V ∈C2(R) and let the special x-values Assumption 4.1 Let V ∈C2(R) and let the special x-values −∞< xa < xcℓ< x0 < xcr < xb−< xb < xb+ < ∞ satisfy the following: 1. Two wells, the left well at value zero: {V ≤0} = {xa} ∪[xb−, xb+]; 1. Two wells, the left well at value zero: {V ≤0} = {xa} ∪[xb−, xb+]; 1. Two wells, the left well at value zero: {V ≤0} = {xa} ∪[xb−, xb+]; 2 i h b f h i h ll V ( ) i V 0 2. xb is the bottom of the right well: V (xb) = minR V < 0; 2. xb is the bottom of the right well: V (xb) = minR V < 0; 3. x0 is the saddle, and the intermediate range lies below it: V (x) ≤V (x0) for xa < x < with V (x) < V (x0) unless x = x0; 3. 4 Compactness In this example the mass will concentrate onto {xa, xd, xb}, with no mass moving from xd to xb [xb−, xb+] need not all end up in xb. Figure 6 shows why: if the right well has a ‘sub-well’ (say xd) such that the transition xd ⇝xb has a higher energy barrier than the transition xa ⇝xd, then the mass leaving xa will be held back at xd, with further transitions to xb happening at an exponentially longer time scale. If we start with all mass concentrated at xa, then the limiting evolution will be concentrated on {xa, xd} instead of on {xa, xb}. [xb−, xb+] need not all end up in xb. Figure 6 shows why: if the right well has a ‘sub-well’ (say xd) such that the transition xd ⇝xb has a higher energy barrier than the transition xa ⇝xd, then the mass leaving xa will be held back at xd, with further transitions to xb happening at an exponentially longer time scale. If we start with all mass concentrated at xa, then the limiting evolution will be concentrated on {xa, xd} instead of on {xa, xb}. Failure type II: Hills at high energy levels Something similar can happen in the ‘wings’ of the energy landscape, as illustrated by Fig. 7. If valleys exist outside of the region {x : V (x) < V (x0)} with energy barriers larger than the xa ⇝xb barrier, then the slowness of transitions between such valleys again will prevent concentration into the sub-zero zone {x : V (x) ≤0}. 4 Compactness x0 is the saddle, and the intermediate range lies below it: V (x) ≤V (x0) for xa < x < xb, with V (x) < V (x0) unless x = x0; 4. The saddle is non-degenerate: V ′′(x0) < 0; g 5. Uniform convexity away from the saddle: there exist A, α > 0 such that A ≥V ′′ ≥α > 0 on (−∞, xcℓ] and [xcr, ∞). 5. Uniform convexity away from the saddle: there exist A, α > 0 such that A ≥V ′′ ≥α > 0 on (−∞, xcℓ] and [xcr, ∞). We also choose two open intervals Ba and Bb containing xa and [xb−, xb+], respectively, and such that supBa∪Bb V < V (x0). The set B0 is deﬁned as the set separating Ba and Bb. Figure 5 illustrates these features. Assumptions 1–4 encode the basic geometry of a two-well potential with unequal wells. Condition number 5 is added to rule out concentration at different points than xa and xb. The following two examples illustrate how concentration at different points may happen if this convexity condition is not imposed. Failure type I: A hilly right well Since the energy barrier is lower for transitions from left to right than vice versa, it is natural to assume that in the limit all mass travels from left to right. Indeed, this is true under weak assumptions, but the mass that arrives in the right well 123 Gamma-convergence of a gradient-ﬂow structure to... Page 17 of 44 103 Page 17 of 44 103 103 Fig. 6 Example of a potential V that is excluded by Assumption 4.1 (failure of ‘type I’ in the text). If the deeper well has internal energy barriers that are larger than the barrier V (x0) −V (xa) for escape from xa, then mass may collect in intermediary valleys instead of at xb. In this example the mass will concentrate onto {xa, xd, xb}, with no mass moving from xd to xb Fig. 6 Example of a potential V that is excluded by Assumption 4.1 (failure of ‘type I’ in the text). If the deeper well has internal energy barriers that are larger than the barrier V (x0) −V (xa) for escape from xa, then mass may collect in intermediary valleys instead of at xb. 4.1 Logarithmic Sobolev inequalties Theidentity(∗)canalsobeseenasarigorousdeﬁnitionoftheleft-handside R∗ ε(ρ, −DEε(ρ)) in terms of the right-hand side R(ρ\|γε, R): this right-hand side is well deﬁned for all ρ, and in addition Lemma 2.2 shows that this is the term that appears in the reformulation of Iε in gradient-system form. Note that the functions E and R are (1, 0)-homogeneous in the pair (μ, ν), i.e. for each μ, ν ∈M≥0(R) and a, b > 0, E(aμ\|bν, A) = aE(μ\|ν, A) and R(aμ\|bν, A) = aR(μ\|ν, A). E(aμ\|bν, A) = aE(μ\|ν, A) and R(aμ\|bν, A) = aR(μ\|ν, A). The following Lemma generalizes classical Logarithmic Sobolev inequalities based on uni- form convexity bounds to the homogeneous functionals E and R and the restriction to subsets A ⊂R. Lemma 4.2 (Logarithmic Sobolev inequality) Let A ⊂R be an interval. If W ∈C2(A) with W ′′ ≥α > 0 on A, then Lemma 4.2 (Logarithmic Sobolev inequality) Let A ⊂R be an interval. If W ∈C2(A) with W ′′ ≥α > 0 on A, then αE(μ\|e−W dx, A) ≤R(μ\|e−W dx, A) for all μ ∈M≥0(A). (27) (27) Proof By e.g. [6, Cor. 5.7.2] or [9, Cor. 1], if W ∈C2(R) with W ′′ ≥α > 0 on R, then the inequality (27) holds for A = R and for all μ ∈P(R). By the homogeneity of E and R the same applies to all μ ∈M≥0(R). 2 Proof By e.g. [6, Cor. 5.7.2] or [9, Cor. 1], if W ∈C2(R) with W ′′ ≥α > 0 on R, then the inequality (27) holds for A = R and for all μ ∈P(R). By the homogeneity of E and R the same applies to all μ ∈M≥0(R). To generalize to the case of A ⫋R and a given potential W ∈C2(A) with W ′′ ≥α on A, ﬁrst smoothly extend W to the whole of R in such a way that W ′′ ≥α on R and R e−W < ∞. Next deﬁne the sequence of C2 potentials y Next deﬁne the sequence of C2 potentials Wk(x) := W(x) + k dist(x, A)4 for x ∈R. Wk(x) := W(x) + k dist(x, A)4 for x ∈R. As k →∞the measures e−Wk(x)dx converge narrowly on R to e−W(x)1A(x)dx. Each Wk satisﬁes W ′′ k ≥α on R, and it follows that for any μ ∈M≥0(R) with μ(R \ A) = 0, As k →∞the measures e−Wk(x)dx converge narrowly on R to e−W(x)1A(x)dx. 4.1 Logarithmic Sobolev inequalties We use logarithmic Sobolev inequalities to capitalize on the uniform convexity bounds in part 5 of Assumption 4.1. Such inequalities are usually formulated for reference measures with unit mass, but in our case it will be convenient to generalize to all ﬁnite positive measures, and also allow for localization to subsets of R. For A ⊂R and μ, ν ∈M≥0(R), we set ≥ E(μ\|ν, A) := ⎧ ⎨ ⎩ A f log f dν −μ(A) log μ(A) ν(A) if μ ≪ν, μ = f ν +∞ otherwise, R(μ\|ν, A) := ⎧ ⎨ ⎩ 2 A ∂x f 2 dν, if μ ≪ν, μ = f ν +∞ otherwise. otherwise, otherwise. With these deﬁnitions, the energy Eε and the ‘slope’ R∗ ε(ρ, −DEε(ρ)) (see (9b)) can be written as With these deﬁnitions, the energy Eε and the ‘slope’ R∗ ε(ρ, −DEε(ρ)) (see (9b)) can be written as ( ) Eε(ρ) = E(ρ\|γε, R) and R∗ ε(ρ, −DEε(ρ)) (∗) = ετεR(ρ\|γε, R). (26) Eε(ρ) = E(ρ\|γε, R) and R∗ ε(ρ, −DEε(ρ)) (∗) = ετεR(ρ\|γε, R). (26) (26) 123 103 Page 18 of 44 M. A. Peletier, M. C. Schlottke Fig. 7 Second example of a potential V that is excluded by Assumption 4.1 (failure of ‘type II’ in the text). If energy barriers exist outside of the range [xa, xb] that are larger than the barrier V (x0) −V (xa) of the escape from xa, then these will prevent mass from moving to xa (and then also from transitioning to xb). In this example the mass will concentrate onto {xe, xa, xb}, with no mass moving from xe to xa or xb Fig. 7 Second example of a potential V that is excluded by Assumption 4.1 (failure of ‘type II’ in the text). If energy barriers exist outside of the range [xa, xb] that are larger than the barrier V (x0) −V (xa) of the escape from xa, then these will prevent mass from moving to xa (and then also from transitioning to xb). In this example the mass will concentrate onto {xe, xa, xb}, with no mass moving from xe to xa or xb Theidentity(∗)canalsobeseenasarigorousdeﬁnitionoftheleft-handside R∗ ε(ρ, −DEε(ρ)) in terms of the right-hand side R(ρ\|γε, R): this right-hand side is well deﬁned for all ρ, and in addition Lemma 2.2 shows that this is the term that appears in the reformulation of Iε in gradient-system form. 4.1 Logarithmic Sobolev inequalties Each Wk satisﬁes W ′′ k ≥α on R, and it follows that for any μ ∈M≥0(R) with μ(R \ A) = 0, 123 Page 19 of 44 103 Gamma-convergence of a gradient-ﬂow structure to... 103 αE(μ\|e−W dx, A) = αE(μ\|e−W 1Adx, R) = lim k→∞αE(μ\|e−Wkdx, R) (27) on R ≤ lim k→∞R(μ\|e−Wkdx, R) = R(μ\|e−W dx, A). αE(μ\|e−W dx, A) = αE(μ\|e−W 1Adx, R) = lim k→∞αE(μ\|e−Wkdx, R) αE(μ\|e−W dx, A) = αE(μ\|e−W 1Adx, R) = lim k→∞αE(μ\|e−Wkdx, R) This proves the claim (27). This proves the claim (27). This proves the claim (27). Bounds on the entropy give rise to concentration estimates of the underlying measure. Lemma 4.3 (Concentration estimates based on E) Let A1 ⊂A2 ⊂R, and let μ, ν ∈ M≥0(A2) with ν(A1) > 0. Then μ(A1) ≤E(μ\|ν, A2) + μ(A2) log ν(A2)/ν(A1) . (28) (28) Proof By homogeneity of E it is sufﬁcient to prove the inequality for the case μ(A2) = ν(A2) = 1. We can also assume that μ ≪ν, and we set μ = f ν. Applying Young’s inequality with the dual pair η(s) = s log s −s +1 and η∗(t) = et −1, we ﬁnd for any a > 0 that μ(A1) = 1 a A1 f a dν ≤1 a A1 η( f ) dν + 1 a A1 ea −1 dν ≤1 a A2 η( f ) dν + ea a ν(A1). Choosing a = \| log ν(A1)\| = −log ν(A1) we ﬁnd Choosing a = \| log ν(A1)\| = −log ν(A1) we ﬁnd Choosing a = \| log ν(A1)\| = −log ν(A1) we ﬁnd Choosing a = \| log ν(A1)\| = −log ν(A1) we ﬁnd μ(A1) ≤ 1 \| log ν(A1)\| E(μ\|ν, A2) + 1 , μ(A1) ≤ 1 \| log ν(A1)\| E(μ\|ν, A2) + 1 , se μ(A2) = ν(A2) = 1. μ(A1) ≤ 1 \| log ν(A1)\| E(μ\|ν, A2) + 1 , h is (28) for the case μ(A2) = ν(A2) = 1. which is (28) for the case μ(A2) = ν(A2) = 1. 4.2 Invariant measures and their normalizations In the introduction we deﬁned the invariant measure γε(dx) := 1 Zε e−V (x)/ε dx, with Zε := R e−V (x)/ε dx. The measure γε is normalized in the usual manner, and is therefore a probability measure on R. Since V has a single global minimum at xb, the measures γε converge to δxb; therefore the mass of γε around xa vanishes. It will also be useful to have a differently normalized measure γ ℓ ε in which the mass around xa does not vanish. For this reason we also deﬁne the left-normalized measures γ ℓ ε by γ ℓ ε (dx) := 1 Zℓε e−V (x)/ε dx, with Zℓ ε := x0 −∞ e−V (x)/ε dx. Figure 8 illustrates the behaviour of γ ℓ ε and γε as ε →0. The following lemma charac- terizes some of their behaviour in precise form. 123 103 Page 20 of 44 103 Page 20 of 44 M. A. Peletier, M. C. Schlottke Fig. 8 Behavior of the left-normalized invariant measure γ ℓε and the fully-normalized measure γε for small values of ε Fig. 8 Behavior of the left-normalized invariant measure γ ℓε and the fully-normalized measure γε for small values of ε 4.3 Auxiliary functions ε and yε To desingularize the functional Iε we will need an auxiliary function φε that is adapted to the singular structure of this system and distinguishes the two wells, in the sense of having constant, but different, values there. For the recovery sequence we will need a related function To desingularize the functional Iε we will need an auxiliary function φε that is adapted to the singular structure of this system and distinguishes the two wells, in the sense of having constant, but different, values there. For the recovery sequence we will need a related function yε, and we deﬁne it here at the same time, and study the properties of φε and yε together. Fix two smooth functions χa, χb ∈C∞ c (R) with χa,b ≥0, supp χa ⊂Ba and supp χb ⊂ Bb, and χa(xa) = 1 = χb(xb). Set yε, and we deﬁne it here at the same time, and study the properties of φε and yε together. Fix two smooth functions χa, χb ∈C∞ c (R) with χa,b ≥0, supp χa ⊂Ba and supp χb ⊂ Bb, and χa(xa) = 1 = χb(xb). Set Fix two smooth functions χa, χb ∈C∞ c (R) with χa,b ≥0, supp χa ⊂Ba and supp χb ⊂ Bb, and χa(xa) = 1 = χb(xb). Set με := e−V /εχa e−V /εχa −e−V /εχb e−V /εχb and Mε(x) := x −∞ με. The function Mε has the following properties: The function Mε has the following properties: 1. 0 ≤Mε ≤1; 1. 0 ≤Mε ≤1; 1. 0 ≤Mε ≤1; 2. Mε is equal to 1 on B0 and equal to zero outside of Ba ∪B0 ∪Bb, and converges in L1 to 1[xa,xb]; 2. Mε is equal to 1 on B0 and equal to zero outside of Ba ∪B0 ∪Bb, and converges in L1 to 1[xa,xb]; Deﬁne φε ∈C2 b(R) and yε ∈C2(R) by Deﬁne φε ∈C2 b(R) and yε ∈C2(R) by e φε ∈C2 b(R) and yε ∈C2(R) by φε(x) := Zℓ ε ετε x x0 eV (ξ)/εMε(ξ) dξ (30) yε(x) := Zℓ ε ετε x x0 eV (ξ)/ε dξ. (31) (30) (31) The functions yε and φε are shown in Fig. 9. Lemma 4.4 Let V satisfy Assumption 4.1. ε a For part 3, we estimate using the superquadratic growth of V that 1 Zℓε V >δ e−V /ε ≤1 Zℓε R exp −1 ε δ ∨C(x2 −1) ε→0 −→0. This proves the claim. Finally, to show that γ ℓ ε ((c, d)) →∞(part 4), note that V (x) ≤−μ < 0 for some constant μ > 0 on an open interval (xb−+ δ, xb−+ 2δ) ⊂(c, d); from this the divergence follows. Lemma 4.4 Let V satisfy Assumption 4.1. and γ ℓ ε are well-deﬁned, and in the limit ε →0, 1. γε and γ ℓ ε are well-deﬁned, and in the limit ε →0, Zε = [1 + o(1)] 2πε V ′′(xb)e−V (xb)/ε, Zℓ ε = [1 + o(1)] 2πε V ′′(xa). (29) (29) Zε = [1 + o(1)] V ′′(xb)e , Zε = [1 + o(1)] V ′′(xa). (29) 2. If ˜x > x0 and V < V (x0) on (x0, ˜x], then Zℓ ε ετε ˜x x0 eV /ε −→1 2 as ε →0. 2. If ˜x > x0 and V < V (x0) on (x0, ˜x], then Zℓ ε ˜x eV /ε −→1 2 as ε →0. 2. If ˜x > x0 and V < V (x0) on (x0, ˜x], then 2. If ˜x > x0 and V < V (x0) on (x0, ˜x], then 2. If ˜x > x0 and V < V (x0) on (x0, ˜x], then Zℓ ε ετε ˜x x0 eV /ε −→1 2 as ε →0. 3. For any δ > 0, limε→0 γ ℓ ε ({V > δ}) = 0. 4. For any xa < c < x0 < xb−< d 4. For any xa < c < x0 < xb−< d, the sequence γ ℓ ε ⌊(−∞, c) converges as measures to δxa, and γ ℓ ε ((c, d)) →∞. 4. For any xa < c < x0 < xb−< d, the sequence γ ℓ ε ⌊(−∞, c) converges as measures to δxa, and γ ℓ ε ((c, d)) →∞. δxa, and γ ℓ ε ((c, d)) →∞. Part 3 above expresses the property that the left-normalized measures concentrate in the limit ε →0 onto the set {V ≤0} = {xa}∪[xb−, xb+]. Part 4 expresses the fact that the ‘left- 123 Gamma-convergence of a gradient-ﬂow structure to... Page 21 of 44 103 103 hand’ part of γ ℓ ε has a well-behaved limit δxa, while the right-hand part of γ ℓ ε has unbounded mass. Proof For part 1, the superquadratic growth of V towards ±∞that follows from uniform convexity implies that Zε and Zℓ ε are ﬁnite for each ε; the scaling of Zε and Zℓ ε then follow directly from Laplace’s method (Lemma A.2). The same holds for part 2, and the convergence of γ ℓ ε ⌊(−∞, c) to δxa (part 4). 4.3 Auxiliary functions ε and yε The deﬁnition of φε is a minor modiﬁcation of [15, Lemma 3.6] and is nearly the same as the committor function, known from potential theory [3] and Transition-Path Theory [42]; see also [26] for a discussion of its use in coarse- graining, which is similar to its function here. The following lemma describes in different ways how φε approximates the function x →sign(x)/2. 2. There exists C > 0 such that \|φε\| ≤C for sufﬁciently small ε, limε→0 φε(−∞) = −1 and limε→0 φε(+∞) = 1/2; Fig. 9 Comparison of the functions φε and yε. Note how the two functions are very similar in the region between and around the two wells; towards ±∞, however, yε is unbounded, while the range of φε is bounded Lemma 4.5 The function φε satisﬁes Lemma 4.5 The function φε satisﬁes 1. φε is non-decreasing on R; 2. There exists C > 0 such that \|φε\| ≤C for sufﬁciently small ε, limε→0 φε(−∞) = −1/2, and limε→0 φε(+∞) = 1/2; 123 103 Page 22 of 44 103 Page 22 of 44 M. A. Peletier, M. C. Schlottke Fig. 9 Comparison of the functions φε and yε. Note how the two functions are very similar in the region between and around the two wells; towards ±∞, however, yε is unbounded, while the range of φε is bounded 3 f l 1/2 d 1/2 3. φε converges uniformly to −1/2 on Ba and to 1/2 on Bb. 4. Zℓ εeV /εμε converges uniformly on R to −χa. 3. φε converges uniformly to −1/2 on Ba and to 1/2 on Bb. 4. Zℓ εeV /εμε converges uniformly on R to −χa. Proof. The non-negativity of Mε proves the monotonicity of φε. The bound on φε and the convergence of the limit values follow from remarking that sup R φε = φε(+∞) = Zℓ ε ετε ∞ x0 eV /εMε. Since on supp Mε ⊂Ba ∪B0 ∪Bb the potential V takes its maximum at the saddle x0, and since Mε is equal to one around the saddle, the integral converges to 1/2 by part 2 of Lemma 4.4. The behaviour at −∞is proved in the same way. Since the expression Zℓ εeV /ε/ετε converges to zero uniformly on Ba and Bb, Eq. (30) implies that φε becomes constant on Ba and Bb and converges uniformly on those sets to its limit values, which are −1/2 and 1/2, respectively. Finally, Zℓ εeV /εμε = Zℓ ε χb e−V /εχb −Zℓ ε χa e−V /εχa =: αε,bχb −αε,aχa. The ﬁrst term vanishes uniformly since the scalar αε,b equals Zℓ ε/ e−V /εχb ∼ eV (xb)/ε →0. The second term converges to −χa, since α−1 ε,a = 1 Zℓε e−V /εχa = χaγ ℓ ε −→χa(xa) = 1. The function yε is very similar to φε, but differs in the tails, and will be used as a coordinate transformation in Section 5. The function yε is very similar to φε, but differs in the tails, and will be used as a coordinate transformation in Section 5. Lemma 4.6 1. The function yε is strictly increasing and bijective. 1 Lemma 4.6 1. The function yε is strictly increasing and bijective. 2. For any x < x0 such that V (x) < V (x0), we have yε(x) →−1 2 as ε →0. 3. For any x > x0 such that V (x) < V (x0), we have yε(x) →+ 1 2 as ε →0. 4. 103 Page 22 of 44 For x < x0 satisfying V (x) < V (x0), we obtain Proof Since y′ ε(x) > 0 for any x ∈R and yε(x) →±∞as x →±∞, the map yε is strictly increasing and bijective. For x < x0 satisfying V (x) < V (x0), we obtain yε(x) = 1 ετε · Zℓ ε · x x0 eV (z)/ε dz = [1 + o(1)] 1 ετε · e−V (xa)/ε 2πε V ′′(xa) · 1 2eV (x0)/ε 2πε \|V ′′(x0)\|(−1) yε(x) = 1 ετε · Zℓ ε · x x0 eV (z)/ε dz = [1 + o(1)] 1 ετε · e−V (xa)/ε 2πε V ′′(xa) · 1 2eV (x0)/ε 2πε \|V ′′(x0)\|(−1) −→−1 2, by using (29) and applying Lemma A.2(b) to the integral. The argument for the case x > x0 is similar. To show that φε ◦y−1 ε converges uniformly on R to id1/2, ﬁrst note that d dy φε y−1 ε (y) = φ′ ε y−1 ε (y) y′ε y−1 ε (y) = Mε y−1 ε (y) for any y ∈R. The function Mε ◦y−1 ε converges in L1(R) to 1[−1/2,1/2]; this can be recognized from the fact that y−1 ε (y) converges to 0 for any −1/2 < y < 1/2, to +∞for y > 1/2, and to −∞ for y < −1/2. The uniform convergence of φε ◦y−1 ε then follows by integration. The function Mε ◦y−1 ε converges in L1(R) to 1[−1/2,1/2]; this can be recognized from the fact that y−1 ε (y) converges to 0 for any −1/2 < y < 1/2, to +∞for y > 1/2, and to −∞ for y < −1/2. The uniform convergence of φε ◦y−1 ε then follows by integration. 103 Page 22 of 44 φε ◦y−1 ε converges uniformly on R to the truncated identity function id1/2, deﬁned by 2 3. For any x > x0 such that V (x) < V (x0), we have yε(x) →+ 1 2 as ε →0. 4. φε ◦y−1 ε converges uniformly on R to the truncated identity function id1/2, deﬁne 2 4. φε ◦y−1 ε converges uniformly on R to the truncated identity function id1/2, deﬁned b id1/2(x) := ⎧ ⎪⎨ ⎪⎩ −1/2 if x ≤−1/2 x if −1/2 ≤x ≤1/2 1/2 if x ≥1/2. 123 Page 23 of 44 103 Gamma-convergence of a gradient-ﬂow structure to... Fig. 10 The structure of the map yε of (31). Points to the left of x0 with V (x) < V (x0) are mapped to −1/2, and similarly, points to the right of x0 are mapped to +1/2. The smaller the value of ε, the sharper is the concentration effect. As ε →0, points far to the left of xa and far to the right of xb are mapped to ∓∞, respectively Proof Since y′ ε(x) > 0 for any x ∈R and yε(x) →±∞as x →±∞, the map yε is strictly increasing and bijective. For x < x0 satisfying V (x) < V (x0), we obtain Fig. 10 The structure of the map yε of (31). Points to the left of x0 with V (x) < V (x0) are mapped to −1/2, and similarly, points to the right of x0 are mapped to +1/2. The smaller the value of ε, the sharper is the concentration effect. As ε →0, points far to the left of xa and far to the right of xb are mapped to ∓∞, respectively Fig. 10 The structure of the map yε of (31). Points to the left of x0 with V (x) < V (x0) are mapped to −1/2, and similarly, points to the right of x0 are mapped to +1/2. The smaller the value of ε, the sharper is the concentration effect. As ε →0, points far to the left of xa and far to the right of xb are mapped to ∓∞, respectively Proof Since y′ ε(x) > 0 for any x ∈R and yε(x) →±∞as x →±∞, the map yε is strictly increasing and bijective. Fig. 10 The structure of the map yε of (31). Points to the left of x0 with V (x) < V (x0) are mapped to −1/2, and similarly, points to the right of x0 are mapped to +1/2. The smaller the value of ε, the sharper is the concentration effect. As ε →0, points far to the left of xa and far to the right of xb are mapped to ∓∞, respectively 4.4 Compactness and lower bound Having deﬁned the auxiliary function φε we can state and prove the main compactness theorem, which includes a lower bound on Iε. Theorem 4.7 (Compactness and lower bound) Let V satisfy Assumption 4.1. Let (ρε, jε) ∈ CE(0, T ) satisfy sup ε Iε(ρε, jε) + εEε(ρε(0)) ≤C < ∞, (32) (32) Page 24 of 44 103 M. A. Peletier, M. C. Schlottke and assume that ρε(0) satisﬁes the narrow convergence and assume that ρε(0) satisﬁes the narrow convergence me that ρε(0) satisﬁes the narrow convergence ρε(0) −⇀ρ◦ 0(dx) := z◦δxa(dx) + (1 −z◦)δxb(dx) as ε →0. (33) ρε(0) −⇀ρ◦ 0(dx) := z◦δxa(dx) + (1 −z◦)δxb(dx) as ε →0. (33) (33) Then there exists a (ρ0, j0) ∈CE(0, T ) and a subsequence along which Then there exists a (ρ0, j0) ∈CE(0, T ) and a subsequence along which ρε −⇀ρ0 narrowly in M([0, T ] × R), where ρ0 ∈M([0, T ] × R) has the structure ρ0(dtdx) = ρ0(t, dx)dt := z(t)δxa(dx)dt + (1 −z(t))δxb(dx)dt, (34) 1. ρε −⇀ρ0 narrowly in M([0, T ] × R), where ρ0 ∈M([0, T ] × R) has the structure ρ0(dtdx) = ρ0(t, dx)dt := z(t)δxa(dx)dt + (1 −z(t))δxb(dx)dt, (34) 1. ρε −⇀ρ0 narrowly in M([0, T ] × R), where ρ0 ∈M([0, T ] × R) has the structure ρ0(dtdx) = ρ0(t, dx)dt := z(t)δxa(dx)dt + (1 −z(t))δxb(dx)dt, (34) (34) and z : [0, T ] →[0, 1] is absolutely continuous. 1 0 and z : [0, T ] →[0, 1] is absolutely continuous. 1 0 converges in duality with C1,0 c ([0, T ) × R) to 2. jε converges in duality with C1,0 c ([0, T ) × R) to j0(dtdx) := j(t)1[xa,xb](x)dxdt, j0(dtdx) := j(t)1[xa,xb](x)dxdt, j0(dtdx) := j(t)1[xa,xb](x)dxdt, Remark 4.8 Note that the two assumptions on the initial data, the convergence (33) and the boundedness Eε(ρε(0)) ≤C/ε of (32), are closely related, but independent: it is possible to satisfy one but not the other. Proof Recall from the discussion in Sect. 2 that by the assumption (32) on the initial data we have the ‘fundamental estimate’ ετε 2 T 0 R ∂x uε(t, x) 2 γε(dx) dt + sup t∈[0,T] Eε(ρε(t)) ≤C ε . (35) (35) Here uε is the density of ρε with respect to the invariant measure γε. Here uε is the density of ρε with respect to the invariant measure γε. 4.4 Compactness and lower bound ε y ρε p γε Step 1: Concentration for the case of the outer half-lines Set Oℓ:= (−∞, xcℓ]. Recall that V ′′ ≥α > 0 on Oℓ; by Lemma 4.2 we therefore have Step 1: Concentration for the case of the outer half-lines Set Oℓ:= (−∞, xcℓ]. Recall t V ′′ ≥α > 0 on Oℓ; by Lemma 4.2 we therefore have Step 1: Concentration for the case of the outer half-lines Set Oℓ:= (−∞, xcℓ]. Recall that V ′′ ≥α > 0 on Oℓ; by Lemma 4.2 we therefore have α ε E(μ\|γε, Oℓ) ≤R(μ\|γε, Oℓ) for all μ ∈M≥0(R) and ε > 0. α ε E(μ\|γε, Oℓ) ≤R(μ\|γε, Oℓ) for all μ ∈M≥0(R) and ε > 0. α ε E(μ\|γε, Oℓ) ≤R(μ\|γε, Oℓ) for all μ ∈M≥0(R) and ε > 0. Then Then T 0 E(ρε(t)\|γε, Oℓ) dt ≤ε α T 0 R(ρε(t)\|γε, Oℓ) dt = ε α T 0 1 2 Oℓ ∂x dρε(t) dγε 2 γε(dx)dt ≤ε α · C ε2τε by (35) = C αετε −→0 as ε →0. = ε α T 0 1 2 Oℓ ∂x dρε(t) dγε 2 γε(dx)dt ≤ε α · C ε2τε by (35) = C αετε −→0 as ε →0. Therefore, if A ⊂Oℓwith dist(A, {xa}) > 0, then by Lemma 4.3, Therefore, if A ⊂Oℓwith dist(A, {xa}) > 0, then by Lemma 4.3, T 0 ρε(t, A) dt ≤ log γε(A) γε(Oℓ) −1 T 0 E(ρε(t)\|γε, Oℓ) + 1 dt ε→0 −→0. It follows that ρε1Oℓconcentrates onto [0, T ] × {xa}. By a similar argument ρε1[xcr,∞) concentrates onto [0, T ] × {xb}. This also implies that ρε is tight on [0, T ] × R. It follows that ρε1Oℓconcentrates onto [0, T ] × {xa}. By a similar argument ρε1[xcr,∞) concentrates onto [0, T ] × {xb}. This also implies that ρε is tight on [0, T ] × R. Step 2: Concentration for the case of the whole domain R We have proved concentration of ρε1(−∞,xcℓ] onto [0, T ] × {xa} and of ρε1[xcr,∞) onto [0, T ] × {xb}. What remains is to bridge the gap between xcℓand xcr. 123 Gamma-convergence of a gradient-ﬂow structure to... Page 25 of 44 103 103 We write uℓ ε for the density of ρε with respect to the left-normalized invariant measure γ ℓ ε , i.e. 4.4 Compactness and lower bound ˆuℓ ε = uεZε/Zℓ ε. We then estimate T 0 R ∂x uℓε 2 (t, x)e(V (x0)−V (x))/ε dxdt = Zℓ ε T 0 R ∂x uℓε 2 (t, x)eV (x0)/εγ ℓ ε (dx)dt = Cτε Zℓ ε T 0 R ∂x √uε 2 (t, x)γε(dx)dt (35) ≤Cε−3/2. Since V ≤V (x0) on [xa, xb+] it follows that Since V ≤V (x0) on [xa, xb+] it follows that T 0 xb+ xa ∂x uℓε 2 (t, x) dxdt ≤Cε−3/2. Applying the generlized Poincaré inequality of Lemma A.1 to f (t, x) = uℓε on [xa, xb+] we ﬁnd Applying the generlized Poincaré inequality of Lemma A.1 to f (t, x) = uℓε on [xa, xb+] we ﬁnd ∥uℓ ε∥L1(0,T;L∞(xa,xb+)) = T 0 ∥uℓ ε(t)∥L∞(xa,xb+) dt ≤C ε−3/2 + T 0 γ ℓ ε ([xa, xb+])−1∥uℓ ε(t)∥L1(xa,xb+;γ ℓε ) dt = C ε−3/2 + γ ℓ ε ([xa, xb+])−1 T 0 ρε(t; [xa, xb+]) dt ≤Cε−3/2 since γ ℓ ε ([xa, xb+]) →∞by Lemma 4.4. To prove concentration, take an interval A such that [xcℓ, xcr] ⊂A ⊂{V ≥δ} for some δ > 0. Then T 0 ρε(t, A) dt = T 0 A uℓ ε(t, x)γ ℓ ε (dx) dt ≤γ ℓ ε (A)∥uℓ ε∥L1(0,T ;L∞(xa,xb+)) ≤Cε−3/2 · e−δ/ε Zℓε ε→0 −→0. Therefore ρε does not charge the region [0, T ] × A in the limit. Therefore ρε does not charge the region [0, T ] × A in the limit. Concluding, ρε concentrates onto [0, T ] × {xa, xb} as ε →0. It follows that the limit ρ0 has support contained in [0, T ] × {xa, xb}, and for almost every t ∈[0, T ], ρ0(t, ·) has mass one on R. This establishes the structure (34), except for the continuity of z; at this stage we only know that z ∈L∞(0, T ) with 0 ≤z ≤1, and the absolute continuity of z will follow in Step 4 below. Remark 4.9 After completing the proof of compactness outlined in the previous two steps, André Schlichting pointed out that by using the Muckenhoupt criterion it is possible to replace the assumption of convex wells by two monotonicity assumptions, one for each well; see Theorem 3.19 in [40] for an example. 4.4 Compactness and lower bound Step 3: Lower bound on Iε From Deﬁnition 2.1 and the bound (32) we have for any b ∈C0,1 c (QT ) the estimate C ≥Iε(ρε, jε) ≥ T 0 R jεb −ετερε ∂xb −1 ε bV ′ + 1 2b2 dxdt. (36) (36) 123 123 103 Page 26 of 44 103 M. A. Peletier, M. C. Schlottke Fix ψ ∈C1([0, T ]) with inf ψ > −1 and ψ(T ) = 0. Deﬁne Fε : [0, T ] × R →R by Fε(t x) := log 1 + ψ(t) ( 1 2 −φε(x)) with φε given by (30) Fix ψ ∈C1([0, T ]) with inf ψ > −1 and ψ(T ) = 0. Deﬁne Fε : [0, T ] × R →R by Fix ψ ∈C1([0, T ]) with inf ψ > −1 and ψ(T ) = 0. Deﬁne Fε : [0, T ] × R →R by Lemma 4.10 Fε and φε have the following properties: Lemma 4.10 Fε and φε have the following properties: 1. Fε ∈C1 b(QT ) and ∂x Fε ∈C1 c (QT ); b c 2. Fε(T , x) = 0 for all x ∈R; b c 2. Fε(T , x) = 0 for all x ∈R; b 2. Fε(T , x) = 0 for all x ∈R; 3. supε,t,x \|Fε(t, x)\| ≤max{log(1 + sup ψ), −log(1 + inf ψ)}; 3. supε,t,x \|Fε(t, x)\| ≤max{log(1 + sup ψ), −log(1 + inf ψ)}; , , 4. φε converges uniformly on [0, T ] × Ba to 1 and on [0, T ] × Bb to zero; , , 4. φε converges uniformly on [0, T ] × Ba to 1 and on [0, T ] × Bb to zero; 5. Fε converges uniformly on [0, T ] × Ba to log(1 + ψ(t)) and on [0, T ] × Bb to zero. These follow directly from Lemma 4.6. These follow directly from Lemma 4.6. If K0(z) = 0, then z(t) = z◦e−t for almost all 0 ≤t ≤T . If K0(z) = 0, then z(t) = z◦e−t for almost all 0 ≤t ≤T . If K0(z) = 0, then z(t) = z◦e−t for almost all 0 ≤t ≤T . We prove this lemma below, and ﬁrst ﬁnish the proof of Theorem 4.7. Note that since J0(z) = K0(z) < ∞, the function z has an absolutely continuous representative and z(0) = z◦; this concludes the proof of part 1 of the theorem. 1 0 p p Step 4 of the proof of Theorem 4.7: Convergence of jε Choose any ϕ ∈C1,0 c (QT ) with ϕ = 0 att = T ,andset(t, x) := x 0 ϕ(t, ξ) dξ;notethat ∈C1 b(QT )and∂x ∈Cc(QT ). We calculate T 0 R jεϕ = T 0 R jε(t, dx)∂x(t, x) dxdt (24) = − T 0 R ρε(t, dx)∂t(t, x) dxdt − R ρε(0, dx)(0, x) ε→0 −→− T 0 R ρ0(t, dx)∂t(t, x) dxdt − R ρ◦ 0(dx)(0, x) (33,34) = − T 0 z(t)∂t(t, xa) + (1 −z(t))∂t(t, xb) dt −z(0)(0, xa) −(1 −z(0))(0, xb) = T 0 z′(t) (t, xa) −(t, xb) dt = T 0 R (−z′(t))ϕ(t, x)1[xa,xb](x) dxdt. This proves the convergence of part 2. Finally, with this deﬁnition of the limit j0 of jε, we have I0(ρ0, j0) = J0(z) = K0(z), d this concludes the proof of Theorem 4.7. and this concludes the proof of Theorem 4.7. and this concludes the proof of Theorem 4.7. Proof of Lemma 4.11 A closely related statement and its proof are discussed in [33, Sec. 3]; for completeness we give a standalone proof. Step 1: If J0(z) < ∞, then z is non-increasing on [0, T ] Fix ϕ ∈C∞ c ((0, T )) with ϕ ≥0. Applying the deﬁnition of J0(z) to f = −λϕ we ﬁnd −λ T 0 zϕ′ ≤J0(z) for allλ > 0, which implies zϕ′ ≥0. Since ϕ ∈C∞ c ((0, T )) is arbitrary, it follows that the equivalence class z ∈L∞has a non-increasing representative, and from now on we write z for this non-increasing representative. We also ﬁnd that −z′ is a positive measure on (0, T ). By the monotonicity of z, the limits of z at t = 0, T exist, and if necessary we redeﬁne z to be continuous at t = 0, T . These follow directly from Lemma 4.6. with znon-increasing and absolutely continuous, and z(0) = +∞ otherwise. ⎧ ⎪⎪⎪⎨2 T 0 S −z′(t)\|z(t) dt if z = z a.e. with znon-increasing These follow directly from Lemma 4.6. We now set bε(t, x) = 2∂x Fε(t, x) = 2ψ(t)φ′ ε(x)/(1 + ψ(t)φε(x)) and ﬁnd that the expression in brackets in (36) equals ∂xbε −1 ε bεV ′ + 1 2b2 ε = 2ψ 1 + ψφε φ′′ ε −1 ε φ′ εV ′(30) = 2ψ 1 + ψφε Zℓ ε ετε eV /εμε. By Lemma 4.5 and the concentration of ρε we therefore ﬁnd that lim ε→0 T 0 R ετερε ∂xbε −1 ε bεV ′ + 1 2b2 ε = lim ε→0 T 0 R 2ψ 1 + ψφε ρε Zℓ εeV /εμε = 2 T 0 R ψ(t) 1 + ψ(t) ρ0(t, dx)χa(x) dt = 2 T 0 ψ(t) 1 + ψ(t) z(t) dt. ( lim ε→0 T 0 R ετερε ∂xbε −1 ε bεV ′ + 1 2b2 ε = 2 T 0 ψ(t) 1 + ψ(t) z(t) dt. (37) (37) We now turn to the ﬁrst term in (36). Applying the Deﬁnition 3.1 of CE, and the assump- tion (33) on the convergence of the initial data, we ﬁnd T 0 R jεbε = −2 T 0 R ρε∂t Fε −2 R ρε(0, dx)Fε(0, x) = −2 T 0 R ρε ψ′φε 1 + ψφε −2 R ρε(0, dx)Fε(0, x) ε→0 −→−2 T 0 z(t) ψ′(t) 1 + ψ(t) dt −2z◦log(1 + ψ(0)). (38) (38) Writing f (t) := −log(1+ψ(t)) we have f (T ) = 0; combining (37) and (38), and observing that ψ/(1 + ψ) = e f −1, we ﬁnd Writing f (t) := −log(1+ψ(t)) we have f (T ) = 0; combining (37) and (38), and observing that ψ/(1 + ψ) = e f −1, we ﬁnd lim inf ε→0 Iε(ρε, jε) ≥J0(z), lim inf ε→0 Iε(ρε, jε) ≥J0(z), with with J0(z) := 2 sup T 0 z(t) f ′(t) −e f (t) + 1 dt + z◦f (0) : f ∈C1 b([0, T ]), f (T ) = 0 . 123 123 Gamma-convergence of a gradient-ﬂow structure to... Page 27 of 44 1 103 Lemma 4.11 Let z ∈L∞(0, T ) with z ≥0, and let z◦≥0. Then J0(z) = K0(z), where Lemma 4.11 Let z ∈L∞(0, T ) with z ≥0, and let z◦≥0. Then J0(z) = K0(z), where K0(z) := ⎧ ⎪⎪⎪⎨ ⎪⎪⎪⎩ 2 T 0 S −z′(t)\|z(t) dt if z = z a.e. and absolutely continuous, and z(0) = z◦ and absolutely continuous, and z(0) = z◦ otherwise. 5 Recovery sequence In this section we state and prove Theorem 5.9, which establishes the existence of a recovery sequence for the -convergence of Theorem 1.3. If K0(z) = 0, then z(t) = z◦e−t for almost all 0 ≤t ≤T . By construction, z now is non-increasing on [0, T ] and −z′ is a positive measure on [0, T ] without atoms at t = 0, T . 103 Page 28 of 44 103 M. A. Peletier, M. C. Schlottke Step 2: Reformulation and matching initial data Since −z′ is a ﬁnite measure and z is continuous at t = 0, T , we can rewrite J0(z) = 2 sup T 0 −z′(t) f (t) −z(t) e f (t) −1 dt + (z◦−z(0)) f (0) : f ∈C1 b([0, T ]), f (T ) = 0 . By choosing functions f with f (0) = λ ∈R and supp f a vanishingly small interval close to t = 0 we ﬁnd J0(z) ≥2λ(z◦−z(0)), and by taking limits λ →±∞it follows that z(0) = z◦. Step 3: Primal form Still under the assumption that J0(z) < ∞, we recognize e f −1 as the dual η∗( f ) of the function η(a) := S(a\|1) (which is equal to a log a −a + 1 for a > 0). We then use the well-known duality characterization of convex functions of measures (see e.g. [1, Lemma 9.4.4]) to ﬁnd, writing μ(dt) := z(t)dt, J0(z) = ⎧ ⎨ ⎩ T 0 η d(−z′) dμ (t) μ(dt) if −z′ ≪μ +∞ otherwise, otherwise, otherwise, and this functional coincides with K0 (see e.g. [35, Lemma 2.3]). The reverse statement, assuming K0(z) < ∞and showing that J0(z) < ∞, follows directly by Young’s inequality for the pair (η, η∗). and this functional coincides with K0 (see e.g. [35, Lemma 2.3]). The reverse statement, assuming K0(z) < ∞and showing that J0(z) < ∞, follows directly by Young’s inequality for the pair (η, η∗). Step 4: Absolute continuity Finally, if J0(z) = K0(z) < ∞, then the superlinearity of η implies that z′ ∈L1(0, T ), and therefore z is absolutely continuous. Step 5: Characterization of minimizers If K0(z) = 0, then z′(t) = −z(t) for almost all t, implying that z(t) = z◦e−t. 5.1 Spatial transformation We start by transforming the system by a nonlinear mapping in space, given by the function yε deﬁned in Sect. 4.3; this function maps R with variable x to R with variable y, and is inspired by a similar choice in [2]. This mapping desingularizes the system. ℓ ℓ We deﬁne the transformed versions ˆρε and ˆγ ℓ ε of ρε and γ ℓ ε by pushing them forward under yε, ˆρε := (yε)#ρε and ˆγ ℓ ε := (yε)#γ ℓ ε , (39a) (39a) which implies that the transformed density ˆuℓ ε is given by which implies that the transformed density ˆuℓ ε is given by ˆuℓ ε(t, yε(x)) := uℓ ε(t, x). (39b) (39b) We transform jε in such a way that the continuity equation is conserved, which leads to the choice ˆjε := (yε)# y′ ε jε , (39c) (39c) 123 Page 29 of 44 10 Gamma-convergence of a gradient-ﬂow structure to... 103 which has an equivalent formulation in the case of Lebesgue-absolutely-continuous ﬂuxes, ˆjε(t, yε(x)) := jε(t, x). (39d) ˆjε(t, yε(x)) := jε(t, x). (39d) (39d) Indeed, if (ρ, j) satisﬁes the continuity Eq. (6a), then the transformed pair ( ˆρε, ˆjε) satisﬁes the corresponding continuity equation in the variables (t, y), ∂t ˆρε + ∂y ˆjε = 0 in R+ × R, ∂t ˆρε + ∂y ˆjε = 0 in R+ × R, which is deﬁned again as in Deﬁnition 3.1, and one can check that (ρ, j) ∈CE(0, T ) ⇐⇒ ( ˆρ, ˆj) ∈CE(0, T ). Since yε is a diffeomorphism, there is a one-to-one relationship between (ρ, j) and ( ˆρ, ˆj). which is deﬁned again as in Deﬁnition 3.1, and one can check that (ρ, j) ∈CE(0, T ) ⇐⇒ ( ˆρ, ˆj) ∈CE(0, T ). Since yε is a diffeomorphism, there is a one-to-one relationship between (ρ, j) and ( ˆρ, ˆj). (ρ j) (ρ j) In terms of ˆjε and the density ˆuℓ ε the rate function formally takes the simpler form Iε(ρ, j) = 1 2 T 0 R 1 ˆuℓε(t, y) ˆjε(t, y) + ∂y ˆuℓ ε(t, y) 2 dydt. Note how the parameters ε and τε are absorbed into the density ˆuℓ ε and the derivative with respect to the new coordinate y. 5.1 Spatial transformation The coordinate transformation yε is the almost the same as in [2]; the only difference is that we use the left-normalized stationary measure, whereas in the symmetric case one can use the stationary measure normalized in the usual manner. This simpler, transformed form is the basis for the construction of the recovery sequence. To make this precise we ﬁrst deﬁne the rescaled versions of Iε and I0. Deﬁnition 5.1 (Rescaled functionals) For given ρ and j, deﬁne ˆρ and ˆj as in (39a) and (39c). We deﬁne ˆEε, Iε, and I0 to be the rescaled versions of Eε, Iε, and I0, ˆEε( ˆρ) := Eε(ρ), Iε( ˆρ, ˆj) := Iε(ρ, j), I0( ˆρ, ˆj) := I0(ρ, j). ˆEε : P(R) →[0, ∞], ˆEε( ˆρ) := Eε(ρ), Iε : CE(0, T ) →[0, ∞], Iε( ˆρ, ˆj) := Iε(ρ, j), I0 : CE(0, T ) →[0, ∞], I0( ˆρ, ˆj) := I0(ρ, j). ˆEε : P(R) →[0, ∞], ˆEε( ˆρ) := Eε(ρ), Iε : CE(0, T ) →[0, ∞], Iε( ˆρ, ˆj) := Iε(ρ, j), I0 : CE(0, T ) →[0, ∞], I0( ˆρ, ˆj) := I0(ρ, j). The following lemma is a direct consequence of the deﬁnition (20), the transformation (39), and part 2 of Lemma 2.2. The following lemma is a direct consequence of the deﬁnition (20), the transformation (39), and part 2 of Lemma 2.2. Lemma 5.2 (Dual formulation of Iε) We have Iε ˆρ, ˆj = sup b∈C∞ c (QT ) QT ˆj b −ˆuℓ ε ∂yb + 1 2b2 ! dydt, (40) (40) provided ˆρ(t, ·) is absolutely continuous with respect to ˆγ ℓ ε with density ˆuℓ ε(t, ·); otherwise we set Iε ˆρ, ˆj = +∞. provided ˆρ(t, ·) is absolutely continuous with respect to ˆγ ℓ ε with density ˆuℓ ε(t, ·); otherwise we set Iε ˆρ, ˆj = +∞. While the left-normalized stationary measure γ ℓ ε in the original variables concentrates onto the set {x : V (x) ≤0} = {xa} ∪[xb−1, xb+], under this transformation the interval [xb−1, xb+] collapses onto a point (see also Fig. 10): Lemma 5.3 (The measures ˆγ ℓ ε concentrate onto {±1/2}) Let a measurable set A ⊂R have positive distance to ±1/2. Then lim ε→0 ˆγ ℓ ε (A) = 0. lim ε→0 ˆγ ℓ ε (A) = 0. Proof Fix 0 < δ < V (x0). 5.1 Spatial transformation Since A has positive distance to {±1/2}, by Lemma 4.6 we have for sufﬁciently small ε that V ≥δ on y−1 ε (A). Therefore p for sufﬁciently small ε that V ≥δ on y−1 ε (A). Therefore ˆγ ℓ ε (A) = γ ℓ ε (y−1 ε (A)) ≤γ ℓ ε ({x : V (x) ≥δ}). ma 4.4, the right-hand side vanishes in the limit ε →0. By Lemma 4.4, the right-hand side vanishes in the limit ε →0. By Lemma 4.4, the right-hand side vanishes in the limit ε →0. 123 103 Page 30 of 44 M. A. Peletier, M. C. Schlottke 5.2 Statement and proof for the transformed system Theorem 5.4 (Upper bound in transformed coordinates) For any ( ˆρ0, ˆj0) ∈CE(0, T ) such that I0( ˆρ0, ˆj0) < ∞, there exist ( ˆρε, ˆjε) ∈CE(0, T ) such that Theorem 5.4 (Upper bound in transformed coordinates) For any ( ˆρ0, ˆj0) ∈CE(0, T ) such that I0( ˆρ0, ˆj0) < ∞, there exist ( ˆρε, ˆjε) ∈CE(0, T ) such that ( ˆρε, ˆjε) CE −→( ˆρ0, ˆj0) and sup ε>0 ε ˆEε( ˆρε(0)) < ∞, (41) (41) and that lim sup ε→0 Iε( ˆρε, ˆjε) ≤I0( ˆρ0, ˆj0). (42) (42) Proof Recall that QT := [0, T ] × R and set Q0 T := [0, T ] × [−1/2, +1/2]. If I0( ˆρ0, ˆj0) is ﬁnite, then by combining Deﬁnitions 5.1 and (16) we ﬁnd that the pair ( ˆρ0, ˆj0) is given by ˆρ0(t, dy) = ˆz0(t)δ−1/2(dy) + (1 −ˆz0(t))δ+1/2(dy), (43) ˆj0(t, dy) = ˆj0(t)1(−1/2,+1/2)(y) dy, (44) (43) (44) (44) where t →ˆz0(t) is absolutely continuous and ˆj0 satisﬁes ˆj0(t) = −∂t ˆz0(t) ≥0. For the later construction of ( ˆρε, ˆjε) we will want to assume that ˆz0 satisﬁes the following regularity assumption. where t →ˆz0(t) is absolutely continuous and ˆj0 satisﬁes ˆj0(t) = −∂t ˆz0(t) ≥0. For the later construction of ( ˆρε, ˆjε) we will want to assume that ˆz0 satisﬁes the following regularity assumption. Assumption 5.5 The density ˆz0 : [0, T ] →[0, 1] satisﬁes ˆz0 ∈C2([0, T ]) and inf t∈[0,T ] ˆz0(t), \|∂t ˆz0(t)\| > 0. (45) (45) Note that this implies that ˆj0 is bounded away from zero and of class C1. Indeed, we can assume that ˆz0 has this regularity since this set is energy-dense: Lemma 5.6 (Energy-dense approximations) If I0( ˆρ0, ˆj0) is ﬁnite, then there are densities ˆzδ 0 satisfying Assumption 5.5 such that the pair ( ˆρδ 0, ˆjδ 0) deﬁned via ˆzδ 0 as in (43) and (44) satisﬁes ( ˆρδ 0, ˆjδ 0) CE −→ δ→0 ( ˆρ0, ˆj0) and lim sup δ→0 I0( ˆρδ 0, ˆjδ 0) ≤I0( ˆρ0, ˆj0). By a standard diagonal argument (e.g. [8, Rem. 1.29]) we can continue under the assump- tion that ˆz0 satisﬁes Assumption 5.5. The bound on the energy (41) follows from the δ-independent estimate in (51e) below. From now on we therefore assume that Assump- tion 5.5 is satisﬁed. The proof of Theorem 5.4 now consists of three steps. Gamma-convergence of a gradient-ﬂow structure to... 103 Fig. 11 The polynomial y →ˆu0(t, y) on [−1/2, +1/2] for the three cases ˆj0(t) > ˆz0(t) (yellow), ˆj0(t) = ˆz0(t) (red) and ˆj0(t) < ˆz0(t) (blue). In particular, the function always satisﬁes ˆu0(t, −1/2) = ˆz0(t) and ˆu0(t, +1/2) = 0 Fig. 11 The polynomial y →ˆu0(t, y) on [−1/2, +1/2] for the three cases ˆj0(t) > ˆz0(t) (yellow), ˆj0(t) = ˆz0(t) (red) and ˆj0(t) < ˆz0(t) (blue). In particular, the function always satisﬁes ˆu0(t, −1/2) = ˆz0(t) and ˆu0(t, +1/2) = 0 The second-order polynomial ˆu0(t, ·) is either concave ( ˆj0 > ˆz0), linear ( ˆj0 = ˆz0) or convex ( ˆj0 < ˆz0). These three cases are sketched in Fig. 11. Note that under Assumption 5.5, ˆb0 and ∂y ˆb0 are bounded on Q0 T . ℓ y T Step 2: Solve an auxiliary PDE for ε > 0 We deﬁne the function ˆuℓ ε : E →[0, ∞) as the weak solution to the auxiliary PDE ˆgℓ ε∂t ˆuℓ ε = ∂yy ˆuℓ ε −∂y(ˆb01Q0 T ˆuℓ ε), (49) (49) where ˆgℓ ε ∈L∞(R) is the Lebesgue density of the left-stationary measure ˆγ ℓ ε from (39a), that is ˆγ ℓ ε (dy) = ˆgℓ ε(y)dy. where ˆgℓ ε ∈L∞(R) is the Lebesgue density of the left-stationary measure ˆγ ℓ ε from (39a), that is ˆγ ℓ ε (dy) = ˆgℓ ε(y)dy. ε ε This choice is inspired by the observation that if we deﬁne the pair ( ˆρε, ˆjε) by ˆρε(t, dy) := ˆuℓ ε(t, y) ˆγ ℓ ε (dy) and ˆjε := −∂y ˆuℓ ε + ˆb01Q0 T ˆuℓ ε, (50) ˆρε(t, dy) := ˆuℓ ε(t, y) ˆγ ℓ ε (dy) and ˆjε := −∂y ˆuℓ ε + ˆb01Q0 T ˆuℓ ε, (50) (50) then by the characterization of weighted L2-norms we have Iε( ˆρε, ˆjε) = sup b QT −∂y ˆuℓ ε + ˆb01Q0 T ˆuℓ ε b −ˆuℓ ε ∂yb + 1 2b2 ! dydt = sup b QT ˆuℓ ε b ˆb01Q0 T − ∂yb + 1 2b2 ! dydt = 1 2 Q0 T ˆuℓ ε ˆb2 0, which is an approximation of I0( ˆρ0, ˆj0) as given by (46). which is an approximation of I0( ˆρ0, ˆj0) as given by (46). 5.2 Statement and proof for the transformed system Step 1: characterization of I0( ˆρ0, ˆj0) By Lemma A.4 the limiting rate function satisﬁes I0( ˆρ0, ˆj0) = 1 2 Q0 T ˆb2 0 ˆu0 dydt, (46) (46) where ˆu0 : Q0 T →[0, ∞) is the function given by where ˆu0 : Q0 T →[0, ∞) is the function given by ˆu0(t, y) = 1 2 −y ˆj0(t) y + 1 2 + ˆz0(t) 1 2 −y (47) ˆu0(t, y) = 1 2 −y ˆj0(t) y + 1 2 + ˆz0(t) 1 2 −y (47) and ˆb0 : Q0 T →R is deﬁned by and ˆb0 : Q0 T →R is deﬁned by ˆb0(t, y) := ˆj0(t) + ∂y ˆu0(t, y) ˆu0(t, y) = 4( ˆj0(t) −ˆz0(t)) ˆj0(t)(y + 1 2) + ˆz0(t)( 1 2 −y) . (48) (48) 123 123 Page 31 of 44 103 Gamma-convergence of a gradient-ﬂow structure to... Page 31 of 44 103 Fig. 11 The polynomial y →ˆu0(t, y) on [−1/2, +1/2] for the three cases ˆj0(t) > ˆz0(t) (yellow), ˆj0(t) = ˆz0(t) (red) and ˆj0(t) < ˆz0(t) (blue). In particular, the function always satisﬁes ˆu0(t, −1/2) = ˆz0(t) and ˆu0(t, +1/2) = 0 Deﬁne the pair ( ˆρε, ˆjε) by (50). Then we have (i) ( ˆρε, ˆjε) ∈CE(0, T ) and (i) ( ˆρε, ˆjε) ∈CE(0, T ) and Iε( ˆρε, ˆjε) = 1 2 Q0 T ˆb2 0 ˆuℓ ε dydt. (53) (53) (ii) supε>0 ε ˆEε( ˆρε(0, ·)) ≤\|V (xb)\| + 1. (ii) supε>0 ε ˆEε( ˆρε(0, ·)) ≤\|V (xb)\| + 1. (ii) supε>0 ε ˆEε( ˆρε(0, ·)) ≤\|V (xb)\| + 1. (ii) supε>0 ε ˆEε( ˆρε(0, ·)) ≤\|V (xb)\| + 1. (iii) The pair ( ˆρε, ˆjε) converges to ( ˆρ0, ˆj0) in the sense of Deﬁnition 3.2. ℓ 2 1 (iii) The pair (ρε, jε) converges to (ρ0, j0) in the sense of Deﬁnition 3.2. (iV) There exists a function ˆuℓ 0 ∈L2(0, T ; H1()) such that (iV) There exists a function ˆuℓ 0 ∈L2(0, T ; H1()) such that ˆuℓ ε1Q0 T ε→0 −−−⇀ˆu0 weakly in L2(Q0 T ), (54) (54) Step 3: Conclude The convergence of ( ˆρε, ˆjε) to ( ˆρ0, ˆj0) in CE(0, T ) is given by part (iii) of Lemma 5.7. The energy bound (41) is satisﬁed by part (ii), and note that this bound is independent of the regularity Assumption 5.5. To prove the limsup-bound (42), we observe that To prove the limsup-bound (42), we observe that lim ε→0 Iε( ˆρε, ˆjε) (53) = lim ε→0 1 2 Q0 T ˆb2 0 ˆuℓ ε dydt (54) = 1 2 Q0 T ˆb2 0 ˆu0 dydt This concludes the proof of Theorem 5.4. Gamma-convergence of a gradient-ﬂow structure to... hen we have Deﬁne the pair ( ˆρε, ˆjε) by (50). Then we have Gamma-convergence of a gradient-ﬂow structure to... W h i iti l d t ˆℓ◦f (49) th t i t ˆ (t 0) i th f ll i which is an approximation of I0( ˆρ0, ˆj0) as given by (46). We choose initial data ˆuℓ,◦ ε for (49) that approximate ˆρ0(t = 0) in the following sense (see Lemma 5 8 for a proof that such initial data can be found): We choose initial data ˆuℓ,◦ ε for (49) that approximate ˆρ0(t = 0) in the following sense (see Lemma 5.8 for a proof that such initial data can be found): 0 < ˆuℓ,◦ ε (y) ≤1 for almost all y ∈R, (51a) ˆuℓ,◦ ε (y) ˆγ ℓ ε (dy) −⇀ˆρ0(0, dy) as ε →0, (51b) R ˆuℓ,◦ ε (y) ˆγ ℓ ε (dy) = 1, (51c) ˆuℓ,◦ ε is constant on (−∞, −1/4) and on (1/4, ∞), (51d) sup ε>0 ε ˆEε ˆuℓ,◦ ε ˆγ ℓ ε ≤\|V (xb)\| + 1. (51e) The following lemma gives the relevant properties of ˆuℓ ε, ˆρε, and ˆjε. The following lemma gives the relevant properties of ˆuℓ ε, ˆρε, and ˆjε. 103 Page 32 of 44 103 M. A. Peletier, M. C. Schlottke Lemma 5.7 (Auxiliary PDE) Assume Assumption 5.5. For any ε > 0 and any initial condi- tion ˆuℓ,◦ ε satisfying (51), there exists a solution ˆuℓ ε to the PDE (49) in the following sense: ˆuℓ ε : E →[0, ∞) is such that ˆuℓ ε > 0 a.e. on QT , ˆuℓ ε ∈C(0, T ; L2(Q0 T )), ∂y ˆuℓ ε ∈L2(0, T ; L2(R)), and ˆγ ℓ ε ˆuℓ ε(t) ∈P(R) for all t, and for any ϕ ∈C1 c (QT ) with ϕ = 0 at t = T , QT ˆgℓ ε ˆuℓ ε∂tϕ + −∂y ˆuℓ ε + ˆb01Q0 T ˆuℓ ε ∂yϕ dydt + R ˆgℓ ε(y)ˆuℓ,◦ ε (y)ϕ(0, y) dy = 0. and for any ϕ ∈C1 c (QT ) with ϕ = 0 at t = T , and for any ϕ ∈C1 c (QT ) with ϕ = 0 at t = T , QT ˆgℓ ε ˆuℓ ε∂tϕ + −∂y ˆuℓ ε + ˆb01Q0 T ˆuℓ ε ∂yϕ dydt + R ˆgℓ ε(y)ˆuℓ,◦ ε (y)ϕ(0, y) dy = 0. (52) ne the pair ( ˆρε, ˆjε) by (50). 5.3 Proof of Lemma 5.7 We now prove the main Lemma 5.7 used for the proof of Theorem 5.4. ℓ We now prove the main Lemma 5.7 used for the proof of Theorem 5.4. Step 1: Existence of the solution ˆuℓ ε Using classical methods such as those in [23] one ﬁnds a function ˆuℓ ε with Step 1: Existence of the solution ˆuℓ ε Using classical methods such as those in [23] one ﬁnds a function ˆuℓ ε with ˆuℓ ε(t) ≥0 and R ˆuℓ ε(t, y) ˆγ ℓ ε (dy) = 1 for all t, 123 123 Page 33 of 44 103 Gamma-convergence of a gradient-ﬂow structure to... 103 that satisﬁes the ε-independent bounds that satisﬁes the ε-independent bounds that satisﬁes the ε-independent bounds ∥ˆuℓ ε∥C([0,T ];L2(Q0 T )) ≤C, (55a) ∥∂y ˆuℓ ε∥L2(QT ) ≤C, (55b) ∥ˆgℓ ε∂t ˆuℓ ε∥L2(0,T;H−1(R)) ≤C, (55c) ∥ˆgℓ ε∂t ˆuℓ ε∥L2(0,T;H−1(R)) ≤C, (55c) d solves Eq. (49) in the weak form (52). olves Eq. (49) in the weak form (52). and solves Eq. (49) in the weak form (52). and solves Eq. (49) in the weak form (52). To brieﬂy indicate the main steps in this existence proof, deﬁne the function B(t, y) := y −1/2 ˆb0(t, ˜y) d ˜y and observe that the transformed function ˆvℓ ε := e−B ˆuℓ ε satisﬁes the equa- tion ˆgℓ ε∂t eB ˆvℓ ε = ∂y eB∂y ˆvℓ ε . (56) (56) Applying the usual method of multiplying by the solution ˆvℓ ε and integrating we obtain this a priori estimate: Applying the usual method of multiplying by the solution ˆvℓ ε and integrating we obtain this a priori estimate: T 0 R eB\|∂y ˆvℓ ε\|2 dydt + sup t∈[0,T] R eB(t) ˆgℓ ε ˆvℓ ε(t)2 dy ≤ R e−B(0) ˆgℓ ε(ˆuℓ,◦ ε )2 (51a) ≤∥e−B(0)∥∞. (57) (51a) ≤∥e−B(0)∥∞. (57) (57) One then constructs by e.g. Galerkin approximation a sequence of approximating solu- tions of (56) that satisfy (57), for which one can extract a subsequence that converges to a limit. Upon transforming back to the function ˆuℓ ε one obtains the weak form (52) and the bounds (55b) and (55c). In order to deduce (55a) from (55b) and (55c) one applies e.g. [41, Th. 5] with the compact embedding H1(Q0 T ) →L2(Q0 T ). 5.3 Proof of Lemma 5.7 Since the function B is bounded, we ﬁnd that ˆjε = −eB∂y ˆvℓ ε is bounded in L2(QT ), because QT \| ˆjε\|2 dydt ≤∥eB∥∞ QT eB\|∂y ˆvℓ ε\|2 dydt (57) ≤C. Hence, taking another subsequence, the ﬂux ˆjεk converges weakly in L2(QT ) to some j0 ∈ L2(QT ). Combining these convergence statements of ˆρεk and ˆjεk, we ﬁnd for any test function ϕ ∈ C1 c (QT ), 0 CE = QT ∂tϕ ˆρεk + ∂yϕ ˆjεk k→∞ −−−→ QT ∂tϕ ρ0 + ∂yϕ j0 . Therefore ( ˆρεk, ˆjεk) converges to (ρ0, j0) in the sense of CE(0, T ). Therefore ( ˆρεk, ˆjεk) converges to (ρ0, j0) in the sense of CE(0, T ). Finally, since ρ0 is concentrated on [0, T ] × {±1/2}, the limiting ﬂux j0 is piecewise constant in y with jumps only at {±1/2}, and j0 ∈L2(QT ) implies that j0 vanishes outside of (−1/2, +1/2). Therefore, the continuity equation 0 = ∂tρ0 + ∂yj0 in the distributional sense implies that the ﬂux is given by j0(t, dy) = −∂tz0(t)1(−1/2,+1/2)(y) dy. (59) (59) Step 4: The limit (ρ0, j0) is equal to ( ˆρ0, ˆj0) We now show that the limit z0 obtained above coincides with the function ˆz0 that characterizes ˆρ0 (see (43)). This proves that (ρ0, j0) = ( ˆρ0, ˆj0) and u0 = ˆu0 on Q0 T . (ρ0 j0) 0 0 QT By further extracting subsequences we can assume that T By further extracting subsequences we can assume that ˆuℓ εk⇀u0 in L2(Q0 T ) and ∂y ˆuℓ εk⇀∂yu0 in L2(QT ). By passing to the limit in (50) we ﬁnd that j0 = −∂yu0 + ˆb01Q0 T u0 almost everywhere in QT . In combination with (59) this means that for almost every t ∈[0, T ], the function y → u0(t, y) is a weak solution of the ODE By passing to the limit in (50) we ﬁnd that j0 = −∂yu0 + ˆb01Q0 T u0 almost everywhere in QT . In combination with (59) this means that for almost every t ∈[0, T ], the function y → u0(t, y) is a weak solution of the ODE −∂yu0(t, y) + ˆb0(t, y)u0(t, y) = −∂tz0(t), for −1 2 < y < 1 2. 5.3 Proof of Lemma 5.7 The missing L2(Q0 T )-estimate can be obtained from (55b) by applying the generalized Poincaré inequality of Lemma A.1 to μ = ˆγ ℓ ε and observing that ˆγ ℓ ε ([−1/2, 1/2]) →∞as ε →0. ε By the strong maximum principle and the positivity (51b) of the initial data the solutions ˆuℓ ε are strictly positive, and since ˆjε ∈L2(QT ) the mass of ˆρε(t) = γ ℓ ε ˆuℓ ε(t) equals the mass of the initial data ˆγ ℓ ε , which is one by (51c). Note that by Assumption 5.5 the function B is not only bounded but also independent of ε, implying that the constants in (55) also are independent of ε. Step 2: Part (i), the value of Iε( ˆρε, ˆjε) The fact that ( ˆρε, ˆjε) ∈CE(0, T ) follows from the regularity (55) of ˆuℓ ε and from the weak form (52) of the equation. The value of Iε( ˆρε, ˆjε) was already calculated before Lemma 5.7. Step 3: Convergence of ( ˆρε, ˆjε) By construction (see (51b)) the initial measures ˆρε(0, ·) converge to ˆρ0(0, ·). To prove convergence of ( ˆρε, ˆjε) we therefore need to show convergence in the continuity equation. y q For any test function ϕ ∈Cb(QT ), For any test function ϕ ∈Cb(QT ), QT ϕ ˆρε 2 (50) = QT ϕ eB ˆgℓ ε ˆvℓ ε dydt 2 CS ≤ QT eB ˆgℓ ε ˆvℓ ε(t)2 dydt ! QT \|ϕeB ˆgℓ ε\| dydt ! (57) ≤C supp(ϕ) ˆgℓ ε(y) dy. 123 103 Page 34 of 44 M. A. Peletier, M. C. Schlottke Hence for any test function with support outside of [0, T ] × {±1/2}, by Lemma 5.3, Hence for any test function with support outside of [0, T ] × {±1/2}, by Lemma 5.3, QT ϕ ˆρε ε→0 −−→0. (58) (58) Take any sequence εk →0. By (58) the family of measures ˆρεk is tight, and therefore it converges weakly on [0, T ] × R, along a subsequence (denoted the same), to a measure ρ0 that is concentrated on [0, T ] × {±1/2}, and therefore has the form ρ0(t, dy) = z0(t)δ−1/2(dy) + (1 −z0(t))δ1/2(dy) some measurable function z0 : [0, T ] →[0, 1]. B ℓ 2 for some measurable function z0 : [0, T ] →[0, 1]. Since +1/2 −1/2 e−B(t,z) dz = −ˆz0(t) ∂t ˆz0(t) > 0, the second boundary condition u0(t, +1/2) = 0 therefore enforces the second boundary condition u0(t, +1/2) = 0 therefore enforces ∂t log z0(t) = ∂t log ˆz0(t). (62) (62) Combined with the convergence assumption on the initial condition ˆρε(0, dy), which implies z0(0) = ˆz0(0), it follows that z0 = ˆz0. This unique characterization of the limit (ρ, j0) also implies that the convergence holds not only along subsequences but in the sense of a full limit ε →0. Step 5: Prove the boundary conditions (61) on u0 To prove the left boundary condition in (61), let Uδ be a small neighborhood around −1/2 of length 2δ > 0. Since ∂y ˆuℓ ε is bounded in L2(QT ) by (55b), there is an α ∈L2(0, T ) such that ˆuℓ ε(t, y) ≤ˆuℓ ε(t, −1/2) + α(t) \|y + 1/2\|1/2 for all ε and a.e. (t, y) ∈Q0 T . We can then estimate for any non-negative ψ ∈C([0, T ]), We can then estimate for any non-negative ψ ∈C([0, T ]), T 0 ψ(t) ˆρε(t, Uδ) dt = T 0 Uδ ψ(t)ˆuℓ ε(t, y) ˆγ ℓ ε (dy)dt ≤ˆγ ℓ ε (Uδ) T 0 ψ(t)ˆuℓ ε(t, −1/2) dt + ∥α∥2∥ψ∥L∞ Uδ \|y + 1/2\|1/2 ˆγ ℓ ε (dy) ≤ˆγ ℓ ε (Uδ) T 0 ψ(t)ˆuℓ ε(t, −1/2) dt + C∥ψ∥L∞δ1/2 Uδ ˆgℓ ε(y) dy. For each δ > 0, Uδ ˆgℓ ε(y)dy converges to 1 as ε →0, and For each δ > 0, Uδ ˆgℓ ε(y)dy converges to 1 as ε →0, and lim ε→0 T 0 ψ(t) ˆρε(t, Uδ) dt = T 0 ψ(t)z0(t) dt. Therefore, T 0 ψ(t)z0(t) dt ≤lim inf ε→0 T 0 ψ(t)ˆuℓ ε(t, −1/2) dt + C′δ1/2. Noting that δ > 0 is arbitrary and repeating the argument for the reversed inequality, we ﬁnd that Noting that δ > 0 is arbitrary and repeating the argument for the reversed inequality, we ﬁnd that T 0 ψ(t)z0(t) dt = lim ε→0 T 0 ψ(t)ˆuℓ ε(t, −1/2) dt. Since the trace map w ∈L2(0, T ; H1(Q0 T )) →w(·, −1/2) ∈L2(0, T ) is weakly continu- ous, the sequence of functions t →ˆuℓ ε(t, −1/2) converges weakly in L2(0, T ) to the limit u0(t, −1/2). This proves the ﬁrst boundary condition in (61). 5.3 Proof of Lemma 5.7 (60) (60) This is a ﬁrst-order ODE in y on the interval (−1/2, 1/2), and we show below that u satisﬁes not one but two boundary conditions, at ±1/2: This is a ﬁrst-order ODE in y on the interval (−1/2, 1/2), and we show below that u satisﬁes not one but two boundary conditions, at ±1/2: u0(t, −1/2) = z0(t) and u0(t, +1/2) = 0 for a.e. t. (61) u0(t, −1/2) = z0(t) and u0(t, +1/2) = 0 for a.e. t. (61) The solution of (60) with left boundary condition u0(t, −1/2) = z0(t) is given by u0(t, y) = eB(t,y) z0(t) + ∂tz0(t) y −1/2 e−B(t,z) dz . (61) The solution of (60) with left boundary condition u0(t, −1/2) = z0(t) is given by B(t ) y B(t ) The solution of (60) with left boundary condition u0(t, −1/2) = z0(t) is given by u0(t, y) = eB(t,y) z0(t) + ∂tz0(t) y −1/2 e−B(t,z) dz . 123 rgence of a gradient-ﬂow structure to... Page 35 of 44 103 Page 35 of 44 103 Gamma-convergence of a gradient-ﬂow structure to... 103 Since 5.4 Proof of Lemma 5.6 The sequence ( ˆρη 0, ˆjη 0 ) therefore satisﬁes the claim of Lemma 5.6. Since The argument for the second boundary condition is similar, using that ˆγ ℓ ε (1/2 −δ, 1/2 + δ) →∞as ε →0. This concludes the proof of Lemma 5 7 Since the trace map w ∈L2(0, T ; H1(Q0 T )) →w(·, −1/2) ∈L2(0, T ) is weakly continu- ous, the sequence of functions t →ˆuℓ ε(t, −1/2) converges weakly in L2(0, T ) to the limit u0(t, −1/2). This proves the ﬁrst boundary condition in (61). The argument for the second boundary condition is similar, using that ˆγ ℓ ε (1/2 −δ, 1/2 + δ) →∞as ε →0. ε This concludes the proof of Lemma 5.7. This concludes the proof of Lemma 5.7. 103 Page 36 of 44 M. A. Peletier, M. C. Schlottke 5.4 Proof of Lemma 5.6 Approximation results of this type are very common; see e.g. [2, Theorem 6.1] or [34, Lemma 4.7]. Fix a pair ( ˆρ0, ˆj0) with I0( ˆρ0, ˆj0) < ∞, and write ˆρ0 in terms of the absolutely continuous function ˆz0 as in (43). We ﬁrst approximate ˆz0 by a sequence of more regular functions ˆzη, for η →0. We do this by ﬁrst extending ˆz0 to R by constants: ˆz0(t) := ⎧ ⎪⎨ ⎪⎩ ˆz0(0) if t ≤0 ˆz0(t) if 0 ≤t ≤T ˆz0(T ) if t ≥T . The extended function ˆz0 again is non-increasing; we then regularize by convolution by setting ˆzη := αη∗ˆz0, where αη(s) := η−1α(s/η) is a regularizing sequence. 1 1 Then ˆzη →ˆz0 in W 1,1(R), and therefore the corresponding pair ( ˆρη, ˆjη) converges in CE to ( ˆρ0, ˆj0) Since the function S in (15) is jointly convex in its two arguments, we have T 0 S(−∂t ˆzη(t)\|ˆzη(t)) dt ≤ R S(−∂t ˆzη(t)\|ˆzη(t)) dt ≤ R αη∗S(−∂t ˆz0\|ˆz0) (t) dt = R S(−∂t ˆz0(t)\|ˆz0(t)) dt = T 0 S(−∂t ˆz0(t)\|ˆz0(t)) dt. Next, deﬁne z(t) := 1/2 −t/4T , and note that z and −∂tz are bounded away from zero on [0, T ]. For each η ∈(0, 1), the convex combination zη(t) := ηz(t) + (1 −η)ˆzη(t), t ∈[0, T ]. also satisﬁes infzη, inf(−∂tzη) > 0. Again using the convexity of S we ﬁnd that T 0 S(−∂tzη(t)\|zη(t)) dt ≤Cη + (1 −η) T 0 S(−∂t ˆz0(t)\|ˆz0(t)) dt. Setting ˆρη 0(t) =zη(t)δ−1/2 + (1 −zη(t))δ1/2 and deﬁning ˆjη 0 accordingly, we then h ˆI0( ˆρη 0, ˆjη 0 ) ≤Cη + (1 −η) ˆI0( ˆρ0, ˆj0). T 0 S(−∂tzη(t)\|zη(t)) dt ≤Cη + (1 −η) T 0 S(−∂t ˆz0(t)\|ˆz0(t)) dt. 0 S(−∂tzη(t)\|zη(t)) dt ≤Cη + (1 −η) 0 S(−∂tz0(t)\|z0(t)) dt. Setting ˆρη 0(t) =zη(t)δ−1/2 + (1 −zη(t))δ1/2 and deﬁning ˆjη 0 accordingly, we then have ˆI0( ˆρη 0, ˆjη 0 ) ≤Cη + (1 −η) ˆI0( ˆρ0, ˆj0). Setting ˆρη 0(t) =zη(t)δ−1/2 + (1 −zη(t))δ1/2 and deﬁning ˆjη 0 accordingly, we then have ˆI0( ˆρη 0, ˆjη 0 ) ≤Cη + (1 −η) ˆI0( ˆρ0, ˆj0). Setting ˆρη 0(t) =zη(t)δ−1/2 + (1 −zη(t))δ1/2 and deﬁning ˆjη 0 accordingly, we then have ˆI0( ˆρη 0, ˆjη 0 ) ≤Cη + (1 −η) ˆI0( ˆρ0, ˆj0). ˆI0( ˆρη 0, ˆjη 0 ) ≤Cη + (1 −η) ˆI0( ˆρ0, ˆj0). 5.5 The initial data in (51) can be realized In the proof of Theorem 5.4 we postulated a choice of initial data with certain properties. The next lemma shows that it possible to construct such initial data. Lemma 5.8 For any given ρ◦= z◦δ−1/2 + (1 −z◦)δ1/2 it is possible to choose a sequence ˆuℓ,◦ ε satisfying the requirements (51). Page 37 of 44 103 Gamma-convergence of a gradient-ﬂow structure to... 103 Proof. For instance one may choose Proof. For instance one may choose Proof. For instance one may choose ne may choose ˆuℓ,◦ ε (y) := ⎧ ⎪⎪⎪⎨ ⎪⎪⎪⎩ z0(0) if y ≤−1/4 smooth monotonic between −1/4 and 1/4 (1 −z0(0) + aε) Zℓ ε Zε if y ≥1/4, ˆuℓ,◦ ε (y) := ⎧ ⎪⎪⎪⎨ ⎪⎪⎪⎩ z0(0) if y ≤−1/4 smooth monotonic between −1 (1 −z0(0) + aε) Zℓ ε Zε if y ≥1/4, if y ≤−1/4 between −1/4 and 1/4 between −1/4 and 1/4 where aε →0 can be tuned in order to achieve the mass constraint (51c). One can verify that the deﬁnitions of γε and γ ℓ ε imply that 1 −z0 + aε ≤1, and because Zℓ ε/Zε < 1 we have the bound ∥ˆuℓ,◦ ε ∥∞≤1. ε To show (51e) for this choice we can write ε ˆEε(ˆuℓ,◦ ε ˆγ ℓ ε ) = ε R η ˆuℓ,◦ ε Zε Zℓε d ˆγε = ε R ˆuℓ,◦ ε Zε Zℓε log ˆuℓ,◦ ε Zε Zℓε d ˆγε. Splitting the integral into parts, the integral over (1/4, ∞) equals ε(1 −z0(0) + aε) log(1 −z0(0) + aε) ˆγε (1/4, ∞) ≤0. e integral over the remaining interval (−∞, 1/4) can be bounded from above by ε ˆρε (−∞, 1/4) log ∥uℓ,◦ ε ∥∞ Zε Zℓε ≤ε log Zε Zℓε (29) ≤\|V (xb)\| + 1 for small ε. ε ˆρε (−∞, 1/4) log ∥uℓ,◦ ε ∥∞ Zε Zℓε ≤ε log Zε Zℓε (29) ≤\|V (xb)\| + 1 for small ε. 5.6 Recovery sequence for the untransformed system for small ε, on the interval (−∞, 1 2(xa + x0)) the function uℓ,◦ ε is equal to a constant aε, with limε→0 aε = z0(0); 1 3. for small ε, on the interval ( 1 2(x0+xb−), ∞) the function uℓ,◦ ε Zε/Zℓ ε is equal to a constant bε, with limε→0 bε = 1 −z0(0). 3. for small ε, on the interval ( 1 2(x0+xb−), ∞) the function uℓ,◦ ε Zε/Zℓ ε is equal to a constant bε, with limε→0 bε = 1 −z0(0). Assuming this lemma for the moment, we calculate for any ϕ ∈Cb(R) that 1 2 (xa+x0) −∞ ρε(0, dx)ϕ(x) = aε 1 2 (xa+x0) −∞ γ ℓ ε (dx)ϕ(x) ε→0 −→z(0)ϕ(xa). ilarly xa+x0) ρε(0, dx)ϕ(x) = aε 1 2 (xa+x0) −∞ γ ℓ ε (dx)ϕ(x) ε→0 −→z(0)ϕ(xa). Similarly, Similarly, ∞ 1 2 (x0+xb−) ρε(0, dx)ϕ(x) = bε ∞ 1 2 (x0+xb−) γε(dx)ϕ(x) ε→0 −→(1 −z(0))ϕ(xb). Finally, by the uniform boundedness of uℓ,◦ ε on R, Finally, by the uniform boundedness of uℓ,◦ ε on R, 1 2 (x0+xb−) 1 2 (xa+x0) ρε(0, dx)ϕ(x) ≤C∥ϕ∥∞ 1 2 (x0+xb−) 1 2 (xa+x0) γ ℓ ε (dx) ε→0 −→0. Therefore ρε(0, ·) satisﬁes the convergence condition (33). Theorem 4.7 then implies that up to extraction of a subsequence, (ρε, jε) converges to a limit (ρ0, j0); the only property to check is that (ρ0, j0) = (ρ0, j0). Let ρ0(t) = z0(t)δxa + (1 −z0(t))δxb; by (33) we have z0(0) = z0(0). Recall from Lemma 4.6 that the function φε ◦y−1 ε converges uniformly on R to the function id1/2. We then calculate for any ψ ∈Cb([0, T ]) that T 0 ψ(t) R ρε(t, dx)φε(x) dt = T 0 ψ(t) R ˆρε(t, dy)φε y−1 ε (y) dt −→ T 0 ψ(t) R ˆρ0(t, dy)id1/2(y) dt = T 0 ψ(t) −1 2z0(t) + 1 2(1 −z0(t)) dt. On the other hand, since φε is uniformly bounded and converges to ∓1/2 in neighbourhoods of xa and xb, we also have On the other hand, since φε is uniformly bounded and converges to ∓1/2 in neighbourhoods of xa and xb, we also have T 0 ψ(t) R ρε(t, dx)φε(x) dt −→ T 0 ψ(t) −1 2z0(t) + 1 2(1 −z0(t)) dt. Since these two should agree for all ψ ∈Cb([0, T ]), it follows that z0 = z0 and therefore ρ0 = ρ0. 5.6 Recovery sequence for the untransformed system Theorem 5.9 Let V satisfy Assumption 4.1. Let (ρ0, j0) ∈CE(0, T ) satisfy I0(ρ0, j0) < ∞. Then there exists a sequence (ρε, jε) ∈CE(0, T ) such that (ρε, jε) CE −→(ρ0, j0), supε>0 εEε(ρε(0)) < ∞, and Iε(ρε, jε) −→I0(ρ0, j0). Proof Since I0(ρ0, j0) < ∞, ρ0 and j0 have the structure (17) in terms of z and j. Deﬁne the corresponding ( ˆρ0, ˆj0) by ˆρ0(t, dx) := z(t)δ−1/2(dx) + (1 −z(t))δ1/2(dx), ˆj0(t, dx) := j(t)1[−1/2,1/2](x) dx. By construction I0( ˆρ0, ˆj0) = I0(ρ0, j0) < ∞, and therefore by Theorem 5.4 there exists a sequence ( ˆρε, ˆjε) that converges to ( ˆρ0, ˆj0) with ˆIε( ˆρε, ˆjε) −→I0(ρ0, j0). By construction I0( ˆρ0, ˆj0) = I0(ρ0, j0) < ∞, and therefore by Theorem 5.4 there exists a sequence ( ˆρε, ˆjε) that converges to ( ˆρ0, ˆj0) with ˆIε( ˆρε, ˆjε) −→I0(ρ0, j0). We deﬁne (ρε, jε) by back-transforming the relation (39): We deﬁne (ρε, jε) by back-transforming the relation (39): ρε := (yε)−1 # ˆρε and jε(t, x) := ˆjε(t, yε(x)). By deﬁnition then Iε(ρε, jε) = Iε( ˆρε, ˆjε) −→I0(ρ0, j0). The only remaining fact to check is the convergence (ρε, jε) CE −→(ρ0, j0). By Theorem 5.4, Iε(ρε, jε) and εEε(ρε(0)) are bounded. We next verify the conver- gence (33) of the initial data. Note that by the properties of push-forwards, ρε(0, dx) = uℓ,◦ ε (x)γ ℓ ε (dx) with uℓ,◦ ε (x) = ˆuℓ,◦ ε (yε(x)). (63) ρε(0, dx) = uℓ,◦ ε (x)γ ℓ ε (dx) with uℓ,◦ ε (x) = ˆuℓ,◦ ε (yε(x)). (63) (63) 103 Page 38 of 44 M. A. Peletier, M. C. Schlottke 103 103 Lemma 5.10 1. uℓ,◦ ε is bounded uniformly in x and ε; Lemma 5.10 1. uℓ,◦ ε is bounded uniformly in x and ε; 1 ℓ Lemma 5.10 1. uℓ,◦ ε is bounded uniformly in x and ε; ε 2. for small ε, on the interval (−∞, 1 2(xa + x0)) the function uℓ,◦ ε is equal to a constant aε, with limε→0 aε = z0(0); ε 2. for small ε, on the interval (−∞, 1 2(xa + x0)) the function uℓ,◦ ε is equal to a constant with limε→0 aε = z0(0); 2. 5.6 Recovery sequence for the untransformed system Since these two should agree for all ψ ∈Cb([0, T ]), it follows that z0 = z0 and therefore ρ0 = ρ0. Finally, to show that also j0 = j0, note that both j0 and j0 are of the form j(t)1[xa,xb], and since they satisfy the continuity equation with the same measure ρ we have ∂y(j0 −j0) = 0 in duality with C1,0 c ([0, T ] × R). It follows that j0 = j0 almost everywhere on [0, T ] × R. W ill h d h f f L 5 10 Proof of Lemma 5.10 Part 1 follows directly from the boundedness of ˆuℓ,◦ ε (see (51a)) and the transformation (63). For part 2, recall from (51d) that ˆuℓ,◦ ε is a constant (say aε) on the interval (−∞, −1/4). Since ˆuℓ,◦ ε ˆγ ℓ ε converges to z0(0)δ−1/2 + (1 −z0(0))δ1/2, the constant aε converges to z0(0). Since y = −1/2 is an interior point of the interval (−∞, −1/4), for 123 Gamma-convergence of a gradient-ﬂow structure to... Page 39 of 44 103 103 sufﬁciently small ε the function yε maps the interval −∞, 1 2(xa + x0) into (−∞, −1/4) (see Lemma 4.6) and therefore uℓ,◦ ε equals aε on −∞, 1 2(xa + x0) . For part 3 the argument is very similar, only replacing the left-normalized ˆγ ℓ ε by the standard normalized ˆγε. Funding The Funding was provided by Nederlandse Organisatie voor Wetenschappelijk Onderzoek (Grant No. 613.001.552) 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/. with a constant C > 0 that only depends on a and b. with a constant C > 0 that only depends on a and b. Proof By density it sufﬁces to prove the inequality for f ∈C1([a, b]). For x, y ∈[a, b] we have f 2(x) = f 2(y) + 2 y x f ′(s) f (s) ds, f 2(x) = f 2(y) + 2 y x f ′(s) f (s) ds, and therefore μ([a, b]) f 2(x) ≤∥f ∥2 L2(μ) + 2μ([a, b])∥f ∥L2(a,b)∥f ′∥L2(a,b) ≤∥f ∥2 L2(μ) + μ([a, b]) 1 α ∥f ′∥2 L2(a,b) + α(b −a)∥f ∥2 L∞(a,b) . The assertion follows by choosing e.g. α = 1/2(b −a). The assertion follows by choosing e.g. α = 1/2(b −a). The assertion follows by choosing e.g. α = 1/2(b −a). A.1 A generalized Poincaré inequality Lemma A.1 For any −∞< a < b < ∞and for all bounded non-negative Borel measures μ on [a, b], we have the following inequality: ∥f ∥2 L∞(a,b) ≤C ∥f ′∥2 L2(a,b) + μ([a, b])−1∥f ∥2 L2(a,b;μ) , for all f ∈H1(a, b) ∩L2(a, b; μ), with a constant C > 0 that only depends on a and b. with a constant C > 0 that only depends on a and b. A.3 Duality characterizations Lemma A.3 (Duality characterization of quadratic entropies) For X = [0, T ] × Rd, f , g : X →R measurable with g > 0, and any nonnegative Borel measure μ, we have the characterization X 1 2 \| f (x)\|2 g(x) dμ(x) = sup b∈C∞ c (X) X −b(x)2 2 ! g(x) + b(x) f (x) dμ(x). A proof is given for instance in [2, Lemma 3.4]. The representation there can be further simpliﬁed by setting a = −b2/2. Lemma A.4 (Duality characterization of S) The function S deﬁned in (15) has the alternative characterization S( j\|z) = 1 4 inf u +1/2 −1/2 1 u(y) j + u′(y) 2 dy, (64) (64) where the inﬁmum is taken over smooth functions u : [−1/2, +1/2] →[0, ∞) satisfying the boundary conditions u(−1/2) = z and u(+1/2) = 0 and the positivity requirement u′(y) > 0 for all y ∈(−1/2, 1/2). The optimal function u is the polynomial where the inﬁmum is taken over smooth functions u : [−1/2, +1/2] →[0, ∞) satisfying the boundary conditions u(−1/2) = z and u(+1/2) = 0 and the positivity requirement u′(y) > 0 for all y ∈(−1/2, 1/2). The optimal function u is the polynomial u(y) = 1 2 −y j y + 1 2 + z 1 2 −y . (65) (65) Proof This result is very similar to that in [24, Prop. A1], which dealt with the slightly different argument ( j2 + u′2)/u with strictly positive boundary conditions for u; the sign of j and the degeneracy of u at the boundary y = 1/2 require some modiﬁcations. 1/2 Proof This result is very similar to that in [24, Prop. A1], which dealt with the slightly different argument ( j2 + u′2)/u with strictly positive boundary conditions for u; the sign of j and the degeneracy of u at the boundary y = 1/2 require some modiﬁcations. 1/2 If j = 0, then the integral equals 1/2 −1/2 4(v′)2 in terms of v = √u, for which the optimal function v is linear and the corresponding value of the integral equals 4z. This proves the identity (64) for the case j = 0. A.2 Laplace’s method Lemma A.2 (Laplace’s method; see e.g. [31, Sec. 7.2]) Let f : [a, b] →R be twice differ- entiable. (a) Suppose that for some xi ∈(a, b), we have f (xi) = inf[a,b] f . Then b a e−nf (x)dx = [1 + o(1)] 2π nf ′′(xi)e−nf (xi) as n →∞. 123 123 103 Page 40 of 44 M. A. Peletier, M. C. Schlottke (b) If xi = a or xi = b, then a or xi = b, then b a e−nf (x)dx = [1 + o(1)] 1 2 2π nf ′′(xi)e−nf (xi) as n →∞. A.4 Proof of Lemma 2.2 Results of this type are fairly standard; similar arguments can be found in [12, Th. 2.3] or [19, Lemmas 8.4 and 8.5]. Since we could not ﬁnd a complete result, we provide a proof here. For the length of this proof we use subscripts t to indicate time slices. Step 1: Alternative duality estimate We have Step 1: Alternative duality estimate We have Iε(ρ, j) ≥ sup f ∈C1,2 b (QT ) R ρT fT −ρ0 f0 − QT ρt ∂t ft + ετε ∂xx ft −1 ε ∂x ftV ′ + 1 2(∂x ft)2 . (66) (66) One ﬁrst obtains this inequality for f ∈C1,2 c (QT ) by substituting b = ∂x f in (8) and using the narrow continuity of ρ with a standard truncation and regularization in time. For general f ∈C1,2 b (QT ) the inequality follows from a regularized truncation in space, using the ﬁniteness of the measure ρ on QT . 0 1 1 2 Step 2: Dual equation Fix ϕ ∈C0,1 c (QT ) and ψ ∈C∞ c (R). Deﬁne g ∈C1,2 b (QT ) as the solution of the backward parabolic equation ∂tgt + ετε ∂xxgt −1 ε ∂xgtV ′ = ετε 2 gt 1 2ϕ2 t −∂xϕt + 1 ε ϕtV ′ gT = eψ/2 ∂tgt + ετε ∂xxgt −1 ε ∂xgtV ′ = ετε 2 gt 1 2ϕ2 t −∂xϕt + 1 ε ϕtV ′ gT = eψ/2 g is bounded. Such a solution exists by e.g. [17, Th. 1.12]. By calculating the derivative explicitly, we ﬁnd that Such a solution exists by e.g. [17, Th. 1.12]. By calculating the derivative explicitly, we ﬁnd that d dt R g2 t eV /ε = 2ετε R e−V /ε ∂xgt + 1 2 gϕ 2 ≥0, implying that implying that R eψe−V /ε = R g2 T e−V /ε ≥ R g2 0e−V /ε. (67) (67) The function f := 2 log g is an element of C1,2 b (QT ) and satisﬁes the equation ∂t ft + ετe ∂xx ft + 1 2(∂x ft)2 −1 ε ∂x ftV ′ = ετe 1 2ϕ2 t −∂xϕt + 1 ε ϕtV ′ with ﬁnal datum fT = ψ. Substituting in (66) yields with ﬁnal datum fT = ψ. A.3 Duality characterizations If j < 0, then one can estimate If j < 0, then one can estimate lim sup a↑1/2 a −1/2 ( j + u′)2 u dy = lim sup a↑1/2 a −1/2 j2 + u′2 + 2 ju′ u dy ≥lim sup a↑1/2 2 j log u(a) −log z = +∞, which establishes the identity (64) for the case j < 0. In the case j > 0 but z = 0, a similar calculation at y = −1/2 yields the same conclusion. The ﬁnal case to consider is j, z > 0. Following the argument of [24], we set f (u, u′) = ( j + u′)2/4u, such that the Euler-Lagrange equation is −(∂u′ f (u, u′))′ + ∂u f (u, u′) = 0. It follows by differentiating (or applying Noether’s theorem) that the Hamiltonian u′∂u′ f (u, u′) −f (u, u′) = (u′2 −j2)/4u is constant on [−1/2, 1/2], say equal to γ/4. By differentiating the resulting equation u′2 = j2+γ u we ﬁnd that all solutions are second-order polynomials, and by applying the boundary conditions on u we obtain (65) and γ = 4(z−j). The identity (64) then follows from a direct calculation. 123 Gamma-convergence of a gradient-ﬂow structure to... Page 41 of 44 103 103 A.4 Proof of Lemma 2.2 Substituting in (66) yields Iε(ρ, j) ≥ R ρT ψ −ρ0 f0 − QT ρετε 1 2ϕ2 t −∂xϕt + 1 ε ϕtV ′ . By reorganizing this inequality and applying the Donsker-Varadhan dual characterization of the relative entropy we ﬁnd R ρT ψ −log R γεeψ + ετε QT ρ 1 2ϕ2 t −∂xϕt + 1 ε ϕtV ′ ≤Iε(ρ, j) + R ρ0 f0 −log R γεeψ ≤Iε(ρ, j) + H(ρ0\|γε) −log R γεeψ R γεe f0 (67) ≤ Iε(ρ, j) + H(ρ0\|γε ≤Iε(ρ, j) + H(ρ0\|γε) −log R γεeψ R γεe f0 (67) ≤ Iε(ρ, j) + H(ρ0\|γε). 123 03 Page 42 of 44 103 M. A. Peletier, M. C. Schlottke Taking the supremum over ψ ∈C∞ c (R) and ϕ ∈C1,2 c (QT ), and applying also the dual formulation of the Fisher Information [16, Lemma D.44], we ﬁnd H(ρT \|γε) + ετε T 0 R(ρt\|γε, R) dt ≤Iε(ρ, j) + H(ρ0\|γε). Summarizing, Iε(ρ, j) < ∞implies that ρ is absolutely continuous on QT with respect to γε(dx)dt, or equivalently to with respect to Lebesgue measure on QT ; the density u := dρ/dγε satisﬁes ∂xu ∈L1 loc(QT ). This proves parts 2 and 3 of Lemma 2.2. Summarizing, Iε(ρ, j) < ∞implies that ρ is absolutely continuous on QT with respect to γε(dx)dt, or equivalently to with respect to Lebesgue measure on QT ; the density u := dρ/dγε satisﬁes ∂xu ∈L1 loc(QT ). This proves parts 2 and 3 of Lemma 2.2. / γ loc(Q ) p p Step 3: Regularity of j To show that j ≪ρ, we use the regularity of u to rewrite Iε(ρ, j) = sup b∈C1c (QT ) QT b j + ετερ ∂xu u −ετε 2 ρb2 . By the dual characterization of L2(QT ; ρ), ﬁniteness of Iε(ρ, j) implies that there exists v ∈L2(QT ; ρ) with j = vρ, and we have the estimate By the dual characterization of L2(QT ; ρ), ﬁniteness of Iε(ρ, j) implies that there exists v ∈L2(QT ; ρ) with j = vρ, and we have the estimate 1 2ετε QT ρ v + ετε ∂xu u 2 ≤Iε(ρ, j). A.4 Proof of Lemma 2.2 (68) (68) Step 4: Rewriting Iε Finally, to show the identity (21), we note that v ∈L2(QT ; ρ) implies that the curve t →ρt is absolutely continuous in the Wasserstein sense [1, Th. 8.3.1]. By [1, Th. 10.4.9], the bound on ∂xut/ut implies that the global Wasserstein slope \|∂Eε\|(ρt) is bounded and ∂xut/ut is an element of the Fréchet subdifferential. Finally, by the chain rule [1, Sec. 10.1.2], we have QT v ∂xu u ρ = Eε(ρ(T )) −Eε(ρ(0)). Expanding the square in (68) establishes (21) with an inequality. Step 5. Inverting the argument The argument up to now can be summarized as “if Iε(ρ, j) is ﬁnite, then ρ and j are regular and identity (21) holds as an inequality”. Vice versa, if the regularity conditions on ρ and j are satisﬁed and the right-hand side in (21) is ﬁnite, then the calculations can be reversed, and we ﬁnd that Iε is ﬁnite and that the inequality is an identity. This concludes the proof. 9. 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https://openalex.org/W3015456572	https://europepmc.org/articles/pmc7147034?pdf=render	English	null	Sonic irrigant activation for root canal disinfection: power modes matter!	BMC oral health	2,020	cc-by	7,839	© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Sonic irrigant activation for root canal disinfection: power modes matter! Florin Eggmann1, Yvonne Vokac2, Sigrun Eick3 and Klaus W. Neuhaus1,4 Florin Eggmann1, Yvonne Vokac2, Sigrun Eick3 and Klaus W. Neuhaus1,4 Abstract Background: Sonic irrigant activation has gained widespread popularity among general dentists and endodontists alike in recent years. This in vitro study aimed to evaluate the impact of three power modes of a sonic activation device (EDDY) on its antimicrobial effectiveness in infected root canals. Methods: The root canals of straight, human roots (n = 120) were prepared to size 40/.06. In a short-term infection experiment, the root canals were inoculated with different microbial species for three days. The following irrigation protocols, using 4 ml of normal saline as irrigant, were performed: negative control, manual rinsing, sonic irrigant activation at power modes “low”, “medium” and “high”. In a second, long-term experiment, testing the same irrigation protocols, inoculation lasted 21 days and sodium hypochlorite was used as irrigant. Sequential infection control samples were assessed using culture assays. The statistical analysis included one-way analysis of variance of log10-scaled counts of colony-forming units (CFU) with post-hoc comparisons using Bonferroni corrections and Chi2 tests (α = 0.05). Results: In the short-term experiment, the sonic irrigation protocols decreased the number of CFUs by 1.88 log10 units compared with the negative control (p < 0.001). The power modes “medium” and “high” achieved the most effective reduction of the microbial load. In the long-term experiment, microbial regrowth occurred after 7 days unless the device was used at its highest power setting. Conclusions: The power modes of the sonic irrigation device have a significant impact on the effectiveness for endodontic disinfection. The sonic irrigation device should always be used at the highest power setting in order to maximize its antimicrobial effectiveness. Keywords: Endodontic disinfection, Oral bacteria, Sonic activation, Needle irrigation, Sodium hypochlorite Background The uninstrumented portions of the root canal system and anatomical complexities, such as accessory canals, ap- ical ramifications, fins and isthmus areas, can harbor tissue debris as well as microorganisms and their deleterious by- products in spite of thorough chemomechanical disinfec- tion [1]. Supplementary irrigation methods such as irri- gant activation techniques are therefore needed to enhance the elimination of endodontic biofilms [5]. Apical periodontitis is a disease caused by endodontic biofilms [1]. To achieve a favorable treatment outcome, it is crucial to eliminate or significantly reduce the mi- crobial load within the root canal system and to prevent any recontamination after the treatment [2, 3]. However, the root canal system has an intricate anatomy, and root canal instrumentation with rotary files leaves, on aver- age, 60% of the root canal wall surface untouched [4]. Ultrasonic irrigant activation is the most commonly used supplementary irrigation method [6]. However, file-to-wall contact of ultrasonic instruments, all but unavoidable under clinical conditions, may lead to the inadvertent removal of small amounts of dentin and significant oscillation dampen- ing [7, 8]. Compared with ultrasonic activation, sonic * Correspondence: florin.eggmann@unibas.ch 1Department of Periodontology, Endodontology and Cariology, University Center for Dental Medicine, UZB, University of Basel, Mattenstrasse 40, CH-4056 Basel, Switzerland Full list of author information is available at the end of the article * Correspondence: florin.eggmann@unibas.ch 1Department of Periodontology, Endodontology and Cariology, University Center for Dental Medicine, UZB, University of Basel, Mattenstrasse 40, CH-4056 Basel, Switzerland Full list of author information is available at the end of the article * Correspondence: florin.eggmann@unibas.ch 1Department of Periodontology, Endodontology and Cariology, University Center for Dental Medicine, UZB, University of Basel, Mattenstrasse 40, CH-4056 Basel, Switzerland Full list of author information is available at the end of the article Eggmann et al. BMC Oral Health (2020) 20:102 https://doi.org/10.1186/s12903-020-01088-5 Eggmann et al. BMC Oral Health (2020) 20:102 https://doi.org/10.1186/s12903-020-01088-5 * Correspondence: florin.eggmann@unibas.ch 1Department of Periodontology, Endodontology and Cariology, University Center for Dental Medicine, UZB, University of Basel, Mattenstrasse 40, CH-4056 Basel, Switzerland Full list of author information is available at the end of the article Eggmann et al. BMC Oral Health (2020) 20:102 Eggmann et al. BMC Oral Health (2020) 20:102 Page 2 of 9 Eggmann et al. BMC Oral Health (2020) 20:102 Page 2 of 9 After shaping the root canals, the external root surfaces of the apices were conditioned for 15 s with a phosphoric acid etchant (Ultra-Etch, Ultradent Products Inc. South Jordan, UT, USA), which was rinsed off with water for 15 s. A multimode adhesive (Scotchbond Universal, 3 M Oral Care, St. Paul, MN, USA) was applied actively with a micro- brush for 20 s, then the adhesive was gently air dried for 5 s. The apices were closed with resin-based composite (Telio CS Inlay, Ivoclar Vivadent AG, Schaan, Principality of Liechtenstein). Light curing of the adhesive and the resin- based composite was performed with a polymerization light at an irradiance of 1200 mW/cm2 (Bluephase [high power mode], Ivoclar Vivadent AG) and as short a distance be- tween the tip of the light probe and the adhesive/resin- based composite as feasible for 10 s and 30 s, respectively. The roots were stored in deionized water at 4 °C until fur- ther use. Prior to the microbial contamination procedure, the roots were autoclaved in water for 20 min at 121 °C (Laboklav ECO, SHB Steriltechnik AG, Detzel Schloss/ Satuelle, Germany). irrigation devices operate at lower frequencies. EDDY (VDW GmbH, Munich, Germany), a flexible, non-cutting polyamide tip that is coupled to an air scaler handpiece, is used with sonic activation at a frequency of 5000 Hz to 6000 Hz [9]. Sonic activation with soft polymer tips promises to be an alternative irrigant activation technique without the risk of unintentional dentin removal, [10] and in vitro evi- dence suggests that the antimicrobial efficacy of sonic activa- tion with EDDY is, at least, on par with ultrasonic irrigant activation in straight and curved root canals [11]. Previous studies on the disinfection effectiveness of irriga- tion acitivation with EDDY employed the sonic device at the highest power mode of the air scaler handpiece [11]. Some dental practitioners may, however, use EDDY with air scaler handpieces set at lower power modes in order to reduce irri- gant spray mist during activation (VDW GmbH, oral com- munication). In ultrasonic irrigant activation, the power setting influences the file oscillation amplitude, which, in turn, has a pronounced effect on irrigant streaming and cleaning effectiveness [12]. Whether or not different power settings have a similar impact on the effectiveness of sonic irrigant activation has not yet been investigated. Short-term infection experiment Microbial contamination The following microorganisms were separately precul- tured and suspended for 18 h: The aim of this in vitro study was therefore to assess, through microbiological testing, if different power modes affect the antimicrobial effectiveness of sonic irrigation ac- tivation with the EDDY device. 1. Enterococcus faecalis ATCC 29212 2. Candida albicans ATCC 76615 3. Streptococcus gordonii ATCC 10558 and Actinomyces oris ATCC 43146 1. Enterococcus faecalis ATCC 29212 2. Candida albicans ATCC 76615 3. Streptococcus gordonii ATCC 10558 and Actinomyces oris ATCC 43146 1. Enterococcus faecalis ATCC 29212 Long-term infection experiment Thirty autoclaved roots were inoculated with S. gordonii ATCC 10558 and A. oris ATCC 43146. The incubation period lasted three weeks at 37 °C with 10% of CO2. Apart from the irrigation solution, the irrigation protocols were analogous to those in the short-term infection experiment. NaOCl (1.5% w/v) was used as irrigation solution instead of 0.9% NaCl. One ml of 0.9% NaCl was only used for the final rise. A rest period of 60 s was added after NaOCl de- livery in the manual rinsing group to standardize irrigant contact time across all groups. Immediately after the irri- gation protocol, a microbiological sample was collected with a sterile paper point as described above. Thereafter, the root canals were filled with nutrient broth and they were incubated at 37 °C as described above. Microbio- logical samples were collected from the root canals after 3, 5 and 7 days. The samples were processed following the same procedures as in the short-term infection experi- ment. A qualitative dichotomous analysis was performed to assess the presence of viable microorganisms after incu- bation periods of 0, 1, 4 and 7 days. 4. Power mode “medium” (n = 6): sonic irrigant activation with a polyamide tip (6000 Hz; EDDY, VDW GmbH) coupled to an air scaler (SONICflex, KaVo Dental GmbH), 3 × 20 s, 4 ml irrigation solution (3 ml for continuous irrigation during activation, 1 ml as a final rinse) 5. Power mode high (n = 6): sonic irrigant activation with a polyamide tip (6000 Hz; EDDY, VDW GmbH) coupled to an air scaler (SONICflex, KaVo Dental GmbH), 3 × 20 s, 4 ml irrigation solution (3 ml for continuous irrigation during activation, 1 ml as a final rinse) 5. Power mode high (n = 6): sonic irrigant activation with a polyamide tip (6000 Hz; EDDY, VDW GmbH) coupled to an air scaler (SONICflex, KaVo Dental GmbH), 3 × 20 s, 4 ml irrigation solution (3 ml for continuous irrigation during activation, 1 ml as a final rinse) The power modes “low”, “medium” and “high” corre- sponded to the markings “1”, “2” and “3”, respectively, on the SONICflex air scaler handpiece. The respective ampli- tudes within the excitation unit of the handpiece were 120 ± 15 μm, 160 ± 15 μm and 240 ± 15 μm according the manufacturer (Ulrike Nagorr, KaVo Dental GmbH, writ- ten communication). Long-term infection experiment All irrigants were delivered by single-use syringes with a capacity of 10 ml (Omnifix Luer Solo, B. Braun Medical AG, Sempach, Switzerland). In the manual rinsing group (group 2) and for the final rinse in groups 3–5, side-vented Gauge 30 irrigation needles (Irri- gation Probe, KerrHawe SA, Bioggio, Switzerland) were inserted to working length. The sonic irrigation tips, like- wise, were placed at working length. The sterile single-use EDDY tips were replaced for the irrigant activation of each root. For continuous irrigation during sonic activation, the side-vented needle was placed within the coronal third of the root canal. All endodontic procedures were performed by one operator (YV) throughout the study, and all irri- gants that were used were at room temperature. Selection and preparation of roots One hundred and twenty mature, straight roots (root curva- ture radius < 15° [13]) of maxillary front teeth, single-rooted maxillary premolars, and palatal roots of maxillary molars were selected from a pool of extracted human permanent teeth stored in a 1% aqueous solution of chloramine-T. The roots had neither resorptive defects nor fracture lines. The local Ethics Committee denied the need for a formal ap- proval for using irreversibly anonymized teeth and issued a written declaration of no-objection. All donors had given their verbal informed consent to the use of their extracted, anonymized tooth/teeth for research purposes. Thirty autoclaved roots were randomly allocated to each group of microorganisms and inoculated for three days at 37 °C. Groups 1 and 2 were cultured under aerobic conditions. Group 3 was cultured with 10% CO2. Tryptic soy agar (TSA) plates with 5% sheep blood were used for the cultivation of the mi- croorganisms. Then, suspensions of E. faecalis ATCC 29212 and C. albicans ATCC 76615 were prepared in a 0.9% sodium chloride solution (McFarland 4), which was then diluted, at a ratio of 1:9, with a brain-heart infusion broth (Oxoid Limited, Basing- stoke, UK). Wilkins-Chalgren anaerobe broth (Oxoid Limited) supplemented with 5 mg/l β-nicotinamide adenine dinucleotide sodium salt (NAD) (Sigma-Al- drich, St. Louis, MO, USA) was used for the suspen- sion of S. gordonii ATCC 10558 and A. oris ATCC 43146 (mixed at a ratio of 1:2). The nutrient broths were renewed daily. At the beginning of the micro- biological experiments, sterility was monitored by sampling and culturing on TSA plates under both aerobic and anaerobic conditions as described above. Cultivation was performed over 4 days. To remove the periodontal tissue, the roots were immersed in 3% (w/v %) sodium hypochlorite (NaOCl) for 10 min. The working lengths were determined using a 10/.02 K file (C-PILOT, VDW GmbH, Munich, Germany). The working length was defined as 0.5 mm shorter than the length at which the instrument was first visible at the apical foramen under 2.5x magnification. After confirmation of canal width by reassuring passive fit and gauging with a 20./02 K file, root canal shaping was performed with a reciprocating single-file system (RECIPROC #40, VDW GmbH); the rinsing solutions, used alternately during the shaping of the root canals, were 3% (w/v %) NaOCl and 17% (w/v %) ethylenedi- aminetetraacetic acid. Page 3 of 9 Eggmann et al. BMC Oral Health (2020) 20:102 Statistical analysis Statistical analyses were performed with SPSS 24.0 (IBM SPSS Statistics, Chicago, IL, USA). Statistical analyses based on log10-scaled microbial counts (total colony forming units ([CFU]) for the short-term experiment. Data were compared using a one-way analysis of vari- ance (ANOVA) with post-hoc comparisons of groups using Bonferroni corrections. For the long-term infec- tion experiment, the Chi2 test was employed. The level of significance was set at α = 0.05. Short-term infection experiment No contamination of the samples occurred throughout the duration of the experiment. All irrigation protocols with sonic activation significantly reduced the number of viable microorganisms, E. faecalis and Candida albicans. The order of magnitude of the reduction of CFUs of E. faecalis was 1.82 log10 units for all sonic activation pro- tocols (groups 3–5) compared with the negative control (group 1) (p < 0.001), and 1.20 log10 units compared with the manual irrigation group (group 2) (p ≤0.006). No Irrigation protocols tubes filled with 1 ml 0.9% NaCl. The tubes were vor- texed for 20 s and sonicated in an ultrasonic bath before 0.1 ml of the solution was dispersed on TSA plates. Incu- bation of E. faecalis ATCC 29212 and C. albicans ATCC 76615 was performed for 2 days. Incubation of S. gordonii ATCC 10558 and A. oris ATCC 43146 lasted 7 days. After the incubation periods, the number of colony-forming units was determined by using an automated colony coun- ter (aCOLyte, Synbiosis, Cambridge, UK). Normal saline, i.e., a solution of 0.9% w/v of sodium chloride, was used as irrigation solution. The root canals, inoculated with microorganisms, were subjected to the following irrigation protocols: 1. Negative control (n = 6): no treatment 2. Manual rinsing (n = 6): manual irrigation, 4 ml irrigation solution 3. Power mode “low” (n = 6): sonic irrigant activation with a polyamide tip (6000 Hz; EDDY, VDW GmbH) coupled to an air scaler (SONICflex, KaVo Dental GmbH, Biberach, Germany), 3 × 20 s, 4 ml irrigation solution (3 ml for continuous irrigation during activation, 1 ml as a final rinse) Counting of colony-forming units Counting of colony-forming units Sterile paper points (R40, RECIPROC Paper Points, VDW GmbH) that matched the taper of the file used for root canal preparation were placed in each root at work- ing length for 30 s. The paper points were then placed in Eggmann et al. BMC Oral Health (2020) 20:102 Page 4 of 9 Fig. 1 Log10 units of CFUs of Candida albicans after different irrigation protocols. “Low”, “medium” and “high” refer to respective the power mode of the air scaler handpiece used for sonic irrigation with the EDDY device. ,## indicate p < 0.05 statistically significant difference was observed between the manual irrigation protocol and the negative control group (p = 0.37). The different power modes showed no statistically significant differences (p = 1.00). The order of magnitude of the reduction of CFUs of Candida albicans was 1.83 log10 units for all sonic activation protocols (groups 3–5) compared with the negative control (group 1) (p < 0.001), and 1.31 log10 units compared with the manual irrigation group (group 2) (p < 0.001). Statistically signifi- cant differences were neither observed between the manual irrigation protocol and the negative control group (p = 0.93) nor between the different power modes (p = 1.00). ) p (p ) In comparison with the negative control group, all irri- gation protocols significantly reduced the number of S. gordonii and A. oris: the order of magnitude of the re- duction of CFUs was 2.72 log10 units for the sonic acti- vation power mode “medium” (p < 0.001), 2.03 log10 units for the sonic activation power mode “high” (p < 0.001) and 1.21 log10 units for the sonic activation power mode “low” (p < 0.001). The manual irrigation protocol reduced the number of CFUs by 0.69 log10 units com- pared with the negative control group (p = 0.022). Over- all, the sonic irrigation protocols decreased the number of CFUs by 1.30 log10 units compared with the manual irrigation protocol. Among the sonic irrigation proto- cols, the power mode “medium” obtained the most sub- stantial CFU reduction: it decreased the number of CFUs by 1.51 log10 units compared with the power mode “low” (p < 0.001) and by 0.69 log10 units compared with the power mode “high” (p = 0.020). The power mode “high” reduced the number of CFUs by 0.82 log10 units compared with the power mode “low” (p < 0.004), Figs. Long-term infection experiment No contamination of the samples occurred throughout the duration of the experiment. After three weeks of in- cubation with S. gordonii and A. oris, no bacteria could be cultivated from the samples taken immediately after irrigation with NaOCl. The addition of nutrient broth and incubation at 37 °C led to bacterial regrowth in all root canals after 7 days except for those that had been ir- rigated with sonic activation at the highest intensity (power mode “high”). Detailed results of the long-term infection experiment are provided in Fig. 4. Counting of colony-forming units 1, 2 and 3 show detailed results of the short-term infection experiment. Fig. 1 Log10 units of CFUs of Candida albicans after different irrigation protocols. “Low”, “medium” and “high” refer to respective the power mode of the air scaler handpiece used for sonic irrigation with the EDDY device. ,## indicate p < 0.05 infection experiment, bacterial regrowth was completely stunted exclusively in root canals that had been irrigated with the sonic irrigation device employed at the highest power setting. Recent in vitro studies have shown that, compared with manual irrigation, the sonic irrigation device EDDY im- proves the dentin debris removal from artificial canal ir- regularities [14], the removal of calcium hydroxide from artificial grooves in the root canal of maxillary incisors [9], the removal of remnants of sealer and guttapercha [15] Fig. 2 Log10 units of CFUs of E. faecalis after different irrigation protocols. “Low”, “medium” and “high” refer to respective the power mode of the air scaler handpiece used for sonic irrigation with the EDDY device. ,## indicate p < 0.05 Discussion This in vitro investigation showed that the power modes of a sonic irrigation device have a significant impact on the antimicrobial efficacy in root canals. In the short- term infection experiment, the two higher power modes achieved a more substantial reduction of viable microor- ganisms than the lowest power mode. In the long-term Fig. 2 Log10 units of CFUs of E. faecalis after different irrigation protocols. “Low”, “medium” and “high” refer to respective the power mode of the air scaler handpiece used for sonic irrigation with the EDDY device. ,## indicate p < 0.05 Fig. 2 Log10 units of CFUs of E. faecalis after different irrigation protocols. “Low”, “medium” and “high” refer to respective the power mode of the air scaler handpiece used for sonic irrigation with the EDDY device. ,## indicate p < 0.05 Page 5 of 9 Eggmann et al. BMC Oral Health (2020) 20:102 Page 5 of 9 Fig. 3 Log10 units of CFUs of S. gordonii and A. oris after different irrigation protocols. “Low”, “medium” and “high” refer to respective the power mode of the air scaler handpiece used for sonic irrigation with the EDDY device. ,##,§§,$ indicate p < 0.05 that study and the present investigation reveals that the order of magnitude of CFU reductions are, in some cases, more than one logarithmic step apart even though the overall trend is qualitatively similar in both studies. For in- stance, Neuhaus et al. [11] reported median log10 CFU counts of 4.03 for the S. gordonii and A. oris co-culture, 2.98 for E. faecalis and 1.7 for C. albicans after sonically ac- tivated irrigation of straight root canals. In the present in- vestigation, on the other hand, the log10 CFU counts in the group with sonic irrigant activation at the highest power setting were 5.16 for the S. gordonii and A. oris co-culture, 5.30 for E. faecalis and 4.94 for C. albicans in the short- term infection experiment. p In this study, microorganisms were used that are fre- quently found in infected root canal and that have the po- tential to invade radicular dentin [19]. The in vitro study of Neuhaus et al. [11] found regrowth of C. albicans and E. faecalis in all samples after seven days, i.e. some viable microorganisms that evaded detection – presumably in dentinal tubules - immediately after irrigation remained in the root canal system. Discussion No microbial regrowth occurred in the long-term infection experiment in the roots that were infected with S. gordonii and A. oris [11]. In that study, however, the sonic irrigation was always performed at the highest power setting [11]. The present investigation re- vealed that microbial regrowth of S. gordonii and A. oris occurred in all samples apart from those that had been sonically irrigated at the highest power setting. The irriga- tion protocols and microbiological assays were carried out in an analogous manner in both studies. However, in the study of Neuhaus et al. [11], the root canals were shaped with a reciprocating file size 25/.08 and both straight and curved roots were included in the long-term infection ex- periment. Contrary to this, the tooth sample of the present study included only straight roots and the root canals were prepared with a reciprocating file size 40/.06. The Fig. 3 Log10 units of CFUs of S. gordonii and A. oris after different irrigation protocols. “Low”, “medium” and “high” refer to respective the power mode of the air scaler handpiece used for sonic irrigation with the EDDY device. **,##,§§,$ indicate p < 0.05 and the median dye penetration depths in dentinal tubules of apical root sections [16]. However, considering the piv- otal etiopathogenetic role of endodontic biofilms in periapi- cal disease, the reduction of the microbial load within root canal systems is arguably the most relevant surrogate marker in experimental irrigation studies [17]. Although microbio- logical studies are fraught with some methodological difficul- ties, the endodontic microbiological status can, at least to some degree, predict healing of apical periodontitis [18]. The setup of the present study is comparable to a previ- ous in vitro study which indicated that, in straight and curved root canals, sonic irrigation with EDDY surpasses manual syringe irrigation in terms of antibacterial efficacy [11]. However, an in-depth comparison of the results of Fig. 4 Bacterial regrowth (log10 units of CFUs, black bars) after irrigation with 1.5% NaOCl (d0, day 0) and after re-incubation and provision of nutrient broth (d1, day 1; d4, day 4, d7, day 7). “Low”, “medium” and “high” refer to respective the power mode of the air scaler handpiece used for sonic irrigation with the EDDY device. “Manual” stands for the manual rinsing group Fig. Discussion Data from a previous in vitro study which assessed how effective sonic irrigant activation with EDDY was against E. faecalis suggest that EDDY is more efficient than syringe needle irrigation against intratubular bac- terial in all portions of the root [22]. Yet, EDDY does not maintain its antibacterial superiority in dentinal tu- bules beyond a depth of 100 μm [22]. Considering the limited reach of sonically activated irrigants in dentinal tubules, it has been argued that the concentration of NaOCl is more important than sonic irrigant activation to eliminate bacteria residing in dentinal tubules [22]. The present investigation confirms that sonic irrigant ac- tivation with EDDY, employed at the highest power set- ting, outperforms the antimicrobial effect of manual syringe irrigation but, in contrast to the studies of Hage et al. [23] and Zeng et al. [22], no viable microorganism were found even after an extended re-incubation period of seven days. In addition to different microbiological as- says and variations in the irrigation protocols, the differ- ent makeup of tooth samples may account, at least to some degree, for these conflicting microbiological re- sults. In the studies of Hage et al. [23] and Zeng et al. [22] exclusively single-rooted premolars were used whereas the sample of the present study included anter- ior teeth, premolars and palatal roots of maxillary mo- lars. Premolars are frequently extracted in adolescence and early adulthood for orthodontic reasons. The anter- ior teeth and molars contained in the sample of the present study, by contrast, were likely to come predom- inately from older patients with tooth loos resulting from periodontal disease. It is thus reasonable to assume that the average age of the teeth in the samples of these studies were different. The degree of root dentin scler- osis increases with advancing age, and consequently bac- terial infection of dentinal tubules is less pronounced in older teeth [17, 24]. This may influence the antimicro- bial effect of irrigation protocols [17, 24]. A tentative conclusion that can be drawn based on the results of the studies which assessed the antibacterial efficacy of the EDDY device is that sonic irrigant activation is advanta- geous compared with manual syringe irrigation but cases with severe intratubular infection may still require ad- junct measures such as the use of higher NaOCl concen- trations and prolonged exposure times [22, 23, 25]. Discussion 4 Bacterial regrowth (log10 units of CFUs, black bars) after irrigation with 1.5% NaOCl (d0, day 0) and after re-incubation and provision of nutrient broth (d1, day 1; d4, day 4, d7, day 7). “Low”, “medium” and “high” refer to respective the power mode of the air scaler handpiece used for sonic irrigation with the EDDY device. “Manual” stands for the manual rinsing group Fig. 4 Bacterial regrowth (log10 units of CFUs, black bars) after irrigation with 1.5% NaOCl (d0, day 0) and after re-incubation and provision of nutrient broth (d1, day 1; d4, day 4, d7, day 7). “Low”, “medium” and “high” refer to respective the power mode of the air scaler handpiece used for sonic irrigation with the EDDY device. “Manual” stands for the manual rinsing group Eggmann et al. BMC Oral Health (2020) 20:102 Eggmann et al. BMC Oral Health (2020) 20:102 Page 6 of 9 Page 6 of 9 In the present investigation, an established in vitro model with extracted teeth with closed-end root canals was used in order to have a standardized and reproducible setup and to replicate in vivo scenarios as closely as pos- sible [5, 11]. It is important to give due consideration to the inherent strengths and limitations of this model and the microbiological methods that were employed. increased apical enlargement and the absence of curved roots provided favorable conditions for improved irrigant delivery to the apical part of the main root canal and more unimpeded oscillation of the EDDY tip in the present study [7, 20, 21]. These advantages notwithstanding, the EDDY device employed at the two lower power settings was insufficiently efficacious in eliminating residual micro- organisms. This underlines that sonic irrigant activation should be performed at the highest power mode even if the conditions for root canal disinfection seem favorable. Decoronated roots were used for this in vitro study in order to increase the level of standardization and to en- sure a straightline access and adequate visual control of the irrigation needles and EDDY tips during the irrigation procedures. However, the use of decoronated roots might have influenced the irrigation protocols and thus the microbiological findings. For example, coronal tooth sub- stance or restoration material can sometimes compromise the straight access to the root canal orifice and the result- ing interference between the crown and the EDDY tip is likely to affect the oscillation behavior of the latter. Discussion y The root curvature affects the effectiveness of activation methods that rely on the insertion of an instrument in the root canal [5, 20]. To decrease the potential for confound- ing, the study material comprised exclusively of roots with no pronounced curvatures. Moreover, as in the majority of in vitro studies, single roots with a simple anatomy were used in the present investigation. Micro-computed tomography data suggest, however, that in the complex mesial root canal systems of mandibular molars sonic irri- gant activation does not improve the removal of accumu- lated hard-tissue debris compared with conventional manual irrigation [10]. Whether or not the same holds true for the antimicrobial efficacy remains currently un- clear. Interestingly, a recent in vitro study that used a stan- dardized isthmus model reported that laser-activated irrigation and sonic irrigant activation with EDDY achieved the most thorough removal of a biofilm- mimicking hydrogel [26]. Corroborating microbiological evidence for this finding is, however, lacking for the time being. Further studies are, therefore, necessary to evaluate if the results of the present investigation are replicable in teeth with multiple root canals, curved roots or more complex root canal anatomies. Endodontic infections are caused by multispecies bio- films. This in vitro investigation, however, used microbial monocultures and two-species co-cultures, consisting of microorganisms commonly found in infected root canals, to assess the effectiveness of different irrigation protocols. Therefore, the results of this study are not directly translat- able to situations where multispecies biofilm are present. Biofilm formation and maturity have a determining influ- ence on the antimicrobial effectiveness of irrigation proto- cols [5]. While biofilms grown from oral bacteria are quite susceptible to antimicrobial agents within the first two weeks of growth, more mature biofilms are more resistant and so the same antimicrobial agents are considerably less Eggmann et al. BMC Oral Health (2020) 20:102 Page 7 of 9 Page 7 of 9 deemed the preferable approach for better comparability. The sonic irrigation tip, too, was inserted to working length and remained there during activation despite the fact that the manufacturer recommends moving the sonic irrigation tip carefully up and down with vertical motions. The manufacturer’s recommendation was deliberately dis- regarded because, again, manually carrying out vertical motions in a standardized way was considered unfeasible. effective after three weeks [27]. Discussion The present study used two different maturation timelines for the biofilm models in order to take account of the different degrees in biofilm re- sistance. In line with methodological recommendations, the microbial purity of the contaminated roots was strictly monitored throughout the experiments to rule out any con- tamination [5]. The extent of microbial growth on the root canal walls and in dentinal tubules was not assessed by microscopic imaging or histology. Nevertheless, the micro- biological results of negative controls provided a framework that allowed to quantitatively evaluate the effect of different irrigation protocols. The irrigant that was used in the short-term infection experiment differed from the one used in the long-term infection experiment. This restricts the comparability of the results of the two experiments and needs to be taken into consideration when contrasting these results. In the short-term infection experiment, normal saline was used as irrigant in order to asess the microbial load reduction that can be achieved through fluid dynamics alone. Bio- films at this stage of maturation are highly susceptible to potent antimicrobial irrigants such as sodium hypochlor- ite and, consequently, relevant differences in the effect- iveness of different irrigations protocols would likely not have been detectable with culture assays if sodium hypo- chlorite had been used. It is essential to bear in mind, though, that normal saline was chosen simply to avoid falling below the microbiological detection threshold. In clinical cases, it is crucial to use an effective antimicro- bial and proteolytic agent as main irrigant regardless of the stage of root canal infection. Conclusive evidence indicating which microbiological sam- pling method is best suited for irrigation studies is currently lacking [5]. Paper points allow straightforward sampling from within the main root canal lumen whereas sampling methods using dentin debris may yield more illuminating insight into dentinal tubule disinfection [5]. The microbio- logical approach of the present investigation was developed to remedy some of the shortcomings of paper point sam- pling [28]. As a response to a scarcity of nutrients and other adverse conditions for life, some microorganisms enter a vi- able but non-culturable state. The reintroduction of nutrients recovers the metabolic activity, albeit quite slowly for some oral bacteria, which can take up to three days for their reacti- vation [29]. ANOVA: Analysis of variance; A. oris: Actinomyces oris; C. albicans: Candida albicans; CFU: Total colony forming units; E. faecalis: Enterococcus faecalis; NAD: β-nicotinamide adenine dinucleotide sodium salt; NaOCl: Sodium hypochlorite; S. gordonii: Streptococcus gordonii; TSA: Tryptic soy agar Discussion The provision of nutrient broth and sequential microbiological sampling over an incubation period that lasted one week ensured that slow and lagged microbial re- growth was detectable in the present investigation. Clinical treatment trials are needed to assess whether the differences in antimicrobial efficacy observed in the present investigation have a measurable impact on patient-centered outcomes such as healing of apical periodontitis. The pre- dictive validity of in vitro irrigation studies for clinical out- come parameters is limited. For instance, although a substantial body of in vitro evidence suggests that ultrasonic irrigant activation is more effective than syringe irrigation, there is a paucity of clinical evidence for the benefit of ultra- sonic irrigant activation in primary root canal treatment of teeth with a single root canal [17]. Syringe irrigation was chosen as control group because it is the most popular irrigation method among general dental practitioners and endodontists alike [6, 30]. To overcome some of the limitations of syringe irrigation with side-vented needles [21, 31], the root canals in this study were shaped with a file that achieved an apical en- largement of an ISO size 40 and a taper of .06 along the most apical millimeters. In addition, small needles, Gauge size 30, that allowed placement at working length without binding were used to facilitate adequate irrigant delivery to the full extent of the main root canal and to improve irrigant exchange through an effective reverse flow [9, 31–33]. It is, however, important to bear in mind that the irrigant flow of side-vented needles is nonuniform and the mechanical cleaning effect thus tends to concentrate on the areas of the root canal walls next to the vent [33]. The orientation of the side-vent during irrigation was not monitored in the present in- vestigation. Furthermore, the tip of the needle remained at working length for the whole duration of the irriga- tion even though up and down movement of needles is likely to be advantageous in terms of mechanical clean- ing. However, as standardized vertical movements of the needles were not possible in the setup of this study, irri- gation with the needle static at working length was Conclusions Within the limitations of this in vitro study, it can be con- cluded that the power modes of the sonic irrigation device EDDY have a significant impact on its effectiveness for root canal disinfection. Lower power settings compromise the antimicrobial effectiveness and they need, therefore, to be avoided. In order to reduce the microbial load within the root canal system as effectively as feasible, the hand- piece that is used to drive the sonic irrigation tip ought to be employed at its highest power setting at all times. Received: 15 January 2020 Accepted: 25 March 2020 23. Hage W, de Moor RJG, Hajj D, Sfeir G, Sarkis DK, Zogheib C. Impact of different irrigant agitation methods on bacterial elimination from infected root canals. Dent J. 2019;7:64. 24. Kakoli P, Nandakumar R, Romberg E, Arola D, Fouad AF. The effect of age on bacterial penetration of radicular dentin. J Endod. 2009;35:78–81. Consent for publication 18. Sathorn C, Parashos P, Messer HH. How useful is root canal culturing in predicting treatment outcome? J Endod. 2007;33:220–5. Abbreviations ANOVA A l i Page 8 of 9 Page 8 of 9 Page 8 of 9 Eggmann et al. BMC Oral Health (2020) 20:102 Eggmann et al. BMC Oral Health (2020) 20:102 Acknowledgements 8. Retsas A, Koursoumis A, Tzimpoulas N, Boutsioukis C. Uncontrolled removal of dentin during in vitro ultrasonic irrigant activation in curved root canals. J Endod. 2016;42:1545–9. The authors are very grateful to Anna Magdoń (Laboratory of Oral Microbiology, Department of Periodontology, University of Bern, Switzerland) for her excellent laboratory work. VDW GmbH (Munich, Germany) provided the sonic irrigation tips for this study. The authors gratefully acknowledge this generous support. VDW GmbH had no involvement whatsoever in the study design, in the collection, analysis and interpretation of data, in the writing of the report and in the decision to submit the article for publication. 9. Urban K, Donnermeyer D, Schafer E, Burklein S. Canal cleanliness using different irrigation activation systems: a SEM evaluation. Clin Oral Investig. 2017;21:2681–7. study design, in the collection, analysis and interpretation of data, in the writing of the report and in the decision to submit the article for publication. 10. Rödig T, Koberg C, Baxter S, Konietschke F, Wiegand A, Rizk M. Micro-CT evaluation of sonically and ultrasonically activated irrigation on the removal of hard-tissue debris from isthmus-containing mesial root canal systems of mandibular molars. Int Endod J. 2019;52:1173–81. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. 15. Kaloustian MK, Nehme W, El Hachem C, Zogheib C, Ghosn N, Mallet JP, et al. Evaluation of two shaping systems and two sonic irrigation devices in removing root canal filling material from distal roots of mandibular molars assessed by micro CT. Int Endod J. 2019;52:1635–44. Funding The work was funded by the participating departments. 14. Plotino G, Grande NM, Mercade M, Cortese T, Staffoli S, Gambarini G, Testarelli L. Efficacy of sonic and ultrasonic irrigation devices in the removal of debris from canal irregularities in artificial root canals. J Appl Oral Sci. 2019;27:e20180045. References 1. 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In vitro evaluation by quantitative real-time PCR and culturing of the effectiveness of disinfection of multispecies biofilms in root canals by two irrigation systems. Clin Oral Investig. 2019;23:913–20. 4. Metzger Z, Zary R, Cohen R, Teperovich E, Paqué F. The quality of root canal preparation and root canal obturation in canals treated with rotary versus self-adjusting files: a three-dimensional micro-computed tomographic study. J Endod. 2010;36:1569–73. 4. Metzger Z, Zary R, Cohen R, Teperovich E, Paqué F. The quality of root canal preparation and root canal obturation in canals treated with rotary versus self-adjusting files: a three-dimensional micro-computed tomographic study. J Endod. Competing interests 19. Stauffacher S, Lussi A, Nietzsche S, Neuhaus KW, Eick S. Bacterial invasion into radicular dentine-an in vitro study. Clin Oral Investig. 2017;21:1743–52. Sigrun Eick is a member of the editorial board for BMC Oral Health, but otherwise had no role in the publication process of this manuscript. The other authors declare that they have no competing interests. 20. Amato M, Vanoni-Heineken I, Hecker H, Weiger R. Curved versus straight root canals: the benefit of activated irrigation techniques on dentin debris removal. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2011;111:529–34. Authors’ contributions KWN d SE d l d h 11. Neuhaus KW, Liebi M, Stauffacher S, Eick S, Lussi A. Antibacterial efficacy of a new sonic irrigation device for root canal disinfection. J Endod. 2016;42:1799–803. KWN and SE developed the conception and design of this study. SE and YV acquired the experimental data. KWN and SE performed the data analysis. FE, KWN, SE and YV interpreted the data. FE and KWN drafted the manuscript. FE, KWN, SE and YV revised the manuscript. All authors read and approved the final manuscript. 12. Jiang L-M, Verhaagen B, Versluis M, Langedijk J, Wesselink P, van der Sluis LWM. The influence of the ultrasonic intensity on the cleaning efficacy of passive ultrasonic irrigation. J Endod. 2011;37:688–92. 13. Schneider SW. A comparison of canal preparations in straight and curved root canals. Oral Surg Oral Med Oral Pathol. 1971;32:271–5. Ethics approval and consent to participate The local Ethics Committee denied the need for a formal approval for using irreversibly anonymized teeth. All donors had given their verbal informed consent to the use of their extracted, anonymized tooth/teeth for research purposes. 16. Galler KM, Grubmüller V, Schlichting R, Widbiller M, Eidt A, Schuller C, et al. Penetration depth of irrigants into root dentine after sonic, ultrasonic and photoacoustic activation. Int Endod J. 2019;52:1210–7. 17. Căpută PE, Retsas A, Kuijk L, Chávez de Paz LE, Boutsioukis C. Ultrasonic irrigant activation during root canal treatment: a systematic review. J Endod. 2019;45:31–44 e13. Author details 21. Boutsioukis C, Gogos C, Verhaagen B, Versluis M, Kastrinakis E, Van der Sluis L. The effect of apical preparation size on irrigant flow in root canals evaluated using an unsteady computational fluid dynamics model. Int Endod J. 2010;43:874–81. 1Department of Periodontology, Endodontology and Cariology, University Center for Dental Medicine, UZB, University of Basel, Mattenstrasse 40, CH-4056 Basel, Switzerland. 2Private practice, Zurich, Switzerland. 3Laboratory of Oral Microbiology, Department of Periodontology, School of Dental Medicine, University of Bern, Bern, Switzerland. 4Department of Preventive, Restorative and Pediatric Dentistry, University of Bern, Bern, Switzerland. 22. Zeng C, Willison J, Meghil MM, Bergeron BE, Cutler CW, Tay FR, et al. Antibacterial efficacy of an endodontic sonic-powered irrigation system: an in vitro study. J Dent. 2018;75:105–12. Received: 15 January 2020 Accepted: 25 March 2020 References Ex vivo assessment of irrigant penetration and renewal during the final irrigation regimen. Int Endod J. 2010;43:663–72. 33. Boutsioukis C, Verhaagen B, Versluis M, Kastrinakis E, Wesselink PR, van der Sluis LWM. Evaluation of irrigant flow in the root canal using different needle types by an unsteady computational fluid dynamics model. J Endod. 2010;36:875–9. References 2010;36:1569–73. 29. de Paz C. Luis E, Hamilton IR, Svensater G. Oral bacteria in biofilms exhibit slow reactivation from nutrient deprivation. Microbiology. 2008;154:1927–38. 5. Nagendrababu V, Jayaraman J, Suresh A, Kalyanasundaram S, Neelakantan P. Effectiveness of ultrasonically activated irrigation on root canal disinfection: a systematic review of in vitro studies. Clin Oral Investig. 2018;22:655–70. 6. Dutner J, Mines P, Anderson A. Irrigation trends among American Association of Endodontists members: a web-based survey. J Endod. 2012;38:37–40. 7. Boutsioukis C, Verhaagen B, Walmsley AD, Versluis M, van der Sluis LWM. Measurement and visualization of file-to-wall contact during ultrasonically activated irrigation in simulated canals. Int Endod J. 2013;46:1046–55. 5. Nagendrababu V, Jayaraman J, Suresh A, Kalyanasundaram S, Neelakantan P. Effectiveness of ultrasonically activated irrigation on root canal disinfection: a systematic review of in vitro studies. Clin Oral Investig. 2018;22:655–70. 6. Dutner J, Mines P, Anderson A. Irrigation trends among American Association of Endodontists members: a web-based survey. J Endod. 2012;38:37–40. 7. Boutsioukis C, Verhaagen B, Walmsley AD, Versluis M, van der Sluis LWM. Measurement and visualization of file-to-wall contact during ultrasonically activated irrigation in simulated canals. Int Endod J. 2013;46:1046–55. 5. Nagendrababu V, Jayaraman J, Suresh A, Kalyanasundaram S, Neelakantan P. Effectiveness of ultrasonically activated irrigation on root canal disinfection: a systematic review of in vitro studies. Clin Oral Investig. 2018;22:655–70. 6. Dutner J, Mines P, Anderson A. Irrigation trends among American Association of Endodontists members: a web-based survey. J Endod. 2012;38:37–40. 7. Boutsioukis C, Verhaagen B, Walmsley AD, Versluis M, van der Sluis LWM. Measurement and visualization of file-to-wall contact during ultrasonically activated irrigation in simulated canals. Int Endod J. 2013;46:1046–55. 5. Nagendrababu V, Jayaraman J, Suresh A, Kalyanasundaram S, Neelakantan P. Effectiveness of ultrasonically activated irrigation on root canal disinfection: a systematic review of in vitro studies. Clin Oral Investig. 2018;22:655–70. 30. Willershausen I, Wolf TG, Schmidtmann I, Berger C, Ehlers V, Willershausen B, Briseno B. Survey of root canal irrigating solutions used in dental practices within Germany. Int Endod J. 2015;48:654–60. 31. Boutsioukis C, Lambrianidis T, Verhaagen B, Versluis M, Kastrinakis E, Wesselink PR, van der Sluis LWM. The effect of needle-insertion depth on the irrigant flow in the root canal: evaluation using an unsteady computational fluid dynamics model. J Endod. 2010;36:1664–8. Page 9 of 9 Eggmann et al. BMC Oral Health (2020) 20:102 Eggmann et al. BMC Oral Health (2020) 20:102 32. Bronnec F, Bouillaguet S, Machtou P. Eggmann et al. BMC Oral Health (2020) 20:102 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W4214631857	https://link.springer.com/content/pdf/10.1007/s41244-022-00246-2.pdf	English	null	Narration Without Narrating	Lili. Zeitschrift für Literaturwissenschaft und Linguistik/Zeitschrift für Literaturwissenschaft und Linguistik	2,022	cc-by	18,067	Sebastian Bücking () Germanistisches Seminar, Universität Siegen, Siegen, Germany E-Mail: sebastian.buecking@uni-siegen.de https://doi.org/10.1007/s41244-022-00246-2 Z Literaturwiss Linguistik (2022) 52:35–64 LABOR LABOR Keywords Fictional Narratives · Narrator · Tense · Epic Preterit · Imagination · Attitudes · Discourse Representation Theory · Attitude Description Theory Erzählen ohne Erzählen Die Rolle von Imagination und die Grammatik des epischen Präteritums Zusammenfassung Dieser Artikel behandelt die Frage, wie man den Unterschied zwischen einem Erzählen, das den Eindruck einer erzählenden Instanz vermittelt, und einem Erzählen, das erzählinstanz-neutral ist, aus linguistischer Perspektive erfassen kann. In einem ersten Schritt werde ich gegen den Ansatz von Eckardt (2015) argumentieren, gemäß dem erzählinstanz-neutrales Erzählen auf dem Fehlen von Wissen über die Erzählsituation beruht. So zeige ich, dass der Ansatz zu ei- nem Existenz-Problem, einem Anthropomorphimus-Problem sowie einem Tempus- Problem führt. In einem zweiten Schritt schlage ich eine imaginationsbasierte alter- native Erfassung von Erzählinstanz-Neutralität vor; dazu kombiniere ich Ideen der Institutional Theory of Fiction nach Walton (1990) und Köppe/Stühring (2011) mit den formalen Werkzeugen der Attitude Description Theory nach Maier (2017). Der alternative Vorschlag erfasst die Unterscheidung zwischen erzählinstanz-erschaffen- dem und erzählinstanz-neutralem Erzählen durch optionale Existenzbindung einer Erzählsituation und Erzählinstanz in der Imaginationskomponente des mentalen Zu- stands von Interpreten. Die Argumentation schenkt der Semantik des deutschen Präteritums in ﬁktionalen Narrativen besondere Aufmerksamkeit. Einerseits werde ich die berühmte Hypothese von Hamburger (31977) und ihren Nachfolger:innen in der germanistischen Linguistik bestätigen, dass das Präteritum eine atemporale Lesart lizenziert und damit eine Interpretation erlaubt, die die grammatische Not- wendigkeit einer Erzählsituation innerhalb der Fiktion aufhebt. Andererseits werde ich die vorherrschende Annahme, das Präteritum markiere in der atemporalen Lesart die Fiktion als solche, zurückweisen. Stattdessen schlage ich vor, dass das Präter- itum Interpreten dazu instruiert, die gegebene Geschichte aus der Perspektive einer distanzierten Beobachtung zu imaginieren. Schlüsselwörter Fiktionale Narrative · Erzählinstanz · Tempus · Episches Präteritum · Imagination · Einstellungen · Discourse Representation Theory · Attitude Description Theory Schlüsselwörter Fiktionale Narrative · Erzählinstanz · Tempus · Episches Präteritum · Imagination · Einstellungen · Discourse Representation Theory · Attitude Description Theory Narration Without Narrating The Role of Imagination and the Grammar of the Epic Preterit Sebastian Bücking Received: 1 December 2020 / Accepted: 31 May 2021 / Published online: 28 February 2022 © The Author(s) 2022 Abstract This paper addresses the question of how to account for the distinction between narrator-creating and narrator-neutral narration from a linguistic perspec- tive. I ﬁrst take issue with the approach by Eckardt (2015), according to which narrator-neutral narration is due to a lack of knowledge about the narrating situa- tion; speciﬁcally, I raise an existence problem, an anthropomorphism problem, and a tense problem. Second, combining ideas of the Institutional Theory of Fiction as described by Walton (1990) and Köppe/Stühring (2011) and formal tools of Attitude Description Theory as developed by Maier (2017), I propose an imagination-based alternative account of narrator-neutrality. According to this, the distinction between narrator-creating and narrator-neutral narration is captured by optional existential binding of a narrating situation and a narrator in an imagination component of an interpreter’s mental state. Particular attention is paid to the semantics of the German preterit in ﬁctional narratives. On the one hand, I conﬁrm the famous hypothesis by Hamburger (31977) and her successors in German linguistics that the preterit licenses an atemporal reading and thus an interpretation that eliminates the gram- matical need for a narrating situation within the ﬁction. On the other hand, I reject the prevailing assumption that the preterit in its atemporal reading marks the ﬁction as such. In lieu thereof, the preterit is argued to instruct interpreters to imagine the story from the perspective of a distant observer. Keywords Fictional Narratives · Narrator · Tense · Epic Preterit · Imagination · Attitudes · Discourse Representation Theory · Attitude Description Theory S. Bücking 36 1 The idiomatic translations are mine. If reasonable, I dispense with word-by-word glosses in long exam- ples for the sake of readability. 1 Introduction Narrative discourse structures in literary texts can typically be distinguished by whether they suggest the ﬁction of a narrator or not. Consider, for instance, the contrast between the following opening sequences of two short stories.1 (1) Ich muß immer an diesen roten Teufel von einer Katze denken, und ich weiß nicht, ob das richtig war, was ich getan hab. Es hat damit angefangen, daß ich auf dem Steinhaufen neben dem Bombentrichter in unserem Garten saß ... (1) Ich muß immer an diesen roten Teufel von einer Katze denken, und ich weiß nicht, ob das richtig war, was ich getan hab. Es hat damit angefangen, daß ich auf dem Steinhaufen neben dem Bombentrichter in unserem Garten saß ... 37 Narration Without Narrating ›I always have to think of that red devil of a cat, and I don’t know whether it was right what I did. It all started with me sitting on a pile of stones next to the bomb crater in our garden ...‹ (Rinser, Die rote Katze, p. 43) ›I always have to think of that red devil of a cat, and I don’t know whether it was right what I did. It all started with me sitting on a pile of stones next to the bomb crater in our garden ...‹ (Rinser, Die rote Katze, p. 43) ›I always have to think of that red devil of a cat, and I don’t know whether it was right what I did. It all started with me sitting on a pile of stones next to the bomb crater in our garden ...‹ (Rinser, Die rote Katze, p. 43) ›I always have to think of that red devil of a cat, and I don’t know whether it was right what I did. It all started with me sitting on a pile of stones next to the bomb crater in our garden ...‹ (2) (2) Plötzlich wachte sie auf. Es war halb drei. Sie überlegte, warum sie aufgewacht war. Ach so! In der Küche hatte jemand gegen einen Stuhl gestoßen. Sie horchte nach der Küche. Es war still ... ›Suddenly she woke up. It was half past two. She thought about why she had woken up. Oh! In the kitchen someone had bumped against a chair. She listened toward the kitchen. It was silent ...‹ (Borchert, Das Brot, p. 1 Introduction 18) The discourse structure in (1) introduces a ﬁrst-person narrator and thereby very clearly creates the ﬁction of a mediating narrating situation with someone telling a story. The discourse structure in (2), by contrast, seems to not provide any clear-cut indication of a narrating situation; it rather suggests immediate access to the given events without interference by a narrating instance. Against this background, this paper addresses the following overarching question: how can the intuitive contrast between narrator-creating and narrator-neutral narration be captured from a linguistic and, more speciﬁcally, grammatical perspective? p y g p p A detailed discussion of this question is provided by Eckardt (2015). She argues that narrator-neutrality of ﬁctional texts follows from a lack of knowledge about the narrator. Accordingly, examples such as (2) do involve a narrator despite intuitions to the contrary; however, they do not license the ﬁction of a corresponding speciﬁc individual because substantial information about it is missing. The approach goes well with what Köppe/Stühring (2011) call pan-narrator theories, that is, with the common assumption among narratologists that ﬁctional narrators are always present in ﬁctional narratives. It also goes well with the common assumption among lin- guists that communication is based on the update of information states by speakers’ assertions; in fact, Eckardt’s implementation builds on corresponding context-sen- sitive formal semantic tools as developed by Stalnaker (1978, 2002), Lewis (1978), and Kaplan (1989). The approach thus also supports a routine account of tense in ﬁctional texts. Speciﬁcally, the German preterit in example (2) simply indicates that the speaker (that is, the implicit narrator) makes assertions about a reference time preceding her utterance time (that is, the narrating time), fully on a par with its function in non-ﬁction. This warrants the conception of narration as a »necessarily retrospective act of sense-making« (Scheffel et al. 2014, p. 873). It is, however, by no means clear whether this neat picture can be upheld upon closer scrutiny. First, Köppe/Stühring (2011) (among others) have argued against pan-narrator theories from the perspective of literary studies. Based on Walton (1990) and the so-called Institutional Theory of Fiction, they instead propose that ﬁctional narratives are invitations to imaginings that can, but need not involve narrators. This suggests beneﬁting from these insights for a new linguistic take on narrator- neutrality that, in contrast to Eckardt’s proposal, can dispense with narrator ubiquity. 1 Introduction I will sketch such an approach here, based on Maier’s (2017) Attitude Description Theory. Second, this paper will call attention to shortcomings of Eckardt’s proposal 38 S. Bücking from a linguistic perspective and thereby complement the critical evaluation of pan- narrator theories in literary studies. Most notably, the underlying routine account of the German preterit as a purely temporal grammatical marker cannot be taken for granted. As famously argued by Hamburger (31977), the narrated story in narratives such as (2) appears to be present; hence, the preterit in these cases – the so-called epic preterit – does not seem to contribute a temporal anteriority relation to an implicit utterance (that is, narrating) situation. Building on Hamburger’s work, linguists such as Thieroff (1992), Welke (2005), and Bredel/Töpler (2007) propose that the preterit in German conveys an underspeciﬁed distance feature that can be speciﬁed as ›past‹ (that is, temporally distant) or as ›ﬁctional‹ (that is, distant from the actual world). This amounts to the intriguing hypothesis that the epic preterit marks the ﬁction as such. I will evaluate this hypothesis by relating it to the more general argument in favor of so-called fake tense in natural language. The most prominent case in point is provided by counterfactual conditionals such as (3) in various languages. As shown by Iatridou (2000) (among others), counterfactual conditionals also involve past morphemes that do not mark temporal anteriority, but distance from the actual world: the speaker of (3) excludes the actual world from the worlds she is talking about. (3) If Mia hadPST a dog, she wouldFUT+PST be happy. (3) If Mia hadPST a dog, she wouldFUT+PST be happy. [see Iatridou 2000, (47a)] While it will be shown that the close link between the preterit and ﬁction as sug- gested within German linguistics is ultimately misleading, the idea that the preterit conveys a distance relation that supports atemporal readings will still turn out to be useful. I will propose that the epic preterit marks distance in the spatial domain. Speciﬁcally, it instructs the interpreter of a ﬁctional narrative to imagine the situation the narrative is about from the perspective of a physically distant observer who is not participating in this situation. The proposal will thus use the ingredients of the imagination-based account of ﬁctional narration in order to shed new light on the contribution of tense within ﬁction, departing from both Eckardt’s account and fake- tense-related approaches. 1 Introduction The paper is organized as follows: in Section 2, I will review Eckardt’s (2015) approach to narrator-neutrality of ﬁctional texts. Section 3 will introduce an imagi- nation-based alternative approach to narrator-neutrality and sketch its formal imple- mentation. Section 4 will zoom in on the analysis of the epic preterit within ﬁction vis-à-vis fake-tense-related approaches. Section 5 offers a conclusion. 2 Relevant expectations can vary between, for instance, recipients (children will have different expecta- tions than adults, etc.) or types of ﬁction (fairytales involve different expectations than historical novels, etc.). Such details will not be important here; see Lewis (1978) and Bonomi/Zucchi (2003) for discussion. 3 According to this standard set-up, the contents of ﬁctional texts are modeled in terms of sets of contexts. It is controversial whether this is a fully intuitive conception. It seems to be more realistic that interpreters have a particular context in mind, which is, then, enriched step by step by the contents the story provides. This controversy is not crucial for the present paper; notably, however, the approach that will be proposed in Section 3 will rely on the imagination of particular contexts and thus on the more realistic conception of interpretation. 2 Relevant expectations can vary between, for instance, recipients (children will have different expect tions than adults, etc.) or types of ﬁction (fairytales involve different expectations than historical novel etc.). Such details will not be important here; see Lewis (1978) and Bonomi/Zucchi (2003) for discussio 2 Eckardt’s (2015) approach to narrator-neutrality and its problems Eckardt (2015) integrates her approach to narrator-neutrality into the following gen- eral framework for the dynamic interpretation of ﬁctional texts; see Stalnaker (1978, 2002), Lewis (1978), and Kaplan (1989) for the relevant background. First, the con- tent of ﬁctional texts is represented by sets of utterance contexts c. The initial set for the reception of a ﬁctional text comprises the expectations of a recipient A about the ﬁctional text at hand before starting to receive it. This set is called STORY(A)0 and 39 Narration Without Narrating described as in (4); see Eckardt (2015, p. 163). WORLD is a function that maps any utterance context to the world in which the utterance is embedded; correspondingly, SPEAKER and TIME single out the speaker and the time of utterance. described as in (4); see Eckardt (2015, p. 163). WORLD is a function that maps any utterance context to the world in which the utterance is embedded; correspondingly, SPEAKER and TIME single out the speaker and the time of utterance. (4) STORY(A)0 = {c: The given story could be told in c about WORLD(c) by SPEAKER(c) at TIME(c) ...} According to Eckardt, these expectations include that WORLD(c) is variably simi- lar to the actual world, but, as ﬁction, never identical to it.2 For the present discussion, the most crucial consequence of the set-up in (4) is that potential utterance contexts c minimally include that there is a telling by a speaker at a time, that is, that there is a narrating situation with a narrator and a narrating time. Second, the interpretation of a ﬁctional text amounts to the successive update of STORY(A)0 by the successively given information p1, p2, etc. That is, p1, p2, etc. constrain the respective preceding information state by intersection, as shown by the general pattern in (5). (6) provides a concrete example, assuming that it is the opening sequence of a ﬁctional text. (The temporal information is simpliﬁed; see the further discussion for more details.) ) STORY(A)0 \ p1 \ p2 \ ... (where pn = sets of utterance contexts) (5) STORY(A)0 \ p1 \ p2 \ ... (6) a. Ich schaue aus dem Fenster. Gestern hat es geregnet. (6) a. Ich schaue aus dem Fenster. Gestern hat es geregnet. I look out the window yesterday has it rained ›I am looking out of the window. It was raining yesterday.‹ ›I am looking out of the window. 4 Roughly, the speaker thus intends to talk about a part of Mia’s looking out of the window, not caring about its beginning and ending. The use of the perfective aspect would, by contrast, indicate that the speaker is talking about a time interval that includes Mia’s looking out of the window as a whole. 2 Eckardt’s (2015) approach to narrator-neutrality and its problems It was raining yesterday.‹ b. STORY(A)0 \ {c: in WORLD(c), SPEAKER(c) is looking out of the window at TIME(c)} \ {c: in WORLD(c) it is raining the day before TIME(c)} c. = {c: the given story could be told in c about WORLD(c) by SPEAKER(c) at TIME(c); in WORLD(c), SPEAKER(c) is looking out of the window at TIME(c) and it is raining the day before TIME(c)} According to this stepwise procedure, each piece of information reduces the set of potential utterance contexts c and thereby enriches our image of the ﬁctional story.3 Most importantly, as the narrating situation is part of the ﬁctional story, the procedure also affects the narrating situation in regular ways. The following sources of information about the narrating situation update our image of the narrator and the narrating time. g Assertions about an explicitly given ﬁrst-person narrator provide the most obvious case in point. For instance, the ﬁrst sentence in (6a) contributes a fairly speciﬁc piece of information about the narrator, namely, that she is looking out of the window while 40 S. Bücking telling her story. A more implicit source of information is given by narrator-oriented interpretations of so-called speaker-oriented expressions such as evaluative adverbs or exclamatives. In (7), for instance, the regret associated with the adverb leider ›regrettably‹ or the surprise associated with the exclamative Wie es regnete! ›How it was raining!‹ can be attributed to the narrator and thereby shape our image of the narrator’s attitude towards the situation at hand. (7) Es regnete. {Leider regnete es. / Und wie es regnete!} it rained {regrettably rained it / and how it rained} ›It was raining. {Regrettably, it was raining. / And how it was raining!}‹ (7) Es regnete. {Leider regnete es. / Und wie es regnete!} it rained {regrettably rained it / and how it rained} ›It was raining. {Regrettably, it was raining. / And how it w However, caution is needed here. It is typical of ﬁctional texts that speaker- oriented expressions are related to a protagonist instead of a potential narrator. In (8), for instance, the surprise is most naturally attributed to Mia, which leads to free indirect discourse; see, for instance, Eckardt (2014) for a comprehensive discussion of free indirect discourse from a linguistic point of view. (8) Mia schaute aus dem Fenster. Wie es regnete! (i) A story is narrator-neutral if for any context c in STORY(A)n with SPEAKER(c) = x, and arbitrary other person y, there is a context c* in STORY(A)n with SPEAKER(c) = y, but WORLD(c) = WORLD(c) and TIME(c) = TIME(c). 2 Eckardt’s (2015) approach to narrator-neutrality and its problems Many options always mean little knowledge, which can come close to no knowledge at all. If the content of a story could have been told by anybody, the illusion of a protag- onist ›somebody‹ simply does not arise.« »In fact, the account does not predict the ﬁction of a narrator. Many options always mean little knowledge, which can come close to no knowledge at all. If the content of a story could have been told by anybody, the illusion of a protag- onist ›somebody‹ simply does not arise.« This knowledge-based approach seems to reconcile the ubiquitous existence of a narrating situation with the appearance of its nonexistence in an elegant way. In the following, however, I will call attention to three problems with the approach: an existence problem, an anthropomorphism problem, and a tense problem. 5 Her formal deﬁnition is given in (i); it is slightly adapted from Eckardt (2015, p. 167). 2 Eckardt’s (2015) approach to narrator-neutrality and its problems Mia looked out the window how it rained ›Mia was looking out of the window. How it was raining!‹ (8) Mia schaute aus dem Fenster. Wie es regnete! Mia looked out the window how it rained ›Mia was looking out of the window. How it was raining!‹ A ﬁnal source of information about the narrating situation is provided by tense. According to standard assumptions in linguistics (building on Reichenbach 1947; see also Klein 1994), ﬁnite clauses combine two layers of temporal information. Tense relates the utterance time and the so-called reference time, where the reference time is deﬁned as the time a speaker talks about. Aspect, by contrast, relates the reference time to the so-called situation time, where the situation time is deﬁned as the run time of the situation described by the verbal predication. For instance, let (9) be a run-of-the-mill non-ﬁctional utterance. (9) Mia schaute aus dem Fenster. (9) Mia schaute aus dem Fenster. Mia looked out the window Mia looked out the window ›Mia was looking out of the window.‹ The tense part of the preterit then conveys that the speaker is talking about a reference time before the utterance time. Assuming that imperfective aspect is used (which is not grammaticalized in German, but is visible in the progressive form of the English translation), the reference time is furthermore said to be a subinterval of the situation time, that is, of the run time of Mia’s looking out of the window.4 Under Eckardt’s set-up, this routine account of tense (and aspect) can be used for ﬁctional texts without any change: the speaker is the narrator and the utterance time is the narrating time; correspondingly, if treated as the ﬁrst sentence of a ﬁctional text, (9) receives the interpretation in (10). 2 Eckardt’s (2015) approach to narrator-neutrality and its problems 41 Narration Without Narrating (10) {c: the given story could be told in c about WORLD(c) by SPEAKER(c) at TIME(c); in WORLD(c), the SPEAKER(c) says about a reference time t be- fore TIME(c) that t is a subinterval of a situation time t00 at which Mia is looking out of the window} (10) {c: the given story could be told in c about WORLD(c) by SPEAKER(c) at TIME(c); in WORLD(c), the SPEAKER(c) says about a reference time t be- fore TIME(c) that t is a subinterval of a situation time t00 at which Mia is looking out of the window} (10) {c: the given story could be told in c about WORLD(c) by SPEAKER(c) at TIME(c); in WORLD(c), the SPEAKER(c) says about a reference time t be- fore TIME(c) that t is a subinterval of a situation time t00 at which Mia is looking out of the window} Crucially, in contrast to speaker-oriented expressions and also in contrast to aspect (see Section 2.3 for protagonist-oriented interpretations of aspect), tense under the routine account is rigidly linked to the narrating time and thus the narrator. Therefore, any ﬁnite clause involves a temporal constraint on the narrating situation. While this information is surely thin contentwise – it merely informs about a temporal relation between narrating time and reference time – it still renders the existence of someone narrating a logical necessity. To summarize the foregoing: There is a principled distinction among informa- tion sources about the narrating situation. Sources such as assertions about a ﬁrst- person narrator and narrator-oriented interpretations of speaker-oriented expressions provide substantial information about the narrator; however, these sources are gram- matically optional. Tense, by contrast, provides rather inconspicuous information about the narrator; under the standard temporal interpretation of tense, however, its contribution to the narrating situation is grammatically encoded and thus an infallible indication of its existence. Against this background, Eckardt deﬁnes her notion of narrator-neutrality as follows. Fictional texts are narrator-neutral if they do not provide narrator-related information except for the inevitable information contributed by tense. That is, the impression that a text appears to not have a narrator follows from the fact that the text does not provide substantial information about the narrator. This makes the narrator arbitrary; see the following quote from Eckardt (2015, p. 182):5 »In fact, the account does not predict the ﬁction of a narrator. 6 Eckardt (2015) could amplify her approach by assumptions that relate to the discourse status of narrators. However, this seems to supersede knowledge as the relevant factor altogether. In fact, in Section 3 I will propose a discourse-based approach to speaker-neutrality. 2.1 Existence problem (12) Ich erzähle dir, was passiert ist: Ein älterer Herr ... ›I am telling you what happened: An elderly man ...‹ (12) Ich erzähle dir, was passiert ist: Ein älterer Herr ... ›I am telling you what happened: An elderly man ...‹ (12) Ich erzähle dir, was passiert ist: Ein älterer Herr ... ›I am telling you what happened: An elderly man ...‹ The ﬁction of a narrator in such examples is surely a categorical trait of ﬁrst-per- son narration. However, following Eckardt, every ﬁctional context c involves a par- ticular narrating situation with a narrator. The narrator should thus be identiﬁable, independently of whether she relates to herself by Ich ›I‹ or not. A purely knowl- edge-based account of narrator-neutrality seems to be at a loss here; the amount of information about narrators does not provide the right tool in order to capture the categorical effect of ﬁrst-person narration.6 2.1 Existence problem The approach by Eckardt builds on the assumption that poor knowledge about an existing narrator creates the impression that the narrator does not exist. However, 42 S. Bücking it is not made transparent by independent linguistic evidence why this assumption should hold; in fact, independent evidence questions the given assumption. I call this the existence problem. it is not made transparent by independent linguistic evidence why this assumption should hold; in fact, independent evidence questions the given assumption. I call this the existence problem. First, lack of knowledge about an entity usually does not create the impression that the entity does not exist. For instance, the example in (11) supports the impression that there is food and a dog although one does not know much about either. (11) Ben saß auf der Veranda, schaute in den Nachthimmel und aß zu Abend. Er hörte ein Bellen. Sonst war es still ... ›Ben was sitting on the porch, gazing at the night sky and having supper. He heard some barking. Otherwise it was silent ...‹ 1) Ben saß auf der Veranda, schaute in den Nachthimmel und aß zu Abend. Er ›Ben was sitting on the porch, gazing at the night sky and having supper. He heard some barking. Otherwise it was silent ...‹ Notably, the relevant entities are introduced as implicit arguments of the predi- cates essen ›eat‹ and Bellen ›barking‹. That is, the impression that the food and the dog exist cannot be attributed to their explicit introduction by suitable referential expressions. The situation is thus comparable to the introduction of implicit narra- tors in ﬁctional texts. It is therefore surprising why only the introduction of implicit narrators should block the impression of their existence. This line of thought can be strengthened by the following consideration. Implicit arguments are usually con- sidered discourse-opaque referents that are barred from discourse-related operations such as cross-sentential anaphoric linking; see, for instance, Farkas/de Swart (2003). That is, implicit arguments support the impression of their existence despite their weak discourse status. It is then all the more surprising why implicit narrators should not support a corresponding impression, given that they occupy a very prominent discourse position according to the invariant set-up in (4), namely, the position of the speaker. Second, the explicit introduction of a narrating situation creates the impression that a narrator exists even if any additional information is lacking, as in (12). 2.2 Anthropomorphism problem The second problem relates to the kind of narrator involved in Eckardt’s proposal. The proposal presupposes an anthropomorphic narrator, that is, someone talking 43 Narration Without Narrating or thinking and living at the narrating time. This presupposition follows from the invariant set-up in (4), repeated in (13), according to which c necessarily includes a regular speech situation. or thinking and living at the narrating time. This presupposition follows from the invariant set-up in (4), repeated in (13), according to which c necessarily includes a regular speech situation. (13) STORY(A)0 = {c: The given story could be told in c about WORLD(c) by SPEAKER(c) at TIME(c) ...} Literary approaches, however, usually provide more cautious descriptions of nar- rators; see, for instance, the following deﬁnition from Martínez/Scheffel (102016, pp. 216f., my translation). »Narrator (narrative instance, narrating subject) [...] E.g., also collectives, ani- mals or inanimate objects can function as narrators; furthermore, the narrating subject can remain more or less disembodied and speak seemingly indepen- dently of any ﬁxed link to space and time.« Similarly, according to the deﬁnition by Margolin (2014), ›narrator‹ primarily denotes an abstract speech position that can only be related to the occupant of this position by interaction of metonymy and anthropomorphization. To a certain extent, Eckardt’s proposal can be adapted to these deﬁnitions easily. Speciﬁcally, in order to integrate stories with narrating animals or inanimate objects, one simply has to assume the ﬁction of a set of contexts c where certain principles of the actual world are suspended. For instance, c could be such that animals, stones, trees, etc. can speak. Nevertheless, as the proposal relies on a narrating situation that is deﬁned as a regular speech situation at a particular time and place within the relevant contexts c, it does not support truely disembodied and abstract narrating instances. In other words, the proposal wrongly predicts that narrators must be conceived of as anthropomorphic concrete entities within the ﬁction. Perhaps the most powerful illustration of the problem is provided by ﬁctions about lifeless space or the end of all life; see (14) for a potential example and Byrne (1993) for a related more general argument against pan-narrator theories from a philosophical perspective. (14) Es gab kein Sprechen, kein Erzählen, kein Leben mehr. Nichts blieb, und das für immer. ›There was no longer any speaking, any narrating, any living. 7 A reviewer criticizes that the following discussion is strongly focused on tense (which also holds for the remainder of the paper as a whole). The reviewer argues that I should pay more attention to the variety of linguistic means by which narrators can become manifest in texts. I disagree with this position for the following reason: The paper is focused on the question of how to account for speaker-neutrality in ﬁctional narratives. More speciﬁcally, it raises the question of whether the knowledge-based account in Eckardt (2015) is on the right track. Tense is of utmost importance for this question because it is grammatically necessary and thereby, according to Eckardt’s set-up, a linguistic means that logically entails a narrating situation with a narrator; see the beginning of Section 2 and the following discussion. Correspondingly, tense is the only linguistic means that threatens a simple account of speaker-neutrality. All other means are grammatically optional. As a consequence, they are not particularly challenging for the account: if a narrative contains such means, the narrative is not speaker-neutral; if they are missing, they do not affect the analysis. In other words, the optional means are of no avail for my primary concern because they do not distinguish between different approaches to speaker-neutrality. 2.3 Tense problem7 Recall that, following Eckardt (2015), tense necessarily expresses a temporal relation to an utterance time, which is the narrating time in ﬁctional narratives. The existence of a narrating situation with a narrator is therefore a logical necessity within her approach. However, the following discussion will show that this argument rests on a controversial premise. Speciﬁcally, prominent literary and linguistic studies argue that tense and, in particular, the preterit need not receive their standard temporal interpretation. p Hamburger (31977) and Weinrich (62001) are the most prominent representatives of this alternative position in literary studies. In a nutshell, they follow the intuition that the content of ﬁctional stories in the preterit appears to be present. Correspond- ingly, they hold that the preterit in narrative ﬁction, the so-called epic preterit, does not relate to a past. There is a common objection to this position within narratology, which, however, I do not consider justiﬁed. This objection is exempliﬁed by the following quote from Martínez/Scheffel (102016, p. 76) [my translation]. »Hamburger neglects the distinction between the imaginary and the real context of the ﬁctional speech – within the imaginary context it is still true that the act of narration temporally follows the events of the narrated story.« »Hamburger neglects the distinction between the imaginary and the real context of the ﬁctional speech – within the imaginary context it is still true that the act of narration temporally follows the events of the narrated story.« The objection is based on the assumption that all ﬁctional narratives involve an imagined retrospective narrating situation. However, in light of our overarching question of how to capture speaker-neutrality within ﬁction, it is precisely this assumption that is up for discussion. Therefore, the given objection is not conclusive. p p g j In German linguistics, Hamburger’s hypothesis has been inspiring for the analysis of the preterit as a grammatical category more generally. Studies such as Thieroff (1992), Welke (2005), and Bredel/Töpler (2007) argue against the view that the preterit is a primarily temporal category. They associate the preterit with an under- speciﬁed distance feature that supports both temporal and atemporal speciﬁcations. The precise nature of the atemporal speciﬁcation need not concern us yet; see Section 4 for an evalution of the intriguing hypothesis that the preterit under its atemporal reading marks the ﬁction as such. 2.2 Anthropomorphism problem Nothing re- mained, and this was so forever.‹ Crucially, the set of contexts c as described by the ﬁction in (14) excludes the existence of a temporally following narrating situation within c. In contrast to the requirements of the invariant set-up in (13), one thus cannot construe a consistent retrospection. I hasten to counter a likely objection to this argument. Of course, it would be consistent for (14) to construe a narrating situation, including a narrator, that is outside of c. However, it is essential for Eckardt’s set-up in (13) that the narrating situation and its narrator are part of c. This is what enables the narrator to talk about her own past in the proposed straightforward way; she cannot talk about her own past in terms of a different c (as this would require additional modal com- ponents). I conclude that ﬁctions such as (14) pose a serious problem for essential aspects of Eckardt’s proposal. 44 S. Bücking 2.3.1 Grammatical reasons for the feasibility of an atemporal preterit From a grammatical perspective, it is a widely received view that the temporal contribution of the preterit in German differs in principled ways from the contribution of the present perfect in German.8 Roughly, the preterit combines past tense with perfective, imperfective, or prospective aspect, whereas the present perfect combines present tense with perfect aspect. As a consequence, a speaker using the preterit talks about a reference time TR before her utterance time TU and locates the situation time TS either in TR, around TR, or after TR. A speaker using the present perfect talks about a reference time TR that coincides with her utterance time TU and locates the situation time TS before TR. A schematic overview is given in (15) and (16). The examples in (17) provide a minimal pair for illustration (assuming imperfective aspect for the preterit); notably, in both examples, Mia’s sleeping takes place before TU, which accounts for their intuitive similarity. (15) preterit: a. TR < TU (past) b. TS TR (perfective) or TS TR (imperfective) or TS > TR (prospective) (16) present perfect: (15) preterit: a. TR < TU (past) b. TS TR (perfective) or TS TR (imperfective) or TS > TR (prospective) (16) present perfect: a. TR = TU (present) b. TS < TR (perfect) (17) a. Mia schlief. Mia sleep:PRET ›Mia was sleeping.‹ Ý9 The speaker talks about her past as a time when Mia is sleeping. b. Mia hat geschlafen. Mia have:PRS sleep:PTCP ›Mia has slept.‹ Ý The speaker talks about her now as a time after Mia’s sleeping. a. TR = TU (present) Ý9 The speaker talks about her past as a time when Mia is sleeping. Ý9 The speaker talks about her past as a time when Mia is sleeping Ý9 The speaker talks about her past as a time when Mia is sleeping. a hat geschlafen. Ý The speaker talks about her now as a time after Mia’s sleeping. This set-up has the following consequences for narratives; see, in particular, Welke (2005, Chapter 7.3) for a corresponding line of thought. As utterances in the present perfect are about the now of the speaker, the reported past events are always con- sidered from the perspective of this now. That is, every use of the present perfect invokes the dependence of the events on the utterance time. 8 It is beyond the scope of this paper to do full justice to the rich research on the preterit and the present perfect in German. However, the main argument is based on fairly uncontroversial basic distinctions. The following rough summary of these distinctions rests upon Eckardt (2014, Chapter 4). 9 In this paper, I employ the symbol Ý as an informal marker for the relation ›leads to the interpretation‹. 2.3 Tense problem7 For the time being, it is merely crucial that any atemporal analysis of the preterit undermines the idea that the use of tensed clauses in ﬁctional narratives renders the existence of a narrating situation a logical 45 Narration Without Narrating necessity. Against this background, it is worthwhile to consider potential evidence for an atemporal analysis of the preterit in German. necessity. Against this background, it is worthwhile to consider potential evidence for an atemporal analysis of the preterit in German. 9 In this paper, I employ the symbol Ý as an informal marker for the relation ›leads to the interpretation 10 It is noteworthy that Welke’s take is also reﬂected in the analysis of the preterit in the formal semantics tradition. Speciﬁcally, Kratzer (1998) (who is not concerned with tense in ﬁction, but with analogies between tenses and pronouns) argues that the reference time TR as introduced by the preterit in German is presupposed and thus linked to discourse-given sequences of events. Correspondingly, in contrast to the present perfect, the preterit cannot be used felicitously in a context that has not established a relevant discourse; see the contrast in (i) (under the assumption that the question initiates a conversation among interlocutors who are looking at churches in Italy). (i) Wer {hat diese Kirche gebaut / baute diese Kirche}? – Borromini. who {have:PRS this church build:PTCP / build:PRET this church} – Borromini. ›Who built this church? – Borromini.‹ [ K t 1998 ( I thank Fabienne Martin for making me aware of this connection. (i) Wer {hat diese Kirche gebaut / baute diese Kirche}? – Borromini. who {have:PRS this church build:PTCP / build:PRET this church} – Borromini. ›Who built this church? – Borromini.‹ [see Kratzer 1998, (40b/ I thank Fabienne Martin for making me aware of this connection. 2.3.1 Grammatical reasons for the feasibility of an atemporal preterit Therefore, the perfect is not a good means to establish narratives that are independent of the utterance time; correspondingly, it does not support narratives that consist of self-contained chains of events. The preterit is different. As utterances in the preterit are about a time that 46 S. Bücking S. Bücking differs from the utterance time, the reported events are only indirectly linked to the now of the speaker. Therefore, the preterit is typical for independent narratives, that is, for chains of events where, instead of the utterance time, the events themselves provide the reference time for respective subsequent events. This independence from the time of utterance sets the stage for an invisible utterance time and thus an atem- poral interpretation of the preterit; Welke (2005, p. 301) [my translation] concludes for ﬁctional narratives in particular: »it [= TU] is nearly abandoned in ﬁctional texts.«10 As far as I know, the previous research has not linked the given grammatical distinction between the preterit and the perfect to the controversy about narrator- neutrality in ﬁctional texts. However, there is an obvious link. As the perfect involves a visible time of utterance and thus, more generally, a visible utterance situation, it continuously indicates that there is someone talking; therefore, it should usually not support narrator-neutral ﬁctional discourse. The preterit, by contrast, can abandon the time of utterance and thus the utterance situation; therefore, it should support narrator-neutral ﬁctional discourse. In fact, this prediction is borne out. Consider the examples in (18), based on the supposition that they each form the beginning of some ﬁctional text. (18) a. Ben hat am Schreibtisch gesessen, einen Kaffee getrunken und Ben have:PRS at the desk sit:PTCP a coffee drink:PTCP and aus dem Fenster geschaut. Es hat geregnet. Da bin ich out of the window look:PTCP it have:PRS rain:PTCP there be:PRS I mir sicher! REFL sure ›Ben has been sitting at the desk, drinking a coffee and looking out of the window. It has been raining. I am sure about it!‹ b. Ben saß am Schreibtisch, trank einen Kaffee und Ben sit:PRET at the desk drink:PRET a coffee and schaute aus dem Fenster. Es regnete. #Da bin ich mir sicher! look:PRET out of the window it rain:PRET there be:PRS I REFL sure ›Ben was sitting at the desk, drinking a coffee and looking out of the window. It was raining. I am sure about it!‹ It was raining. 2.3.1 Grammatical reasons for the feasibility of an atemporal preterit I am sure about it!‹ [see Kratzer 1998, (40b/c)] [see Kratzer 1998, (40b/c)] 47 Narration Without Narrating In both examples, the text ﬁrst provides some information about Ben and the weather and then introduces a ﬁrst-person narrator, which makes explicit that the given information originates with this narrator. Crucially, the choice of temporal forms in the ﬁrst two sentences yields a subtle contrast. In (18a), where the present perfect is used, the sudden introduction of an explicit narrator comes as little surprise. This is expected under the assumption that the existence of a narrating situation is already marked by the perfect itself. By contrast, in (18b) where the preterit is used, the sudden introduction of a narrator is surprising and thus renders the discourse structure slightly incoherent. This effect can be accounted for by the assumption that the preterit supports an atemporal use that is independent of a narrating situation.11 The given reasoning is also compatible with the examples from the introduction, repeated in (19) and (20). Subscripts on ﬁnite verbs specify which temporal form is used in the respective sentence. (19) Ich mußPRS immer an diesen roten Teufel von einer Katze denken, und ich weißPRS nicht, ob das richtig warPRET, was ich getan habPRS PRF. Es hatPRS PRF damit angefangen, daß ich auf dem Steinhaufen neben dem Bombentrichter in un- serem Garten saßPRET ... ›I always have to think of that red devil of a cat, and I don’t know whether it was right what I did. It all started with me sitting on a pile of stones next to the bomb crater in our garden ...‹ (Rinser, Die rote Katze, p. 43) (20) Plötzlich wachtePRET sie auf. Es warPRET halb drei. Sie überlegtePRET, warum sie aufgewacht warPST PRF. Ach so! In der Küche hattePST PRF jemand gegen einen Stuhl gestoßen. Sie horchtePRET nach der Küche. Es warPRET still ... ›Suddenly she woke up. It was half past two. She thought about why she had woken up. Oh! In the kitchen someone had bumped against a chair. She listened toward the kitchen. It was silent ...‹ (Borchert, Das Brot, p. 18) The example in (19) introduces a ﬁrst-person narrator and thereby creates the ﬁction of a mediating narrating situation. As expected, this narrator-creating example licenses the use of the present perfect. Furthermore, the example clearly advances the impression that the narrator is reporting on events of her past. 11 A reviewer considers the effect barely comprehensible. Speciﬁcally, the reviewer argues that the ex- amples in (18) are too artiﬁcial to be convincing. From a linguistic perspective, I ﬁnd the construction of (artiﬁcial) minimal pairs legitimate. They allow one to zoom in on the particular issue at hand and to abstract away from the complexities that come with authentic examples. Of course, this method also has its downsides; correspondingly, it is often up for discussion whether the examples as constructed in favor of a certain argument are in fact acceptable and show what they are supposed to show. In spite of the reviewer’s sceptical position, I still believe in the presumptive subtle contrast between (18a) and (18b). In order to substantiate it by empirical means, one probably needs psycholinguistic methods (e.g., reading time experiments); this, however, goes beyond the scope of the present paper. 2.3.1 Grammatical reasons for the feasibility of an atemporal preterit That is, there is a narrating situation in which previous events are treated as factual, which conforms to the standard conception of ﬁctional narration as a truly retrospective act by an imagined narrator. There are also two preterit forms in (19); however, as they receive 48 S. Bücking a standard temporal interpretation in the given context, they do not challenge the suggested line of interpretation. a standard temporal interpretation in the given context, they do not challenge the suggested line of interpretation. The grammatical set-up of the narrator-neutral example in (20) is very different. Most importantly, the predominant temporal form here is the preterit, while the present perfect is not used at all. In this case, the impression of a retrospective narrating act is completely missing. This effect can easily be explained by the current approach, according to which the preterit licenses atemporal interpretations. It is however at odds with approaches such as Eckardt’s, according to which the preterit should always receive its standard temporal interpretation and thus unequivocally evoke a readily available narrating situation. Notably, the use of the past perfect in (20) is fully regular as well. According to standard assumptions, the past perfect combines the interpretation of the preterit with the interpretation of perfect aspect; see Reichenbach (1947), Klein (1994), and Eckardt (2014). The current approach thus predicts for (20) that it combines an atemporal interpretation of the preterit with anteriority of event times as conveyed by aspect; this is intuitively correct: the past perfect in (20) merely indicates that the relevant events – the waking up and the bumping against the chair – happen before the events in the main chain of situations, which is not further speciﬁed timewise. One should ﬁnally note that the relevant retrospection in this case is not attributed to a narrator (which would undermine narrator-neutrality), but to a protagonist, that is, to the person sie ›she‹ refers to. This leads to (free) indirect discourse and the impression that the protagonist is reﬂecting on events in her immediate past (compare also the use of the attitude verb überlegen ›think‹ and the interjection Ach so! ›Oh!‹). 12 Fabienne Martin pointed out to me that, intuitively, preterit-based texts such as (20) are more proto- typical representatives of ﬁctional narratives than texts such as (19) are. I share this intuition and offer the following explanation. In light of the hypothesis that the preterit supports an interpretation of ﬁctional narratives without any actual narrating situation within the ﬁction, there is no one in (20) who commits to the truth of the story being told. In (19), by contrast, there is an actual narrating situation within the ﬁction and thus a narrator who commits to the truth of the story. The story as such is thereby presented as factual, which reduces the impression of its being ﬁctional. 13 According to a reviewer, the vernacular literature of Southern German generally makes use of the perfect instead of the preterit; the reviewer also notes that hab instead of habe for the ﬁrst-person singular of the auxiliary haben ›have‹ can be used in order to mark informal language. Given the general use of the perfect, one might speculate that the relevant vernacular literature always comes with the impression that there is someone narrating. Relatedly, Eckardt (2014, p. 91) claims that Southern German dialects do not support free indirect discourse, and that this restriction might be due to the fact that the dialects lack the preterit and the relevant elaborate system of tense and aspect of the standard variety. I would like to leave to future research whether these speculations are on the right track. It might of course also be that the relevant tenses and underlying grammatical systems cannot be compared in such a simple way. 12 Fabienne Martin pointed out to me that, intuitively, preterit-based texts such as (20) are more proto- typical representatives of ﬁctional narratives than texts such as (19) are. I share this intuition and offer the following explanation. In light of the hypothesis that the preterit supports an interpretation of ﬁctional narratives without any actual narrating situation within the ﬁction, there is no one in (20) who commits to the truth of the story being told. In (19), by contrast, there is an actual narrating situation within the ﬁction and thus a narrator who commits to the truth of the story. The story as such is thereby presented as factual, which reduces the impression of its being ﬁctional. 13 According to a reviewer, the vernacular literature of Southern German generally makes use of the perfect instead of the preterit; the reviewer also notes that hab instead of habe for the ﬁrst-person singular of the auxiliary haben ›have‹ can be used in order to mark informal language. Given the general use of the perfect, one might speculate that the relevant vernacular literature always comes with the impression that there is someone narrating. Relatedly, Eckardt (2014, p. 91) claims that Southern German dialects do not support free indirect discourse, and that this restriction might be due to the fact that the dialects lack the preterit and the relevant elaborate system of tense and aspect of the standard variety. I would like to leave to future research whether these speculations are on the right track. It might of course also be that the relevant tenses and underlying grammatical systems cannot be compared in such a simple way. 2.3.2 Temporal adverbials and atemporal preterit The most prominent argument in favor of atemporal tenses originates with Ham- burger (31977) and her consideration of tense forms in combination with temporal adverbials; see (21a) and the analogous simpliﬁed variant in (21b). (21) a. Aber am Vormittag hatte sie den Baum zu putzen. Morgen warPRET Weih- nachten. (21) a. Aber am Vormittag hatte sie den Baum zu putzen. Morgen warPRET Weih- nachten. ›But in the morning she had to clean the tree. It was Christmas tomorrow.‹ [see Hamburger (31977, p. 65), citing from Die Bräutigame der Babette Bomberling ›The bridegrooms of Babette Bomberling‹ by Alice Berend] b. Mia lächelte. Morgen war Weihnachten. Mia smile:PRET tomorrow be:PRET Christmas (21) a. Aber am Vormittag hatte sie den Baum zu putzen. Morgen warPRET Weih- nachten. ›But in the morning she had to clean the tree. It was Christmas tomorrow.‹ [see Hamburger (31977, p. 65), citing from Die Bräutigame der Babette Bomberling ›The bridegrooms of Babette Bomberling‹ by Alice Berend] b. Mia lächelte. Morgen war Weihnachten. Mia smile:PRET tomorrow be:PRET Christmas ›Mia was smiling. It was Christmas tomorrow.‹ nachten. ›But in the morning she had to clean the tree. It was Christmas tomorrow.‹ [see Hamburger (31977, p. 65), citing from Die Bräutigame der Babette Bomberling ›The bridegrooms of Babette Bomberling‹ by Alice Berend] ›But in the morning she had to clean the tree. It was Christmas tomorrow.‹ [see Hamburger (31977, p. 65), citing from Die Bräutigame der Babette Bomberling ›The bridegrooms of Babette Bomberling‹ by Alice Berend] b. Mia lächelte. Morgen war Weihnachten. Mia smile:PRET tomorrow be:PRET Christmas ›Mia was smiling. It was Christmas tomorrow.‹ The argument is straightforward: The adverbial morgen ›tomorrow‹ locates Christ- mas in the future. If the preterit received its standard temporal interpretation, it should locate Christmas in the past and thus yield a contradiction. As no contradic- tion arises, the conclusion is that the preterit must receive a non-standard atemporal interpretation here. However, once a more nuanced view on the semantics of tem- poral information and perspective-taking is adopted, the given argument loses its validity; see, for instance, Rauh (1985) and Eckardt (2014, Chapter 4) for detailed discussions. In order to show why the examples are considered compatible with a purely temporal interpretation of the preterit, I will sketch an analysis of (21b) along the lines of Eckardt’s proposal. 2.3.1 Grammatical reasons for the feasibility of an atemporal preterit Generally, this option is due to the fact that, in contrast to tense, aspect is a shiftable indexical category; see Eckardt (2014) for an elaborate discussion of the past perfect as a trigger for free indirect discourse and the next section for further information on the sensitivity of aspect to the perspective of a protagonist.12 In sum, the preterit in fact supports the interpretation of ﬁctions where an imag- ined retrospective narrating situation is missing. This substantiates the assumption that the preterit can have an atemporal reading.13 13 According to a reviewer, the vernacular literature of Southern German generally makes use of the perfect instead of the preterit; the reviewer also notes that hab instead of habe for the ﬁrst-person singular of the auxiliary haben ›have‹ can be used in order to mark informal language. Given the general use of the perfect, one might speculate that the relevant vernacular literature always comes with the impression that there is someone narrating. Relatedly, Eckardt (2014, p. 91) claims that Southern German dialects do not support free indirect discourse, and that this restriction might be due to the fact that the dialects lack the preterit and the relevant elaborate system of tense and aspect of the standard variety. I would like to leave to future research whether these speculations are on the right track. It might of course also be that the relevant tenses and underlying grammatical systems cannot be compared in such a simple way. 49 Narration Without Narrating 2.3.2 Temporal adverbials and atemporal preterit (22) Morgen war Weihnachten. tomorrow be:PRET Christmas a. past tense Ý TR < TU: the narrator is talking about a past time TR. b. prospective aspect Ý TS > TR (and < TU): Christmas is after TR (and before TU). c. adverb morgen Ý TS is one day after TUprot; TUprot = TR: Christmas is one day after TUprot/TR (and before TU). 2.3.2 Temporal adverbials and atemporal preterit The proposal is based on the familiar distinction between tense and aspect on the one hand and well-known shifts between narrator perspective and protagonist perspective on the other. First, the preterit in (22) conveys its standard past meaning; see (22a). Accordingly, the narrator is talking about a reference time TR before her utterance time TU. Second, the preterit is assumed to be underspeciﬁed with regard to aspect (recall (15) from above). In this particular case, it can thus license prospective aspect, according to which the relevant situation time TS is located after TR (and before TU); see (22b). Finally, the proposal makes use of established facts about perspective-taking. Adverbials such as morgen are shiftable indexicals, which supports their interpretation relative to the thinking of a protagonist TUprot and the creation of free indirect discourse. Furthermore, following Doron (1991), free indirect discourse is characterized by the generalization that reference time TR and the protagonist’s thinking TUprot coincide; see (22c). (22) Morgen war Weihnachten. tomorrow be:PRET Christmas a. past tense Ý TR < TU: the narrator is talking about a past time TR. b. prospective aspect Ý TS > TR (and < TU): Christmas is after TR (and before TU). c. adverb morgen Ý TS is one day after TUprot; TUprot = TR: Christmas is one day after TUprot/TR (and before TU). 50 S. Bücking The overall result for (21b) is intuitively correct (see, however, below for quali- ﬁcations regarding the narrator). A narrator says about a time before her utterance time that Mia is smiling while thinking that it will be Christmas the next day. In a nutshell, the temporal contradiction invoked by Hamburger’s original argument does not arise because only tense is rigidly bound to the perspective of the narrator; aspect and adverbs, by contrast, can be bound to the perspective of a protagonist. The result, then, is that Hamburger’s prominent examples do not provide con- clusive evidence in favor of an atemporal preterit. Crucially, however, the relevant examples do not provide conclusive evidence against it either. Eckardt’s approach merely shows in which way future-oriented adverbials can be reconciled with a stan- dard temporal interpretation of the preterit; it does not show that this type of ap- proach is mandatory. 2.3.2 Temporal adverbials and atemporal preterit Speciﬁcally, only the reference time TR, corresponding to the time of Mia’s thinking TUprot, and the situation time TS of Christmas are necessary for a temporally adequate representation of the scenario under discussion. There is simply no intuition according to which recipients must imaginatively represent a retrospective narrating situation. Correspondingly, the core meaning of example (22) would also be captured without condition (22a) and, thus, without the standard temporal interpretation of the preterit. The obvious follow-up question is whether there are adverbial examples that could distinguish between both approaches at all. Examples with adverbials that are interpreted relative to a protagonist will be of no avail. The protagonist perspective is bound to aspect, which is underspeciﬁed in the case of the preterit and as such uninformative for the controversial part of the preterit’s contribution. It is more promising to consider the interaction between the preterit and temporal adverbials that must be interpreted relative to a narrator; see (23), where the relevant sentence is marked by italics. (23) Ben wachte früh morgens auf und machte sich an die Arbeit am Boot. Er hatte, seit er auf der Insel gestrandet war, jedes Zeitgefühl verloren. Morgen hattePRET er Geburtstag, ohne dass er davon wusstePRET. ›Ben woke up early in the morning and set to work on the boat. Since he had become stranded, he had lost all sense of time. It was his birthday tomorrow without his knowing it.‹ ›Ben woke up early in the morning and set to work on the boat. Since he had become stranded, he had lost all sense of time. It was his birthday tomorrow without his knowing it.‹ In the given context, the future-oriented adverbial morgen is interpreted relative to a narrator; nevertheless the preterit is used. This reading is at odds with the standard temporal approach to the preterit; see (24) with contradictory requirements: Ben’s birthday should be both before and after the narrating time TU. (24) Morgen hatte er Geburtstag. tomorrow have:PRET he birthday a. past tense Ý TR < TU: the narrator is talking about a past time TR. b. prospective aspect Ý TS > TR (and < TU): Ben’s birthday is after TR (and before TU). c. adverb morgen Ý TS is one day after TU: Ben’s birthday is one day after a. past tense Ý TR < TU: the narrator is talking about a past time TR. b. 2.4 Interim conclusions Eckardt (2015) argues that ﬁctional narratives invariably involve a narrating situ- ation with a narrator, and that any impression to the contrary is due to a lack of knowledge about these components. I have called attention to three problems with this approach. First, it remains unclear why the existence of an arbitrary narrator should create the impression of its nonexistence. Second, the narrating instance is potentially more abstract than predicted by the assumption that there is always an anthropomorphic narrator within the story being told. Third, it cannot be taken for granted that the preterit must relate utterance time and reference time and thereby make the existence of a narrating situation a logical necessity. In particular, evi- dence drawn from contrasts between the preterit and the present perfect and from the combinatorics of the preterit with temporal adverbials complies with the oppos- ing view that the preterit also licenses an atemporal use. In the following sections, I will tackle an alternative approach to narrator-neutrality in ﬁctional narratives and specify in more detail the use potential of the preterit. 2.3.2 Temporal adverbials and atemporal preterit prospective aspect Ý TS > TR (and < TU): Ben’s birthday is after TR (and before TU). c. adverb morgen Ý TS is one day after TU: Ben’s birthday is one day after TU. 51 Narration Without Narrating By contrast, the contradiction does not arise under an atemporal interpretation of the preterit. There could be a co-temporal narrator such that her reference time TR coincides with her utterance time TU; correspondingly, Ben’s birthday is simply located on the subsequent day. The example in (23), then, provides evidence in favor of the assumption that the preterit can receive atemporal readings. While I consider this line of thought sound, it comes with a ﬂaw. I speculate that it is more common for narratives to either not have a narrator (which goes well with an atemporal preterit) or to have a truly retrospective narrator (which goes well with a temporal preterit). Therefore, the peculiar conﬁguration that involves a co-temporal narrator and can thereby bring out a substantial difference of the standard approach to the preterit from Hamburger’s position is probably untypical. This calls into question whether the given argument is in fact fully reliable. (25) a. ›Imagine that a narrator narrates that ...‹ b. ›Imagine that ...‹ 14 I will also not address alternative approaches to the paradox here; see Maier (2017) for a detailed review of their merits and problems. 3 Outline of an imagination-based approach to speaker-neutrality Fictional narratives can be understood as invitations to imagine; see, in particular, Walton (1990) and the Institutional Theory of Fiction as summarized by Köppe/ Stühring (2011, Section 2). Against this background, Köppe/Stühring (2011, p. 74, p. 62) argue against the ubiquity of a narrator: »The question whether a text has a ﬁctional narrator comes down to whether the text authorizes imaginings about a ﬁctional narrator.« They thus distinguish between the two options in (25). They thus distinguish between the two options in (25). They thus distinguish between the two options in (25). 52 S. Bücking I will essentially follow their position here. In order to spell out its linguistic implementation, I will take advantage of formal tools as independently argued for by Maier (2017). The main concern of Maier (2017) is a new solution to a long-standing puzzle about the semantics of ﬁctional names, namely, the paradox that their referents can simultaneously be said to bear properties of individuals of ﬂesh and blood and to not exist; see the ﬁctional and the metaﬁctional statement in (26) for exempliﬁcation. (26) a. Frodo is a hobbit born in the Shire. b. Frodo is a ﬁctional character invented by Tolkien. b. Frodo is a ﬁctional character invented by Tolkien. 15 This is a strongly simpliﬁed assumption. Maier (2017) discusses in detail the interpretation of proper names that refer to individuals in the actual world without being perceptually anchored by most speakers. These details, which build on the classic analysis of proper names by Kripke (1980) via causal chains, do not matter for our discussion. 16 This does not mean that ﬁctional narratives single out just one particular context. For one, the succes- sively given information is surely not exhaustive. Furthermore, what kind of particular c emerges depends on the individual interpreter. There is also room for variation within interpreters once the dynamics of mental states during reception is taken into account. [see Maier 2017, (1)] [see Maier 2017, (1)] For our purposes, a very rough sketch of Maier’s solution sufﬁces.14 His solu- tion builds on a formalization of an imagination-based approach to ﬁction in terms of Discourse Representation Theory (= DRT; see Kamp/Reyle 2011 for a general overview of Discourse Representation Theory). In DRT, the mental states of in- terpreters form an integral part of the semantic representations for linguistic units. Correspondingly, the interpretation of a discourse is modeled by dynamic updates of mental representations. The integration of mental states is particularly evident in what Maier calls psychologistic versions of DRT, where mental states receive explicit structural representations; see Kamp (1990, 2015) for the relevant extension of standard DRT. Speciﬁcally, Maier (2017) takes advantage of the idea that the mental state of an interpreter uniﬁes distinct but interacting attitudes. Formally, each attitude description consists of a pair hATTITUDE; BOXi, where ATTITUDE labels the attitude under discussion and BOX represents its content in the form of the standard box notation for discourse representation structures. For (26), Maier proposes the set of attitude descriptions in (27). [see Maier 2017, (29)] The boxes can be read as usual: the upper part introduces existential quantiﬁcation over variables, while the lower part introduces conditions on these variables; for instance, the ﬁrst box thus says that there is a x such that x is an author with the name Tolkien. The structure of the mental state of the interpreter is given by the attitudes that embed the pieces of information as captured in the boxes. The ﬁrst attitude description, labeled ANCH, is a so-called internal anchor that describes in which mind-internal way an interpreter is acquainted with a discourse referent Narration Without Narrating 53 that is anchored to the external world and thus external to the mental state of the interpreter. The given anchor for Tolkien captures that he is known to interpreters by virtue of being an author.15 For the present discussion, the distinction between the second and the third attitude descriptions is the most important one. The attitude labeled IMAG comprises what interpreters imagine. Speciﬁcally, if interpreters accept a text they receive as ﬁction, they follow the invitation to imagine as suggested by Walton (1990) and successively update their IMAG-component. [see Maier 2017, (1)] In other words, the IMAG-component of a speaker-neutral ﬁctional narrative r existentially binds a context c, while it lacks the variables and conditions marked by pq in (28). From a grammatical perspective, the most urgent follow-up question is in which way the given approach can cover the obligatory contribution by tense, in particular, the contribution by the epic preterit. An answer will be pursued in the next section. However, before turning to the epic preterit, I will brieﬂy comment on a principled objection to the given approach raised by a reviewer. The reviewer argues that the reduction of ﬁctional narratives to an invitation to imagine is a clear oversimpli- ﬁcation. The approach would hardly do justice to the variety of narrative genres, narrative functions, and narrative instances. Speciﬁcally, the reviewer calls attention to narratives about real events that could be conceived of as invitations to imagine as well. While I agree with the reviewer that the given picture is simpliﬁed, I do not think that this simpliﬁcation disqualiﬁes the given approach from being useful. The main aim of this section is to highlight the principled merits of an approach to ﬁctional names and speaker-neutrality in terms of interacting attitude descriptions (in lieu of alternative formal frameworks). In order to abstract away from potential complexities, the given illustration intentionally builds on ordinary ﬁctional narra- tives; for such simple cases, it is sufﬁcient to distinguish between attitudes such as IMAG, BEL, and ANCH and to use coarse-grained predicates such as ›ﬁctional‹ or ›ﬁctional narrative‹. That said, it is surely legitimate to ask whether the given approach could handle other cases as well. I think that the model in fact has the relevant potential. The main reason for this belief is that the model is designed to capture potentially ﬁne-grained mental states. Let us, for instance, take a narrative for which an interpreter assumes that it is narrated by a real narrator and that it com- prises real events. In this case, neither the narrating situation including the narrator nor the given events should be bound within the IMAG-component. In lieu thereof, the relevant entities would be anchored to the external world of the interpreter or be bound to her belief; that is, the upper box of the discourse representation structure in the IMAG-component would remain empty. [see Maier 2017, (1)] This is the case for the ﬁctional statement in (26a); its representation in (27) says that the interpreter is authorized to imagine that there is a y and a z (compare the upper box in the discourse representation structure) and that y is called Frodo, is a hobbit, and is born in z called the Shire. The attitude labeled BEL comprises what interpreters believe to be true. Speciﬁcally, if interpreters receive ordinary assertions about the actual world and trust the respective speaker, they update their BEL-component. This is the case for the metaﬁctional statement in (26b); its representation in (27) says that interpreters are authorized to believe that y is ﬁctional and invented by x. Crucially, there is no existential quantiﬁcation over y (that is, Frodo) in the BEL-component (compare the lack of an upper box) capturing that interpreters do not subscribe to the contradictory belief that a non-existent entity exists. In a nutshell, metaﬁction is thus characterized by the fact that a referent described in the BEL-component is bound within the IMAG-component such that someone’s belief becomes dependent on imagination. Maier (2017) is not concerned with speaker-neutrality within ﬁctional narratives. However, his framework offers a straightforward approach to it. It simply suggests that optional existential binding within the IMAG-component represents whether a narrative licenses the ﬁction of a narrating situation with a narrator or not. Specif- ically, I suggest the schematic representation in (28), where pq marks optionality. According to the ANCH-component, there is an r that an interpreter considers as anchored to the actual external world by virtue of being a ﬁctional narrative about some context c. In contrast to the construal of narrative contents as sets of contexts (recall the set-up in Eckardt 2015), I thus assume that ﬁctional narratives describe particular contexts c. This is in line with the common intuition that recipients of a ﬁctional narrative have a particular context in mind during reception.16 Corre- 54 S. Bücking spondingly, c is not anchored to the external world, but existentially bound within the IMAG-component, which, in turn, comprises all information that the ﬁctional narrative provides about c. According to the set-up in (28), c can, but need not include a situation e of a narrator x narrating c. A ﬁctional narrative r about c can then be deﬁned as speaker-neutral iff c does not include a narrating situation e with a narrator x narrating c. 17 I thank Sarah Zobel for suggesting to me a systematic comparison between the epic preterit and fake tense. 18 Iatridou (2000, p. 246) follows the standard assumption that temporal tense is either past or present. The future, by contrast, is considered a basically modal category. This is why the temporal exclusion in (31a) does not yield a future reading. [see Maier 2017, (1)] One could also make more subtle distinctions by assigning some entities to the IMAG-component and others to ANCH or BEL. In fact, Maier (2017, pp. 23–24) already discusses the case of non-ﬁctional names in ﬁctional narratives. His example is the interpretation of Napoleon in Tol- stoj’s War and Peace; he proposes that the events in which Napoleon participates are bound within the IMAG-component, while Napoleon himself is interpreted via ANCH. I am agnostic as to the question of whether this approach to narratives that are (partially) about real events and real participants sufﬁces. Another option would be to distinguish between different kinds of imagination such that the attitudes them- selves come with different implications. In any case, a logical model that is sensitive to the mental states of interpreters seems to have the necessary expressive power. Narration Without Narrating 55 4 The semantics of the epic preterit Building on Hamburger (31977), Thieroff (1992), Welke (2005), and Bredel/Töpler (2007), I have argued in Section 2.3 that the preterit in German licenses an atempo- ral use where no anteriority relation is contributed to an implicit narrating situation. Recall that this is a logical prerequisite for an account of speaker-neutrality in terms of an optional narrating situation in the imagination component of an interpreter’s mental state. But what, then, is the semantics of this atemporal epic preterit? As already noted in Section 2.3, the most prominent claim is that the preterit in German conveys an underspeciﬁed distance feature that can be speciﬁed as either temporally distant or as distant from the actual world. The ﬁrst speciﬁcation yields the temporal past meaning; the second speciﬁcation is the crucial atemporal meaning, according to which the epic preterit marks an utterance as ﬁction. If applied to the imagination- based approach to ﬁction as outlined in Section 3, the epic preterit thus denotes the invitation to an update of the imagination component. As far as I see, however, the consequences of this intriguing claim have not been discussed in substantial depth in the previous literature. In Section 4.1, I will comply with this desideratum by evaluating it against the background of more general discussions about atemporal tense, so-called fake tense, within formal semantics.17 The upshot will be that the epic preterit cannot be treated as a marker for ﬁction. In Section 4.2, I will outline an alternative analysis according to which the preterit marks imagination from a dis- tance and thus instructs interpreters on how they should imagine the contents of the ﬁctional narrative they receive. 18 Iatridou (2000, p. 246) follows the standard assumption that temporal tense is either past or present. Th future, by contrast, is considered a basically modal category. This is why the temporal exclusion in (31 does not yield a future reading. 4.1 The epic preterit as fake tense? But she does not have a dog.‹ This is in line with the modal reading as sketched in (31b). On the one hand, the given semantics merely says that a CF of the form if p, q is about worlds that exclude the actual world; therefore, semantically speaking, the CF is not about the actual world w@ and thus does not make explicit whether p holds in w@ or not. On the other hand, the counterfactual intuition for CFs can easily be derived from (31b) by the standard pragmatic reasoning in (33); see Iatridou (2000, pp. 247-249). (33) Let a speaker S utter a CF if p, q. Then, if S believed that p is true in w@, S would not exclude w@ from the worlds she is talking about. As she does precisely this by using ExclF, she implicates that p is false in w@. (33) Let a speaker S utter a CF if p, q. Then, if S believed that p is true in w@, S would not exclude w@ from the worlds she is talking about. As she does precisely this by using ExclF, she implicates that p is false in w@. Against this background, one might suggest that the epic preterit agrees with the exclusion feature ExclF as well and thereby identiﬁes the ﬁction as such. In fact, Schulz (2014, p. 133) hints at this direction without, however, discussing a concrete proposal and its consequences. A rough sketch along the lines of Iatridou’s analysis for CFs could look as in (34). (34) a. The worlds that a given narrative is talking about exclude the worlds that for all we know are the worlds of the narrative (= w@). (34) a. The worlds that a given narrative is talking about exclude the worlds that for all we know are the worlds of the narrative (= w@). g g all we know are the worlds of the narrative (= w@). b. Implicature: The propositions p of the narrative (that is, its content) are false in w@ and thus ﬁction. b. Implicature: The propositions p of the narrative (that is, its content) are false in w@ and thus ﬁction. b. Implicature: The propositions p of the narrative (that is, its content) are false in w@ and thus ﬁction. 4.1 The epic preterit as fake tense? The discussion about fake tense has primarily evolved from the observation that, crosslinguistically, counterfactual conditionals (= CFs) make use of past-morphemes; see the examples in (29) from English. (29) a. If Mia hadPST a dog, she wouldFUT+PST be happy. (29) a. If Mia hadPST a dog, she wouldFUT+PST be happy. b. If Ben tookPST this syrup, he wouldFUT+PST get better. [see Iatridou 2000, (47a)/(8)] The standard analysis in formal semantics originates with Iatridou (2000) (see Schulz 2014 for reﬁnements and Romero 2014 for an alternative formal perspective). Its key ingredients are the following. First, the past-morpheme is assumed to agree with an exclusion feature ExclF as given in (30). Depending on whether the exclusion operates on the temporal or modal domain, ExclF gives rise to either the temporal and thus standard past reading in (31a) or to the modal and thus fake tense reading in (31b).18 56 S. Bücking (30) The x that the speaker is talking about excludes the x that for all we know is the x of the speaker; x can be times or worlds. (31) a. Temporal reading: The topic time excludes the time of the speaker (= TU). Ý standard past b. Modal reading: The topic worlds exclude the worlds of the speaker (= the actual world w@). Ý fake past b. Modal reading: The topic worlds exclude the worlds of the speaker (= the actual world w@). Ý fake past [see Iatridou 2000, (52)/(57)] Second, the interpretation of CFs builds on the modal reading in (31b) in the following way. A crucial trait of CFs of the form if p, q is that they pragmatically implicate (in other words, suggest) that p is false without, however, semantically entailing it. For instance, as shown by (32), the counterfactual implicature of CFs can be made explicit without being redundant (which is a standard test for implicatures in pragmatic analyses following Grice 1975). (32) Wenn Mia einen Hund hätte, wäre sie glücklich. Aber sie if Mia a dog have:PST:SBJV be:PST:SBJV she happy but she hat keinen Hund. have:PRS no dog ›If Mia had a dog, she would be happy. But she does not have a dog.‹ (32) Wenn Mia einen Hund hätte, wäre sie glücklich. Aber sie if Mia a dog have:PST:SBJV be:PST:SBJV she happy but she hat keinen Hund. have:PRS no dog ›If Mia had a dog, she would be happy. 4.1 The epic preterit as fake tense? The given suggestion thus seems to pave the way for a precise and systematic take on the contribution of the atemporal preterit in ﬁctional narratives. However, for reasons to be discussed next, I consider the presumptive close link between the epic preterit and the marking of ﬁction ﬂawed in fundamental ways. 57 Narration Without Narrating First, there is a morphological problem. The contrast in (35) shows that CFs in German require additional subjunctive marking, while the epic preterit prohibits this.19 (35) a. Wenn Mia den Sirup nähme, (35) a. Wenn Mia den Sirup nähme, würde es ihr besser if Mia the syrup take:PST:SBJV AUX:PST:SBJV it her better gehen. go ›If Mia took the syrup, she would get better.‹ b. #Mia säße am Fenster und würde seufzen. Es Mia sit:PST:SBJV at the window and AUX:PST:SBJV sigh it würde regnen. AUX:PST:SBJV rain ›Mia would be sitting at the window sighing. It would be raining.‹ (35) a. Wenn Mia den Sirup nähme, würde es ihr besser if Mia the syrup take:PST:SBJV AUX:PST:SBJV it her better gehen. go ›If Mia took the syrup, she would get better.‹ b #Mi ß F d d f E ›If Mia took the syrup, she would get better.‹ b. #Mia säße am Fenster und würde seufzen. Es Mia sit:PST:SBJV at the window and AUX:PST:SBJV sigh it würde regnen. AUX:PST:SBJV rain ›Mia would be sitting at the window sighing. It would be raining. ›Mia would be sitting at the window sighing. It would be raining.‹ ›Mia would be sitting at the window sighing. It would be raining.‹ In order to predict that only CFs require the subjunctive, one might argue that the exclusion feature ExclF agrees with the subjunctive only as a local sentential feature, but not as a global discourse feature. However, this assumption would at the same time undermine the crucial idea that ExclF can agree with the epic preterit. If one conceived of ExclF as a global discourse feature for the identiﬁcation of ﬁction, it could not interact with this local morphological category either. The use of present tense reveals a second problem. Iatridou (2000, pp. 252-253) argues that the present supports a modal reading as well. This fake present differs from the fake past by marking the lack of exclusion. 19 In the analysis by Iatridou (2000), the subjunctive marking in CFs is a well-formedness condition that is semantically irrelevant. This background does not affect the problem that I raise here. 20 The beliefs of authors play a role in determining implicit truths in ﬁctional narratives; see Bonomi/ Zucchi (2003) for discussion and the following example from their work for illustration. Let there be a realistic contemporary novel where nothing is explicitly said about whether the world is ﬂat or round. Readers will usually assume that the world is round in this novel, which follows from the assumption that the author shares this common knowledge about the actual world and implicitly attributes it to the invented story as well. Notably, however, this reasoning concerns the derivation of ﬁctional truths based on w@; it does not concern truths in w@ as derived from ﬁctional truths. 4.1 The epic preterit as fake tense? By using a corresponding example such as (36), a speaker thus conveys that she does not exclude the actual world w@ from the worlds she is talking about. (36) Wenn Mia den Sirup nimmt, wird es ihr besser gehen. if Mia the syrup take:PRS AUX:PRS it her better go ›If Mia takes the syrup, she will get better.‹ (36) Wenn Mia den Sirup nimmt, wird es ihr besser gehen. if Mia the syrup take:PRS AUX:PRS it her better go The problem now is this: If the epic preterit were an instance of fake past, the epic present should also be an instance of fake present. Therefore, it should also mark the lack of exclusion. However, the use of present tense in ﬁctional narratives does not suspend the ﬁction; for instance, despite the use of the present, (37) can still be a regular ﬁctional discourse that excludes w@ from the worlds it is about. (37) Mia sitzt am Fenster und seufzt. Es regnet ... Mia sit:PRS at the window and sigh:PRS it rain:PRS ... ›Mia is sitting at the window sighing. It is raining ...‹ ›Mia is sitting at the window sighing. It is raining ...‹ In other words, it is probably impossible to derive whether a text is ﬁction or not from its morphological features (at least for German). A third problem is that fake past and ﬁction instantiate very different types of distance from the actual world. Fake past conveys a distance from w@ that pertains 58 S. Bücking to a speaker’s belief about w@ and that is gradual due to uncertainties of the speaker (see Schulz 2014 for details on the role of epistemically optimal worlds in CFs). Fiction, by contrast, conveys a distance from w@ that does not pertain to the belief (of the author) about w@ in a comparable way. The distance in this case is categorical, as the ﬁction is not the reality, independently of whether the ﬁctional propositions are true in w@ or not.20 to a speaker’s belief about w@ and that is gradual due to uncertainties of the speaker (see Schulz 2014 for details on the role of epistemically optimal worlds in CFs). Fiction, by contrast, conveys a distance from w@ that does not pertain to the belief (of the author) about w@ in a comparable way. 21 Two remarks are in order here. First, I am not saying that ﬁctional narratives are always consistent. In particular, there can be unreliable narration. However, corresponding inconsistencies are not due to the direct juxtaposition of statements, as crucial for (38) and (39). Second, examples such as (39) can be felicitous in cases with stacked ﬁctions; see also the discussion of metaleptic narratives of various forms. For instance, the following example suggests an analysis in terms of ﬁction within ﬁction. (i) I have spoken of a voice telling me things. [...] It told me to write the report. Does this mean I am freer now than I was? I do not know. I shall learn. Then I went back into the house and wrote, It is midnight. The rain is beating on the windows. It was not midnight. It was not raining. 20 The beliefs of authors play a role in determining implicit truths in ﬁctional narratives; see Bonomi/ Zucchi (2003) for discussion and the following example from their work for illustration. Let there be a realistic contemporary novel where nothing is explicitly said about whether the world is ﬂat or round. Readers will usually assume that the world is round in this novel, which follows from the assumption that the author shares this common knowledge about the actual world and implicitly attributes it to the invented story as well. Notably, however, this reasoning concerns the derivation of ﬁctional truths based on w@; it does not concern truths in w@ as derived from ﬁctional truths. 21 Two remarks are in order here. First, I am not saying that ﬁctional narratives are always consistent. In particular, there can be unreliable narration. However, corresponding inconsistencies are not due to the direct juxtaposition of statements, as crucial for (38) and (39). Second, examples such as (39) can be felicitous in cases with stacked ﬁctions; see also the discussion of metaleptic narratives of various forms. For instance, the following example suggests an analysis in terms of ﬁction within ﬁction. 20 The beliefs of authors play a role in determining implicit truths in ﬁctional narratives; see Bonomi/ Zucchi (2003) for discussion and the following example from their work for illustration. Let there be a realistic contemporary novel where nothing is explicitly said about whether the world is ﬂat or round. Readers will usually assume that the world is round in this novel, which follows from the assumption that the author shares this common knowledge about the actual world and implicitly attributes it to the invented story as well. Notably, however, this reasoning concerns the derivation of ﬁctional truths based on w@; it does not concern truths in w@ as derived from ﬁctional truths. 21 Two remarks are in order here. First, I am not saying that ﬁctional narratives are always consistent. In particular, there can be unreliable narration. However, corresponding inconsistencies are not due to the direct juxtaposition of statements, as crucial for (38) and (39). Second, examples such as (39) can be felicitous in cases with stacked ﬁctions; see also the discussion of metaleptic narratives of various forms. For instance, the following example suggests an analysis in terms of ﬁction within ﬁction. (i) I have spoken of a voice telling me things. [...] It told me to write the report. Does this mean I am freer now than I was? I do not know. I shall learn. Then I went back into the house and wrote, It is midnight. The rain is beating on the windows. It was not midnight. It was not raining. (End of Beckett, Molloy). However, the comparison between (38) and (39) above is based on the assumption that they do not (End of Beckett, Molloy). 4.1 The epic preterit as fake tense? other words, taken as a sincere response, the text-external objection by A leads to the conclusion that A misconceives the crucial point of ﬁction. (40) Kohl seufzte. Er hatte von Napoleon geträumt. Kohl sigh:PRET he have:PST about Napoleon dream:PTCP ›Kohl sighed. He had dreamed about Napoleon.‹ (41) a. A: »How should one know? That’s nonsense!« b. B: »Eh? But that doesn’t matter; it’s ﬁction!« Another difference arises with regard to the handling of factual information. CFs are infelicitous in contexts where the speaker knows that the antecedent is true, as in (42). (42) a. Context: Speaker ﬁnds out that Mia has a dog: wäre if Mia a dog have:PST:SBJV be:PST:SBJV she happy ›If Mia had a dog, she would be happy.‹ This is again different for ﬁctional narratives. They can integrate facts of the actual world. The transfer to ﬁction just renders the distinction between ±factual essentially irrelevant. For instance, if (43) is used as part of a newspaper report, ±factual in the actual world is surely relevant. If the same sentence is used as part of a ﬁctional narrative, its being true or false in the actual world does not bear on the felicity conditions of the ﬁction. That is, the ﬁction would clearly not become infelicitous once we must assume that the author in fact knows that Kohl went up to Mitterand. (43) Kohl ging auf {Mitterand / Napoleon} zu. Koch go:PRET to {Mitterand / Napoleon} up ›Kohl went up to {Mitterand / Napoleon}.‹ Kohl went up to {Mitterand / Napoleon}.‹ Finally, the standard temporal reading of the preterit has different effects on conditionals and ﬁction. In conditionals, the use of a temporal preterit yields so- called factual conditionals, as in (44). Factual conditionals do not exclude the actual world from the worlds they are about; the temporal reading thus automatically suspends the modal effect of CFs, which is expected, as the exclusion feature cannot operate on both the temporal and the modal domain. (44) Wenn Ben gestern in Rom war, ist er vermutlich heute if Ben yesterday in Rome be:PRET be:PRS he presumably today in Mailand. in Milan ›If Ben was in Rome yesterday, he is presumably in Milan today.‹ ›If Ben was in Rome yesterday, he is presumably in Milan today.‹ By contrast, the example in (45) shows that a temporal reading of the preterit within ﬁction does not suspend the ﬁction. 4.1 The epic preterit as fake tense? The distance in this case is categorical, as the ﬁction is not the reality, independently of whether the ﬁctional propositions are true in w@ or not.20 This intuitive distinction between both types of distance can be conﬁrmed by several linguistic observations. For one, recall that the counterfactual implicature introduced by CFs can be made explicit, as in (38) (= (32)). (38) Wenn Mia einen Hund hätte, wäre sie glücklich. Aber sie if Mia a dog have:PST:SBJV be:PST:SBJV she happy but she hat keinen Hund. have:PRS no dog ›If Mia had a dog she would be happy But she does not have a dog ‹ ›If Mia had a dog, she would be happy. But she does not have a dog.‹ Fictional narratives, however, cannot create a true alternative to the ﬁction within the ﬁction. In sharp contrast to (38), examples such as (39) are clearly inconsistent.21 (39) #Mia saß am Fenster und seufzte. Aber Mia {sitzt / saß} Mia sit:PRET at the window and sigh:PRET but Mia {sit:PRS / sit:PRET} nicht am Fenster und {seufzt / seufzte}. not at the window and {sigh:PRS / sigh:PRET} ›Mia was sitting at the window sighing. But Mia {is / was} not sitting at the window sighing.‹ ›Mia was sitting at the window sighing. But Mia {is / was} not sitting at the window sighing.‹ Furthermore, text-external objections to some ﬁctional truth are infelicitous as well. For instance, if A and B received a ﬁctional discourse such as (40) and A responded to this by (41a), B would probably be irritated and thus utter (41b). In (i) I have spoken of a voice telling me things. [...] It told me to write the report. Does this mean I am freer now than I was? I do not know. I shall learn. Then I went back into the house and wrote, It is midnight. The rain is beating on the windows. It was not midnight. It was not raining. (End of Beckett, Molloy). However, the comparison between (38) and (39) above is based on the assumption that they do not involve such stacked discourses. Narration Without Narrating 59 other words, taken as a sincere response, the text-external objection by A leads to the conclusion that A misconceives the crucial point of ﬁction. 4.1 The epic preterit as fake tense? Speciﬁcally, the temporal forms in the 60 S. Bücking second clause are related to Ben’s past and thus interpreted in a temporal way. However, this does not affect the ﬁctional character of the narrative as a whole.22 second clause are related to Ben’s past and thus interpreted in a temporal way. However, this does not affect the ﬁctional character of the narrative as a whole.22 (45) Ben dachte über den Tag zuvor nach. Nachdem er geputzt Ben think:PRET about the day before after he clean:PTCP hatte, war er im Garten. Dann ... have:PST be:PRET he in the garden then ... ›Ben was thinking about the day before. After he had ﬁnished cleaning, he was in the garden. Then ...‹ ›Ben was thinking about the day before. After he had ﬁnished cleaning, he was in the garden. Then ...‹ In sum, upon closer scrutiny, it turns out to be false that there is a close link between the epic preterit as a local morphological category and the marking of ﬁction as a global feature of a narrative as a whole. 4.2 The epic preterit as an instruction to imagine from a distance The previous section has shown that the epic preterit does not contribute a distance feature that marks ﬁctionality. In light of the imagination-based approach to ﬁctional narratives as sketched in Section 3, I propose as an alternative that the epic preterit marks imagination from a distance. That is, it instructs an interpreter of a ﬁctional sentence to imagine the situation that the sentence is about in a way as if she were not part of this situation. To illustrate, let (46) be the beginning of a speaker-neutral narrative that an interpreter is reading and that she acknowledges as ﬁction. (46) Ben saß am Schreibtisch. Es regnete. Ben sit:PRET at the desk it rain:PRET ›Ben was sitting at the desk. It was raining.‹ (46) Ben saß am Schreibtisch. Es regnete. Ben sit:PRET at the desk it rain:PRET ›Ben was sitting at the desk. It was raining.‹ According to the imagination-based approach to ﬁction, the interpreter is invited to imagine a context with a topic situation in which there is a person called Ben sitting at a desk while it is raining. The proposed semantics of the epic preterit adds to this the constraint that the interpreter should not imagine being part of this topic situation. In other words, the interpreter remains an observer from the outside of the situation that she is invited to imagine. More formally, this can be implemented by the representation in (47). 22 It, of course, bears on what is considered true within the ﬁction. But this is not relevant for the given argument. 61 Narration Without Narrating The ANCH-component in this case captures that the narrative r about context c is anchored to the actual world in virtue of being read by an interpreting I (= i) at her now (= ni). As outlined in Section 3, c is existentially bound to the IMAG- component. As we assume that (46) is a speaker-neutral narrative, c does not contain any narrator-related pieces of information (that is, it lacks the components of (28) that are marked by pq). However, it contains the following information. There is a topic situation s that is part of c, an event e0 of some y called Ben sitting at the desk, and a raining event e00. 4.2 The epic preterit as an instruction to imagine from a distance Furthermore, the time of the topic situation s* is a subinterval of both event times, which follows from the plausible assumption that the aspect in (46) is imperfective. The crucial condition is the ﬁnal condition, which captures the semantics of tense as contributed by the preterit. This condition says that, semantically, the preterit contributes distance between two underspeciﬁed variables, namely, a variable f (s) that is determined by a variable function f applied to the topic situation s and an indexical variable vindex. This echoes the standard assumption that tense relates a topic constituent to an indexical. The variables must be speciﬁed by context-dependent conceptual means, which I will discuss in more detail next. I suggest that there are two principled options for the speciﬁcation, as sketched in (48). In a nutshell, the temporal preterit is based on distance in the temporal domain, while the atemporal preterit is based on distance in the spatial domain.23 (48) a. distal(f(s), vindex) Ý <(τ(s), ni) (temporal preterit) b. distal(f(s), vindex) Ý ¬overlap(σ(s), pi) (atemporal preterit) (48) a. distal(f(s), vindex) Ý <(τ(s), ni) (temporal preterit) b. distal(f(s), vindex) Ý ¬overlap(σ(s), pi) (atemporal preterit) (temporal preterit) (atemporal preterit) The option in (48a) corresponds to the usual temporal reading of the preterit. The distance is resolved to temporal precedence <, f is the time function singling out the topic time, and vindex is identiﬁed with a relevant now. Usually, this now would be the time of a speaker’s utterance. That is, if the ﬁctional narrative licensed a narrator in the IMAG-component, the now would be identiﬁed with the narrating time, yielding the standard conception of ﬁctional narratives with a retrospective narrator. For (47), however, there is no narrating situation in the IMAG-component (as we assume a speaker-neutral ﬁctional discourse). Therefore, the only indexical time available is the now of the interpreter ni. The interpreter would thus be instructed to imagine that the topic situation the narrative is about occurred before her actual now while reading. I assume that this is a particularly feasible option for ﬁctional narratives that are clearly related to former times. In many cases, however, the relation of the ﬁctional content to the actual timeline does not play any crucial role; as a consequence, the temporal reading in (48a) is not used. The alternative option in (48b) implements the idea that the preterit can license imagination from a distance without involving time features. 23 I thank Claudia Maienborn for a query about a slightly different speciﬁcation of the atemporal preterit in an earlier version of this paper. This query inspired me to use explicitly spatial notions for the speciﬁcation in (48b) and to thereby highlight the principled analogy between both readings. 4.2 The epic preterit as an instruction to imagine from a distance Its set-up is analogous to the temporal reading, but it operates on spatial instead of temporal parameters. Speciﬁcally, the distance is resolved to the negation of a spatial overlap. Corre- 62 S. Bücking spondingly, σ is the space function singling out the space that is occupied by the topic situation s*, and vindex is identiﬁed with the place pi where the interpreter i herself is located. As desired, the interpreter is thus instructed to look at the topic situation from the perspective of a physically distant observer who is not partic- ipating in this situation. Notably, according to this speciﬁcation, the preterit has a very light function. It merely concerns a certain perspective on the scenario that is described. Furthermore, I consider this perspective the unmarked case in ﬁctional narratives, simply because the interpreter usually takes for granted that she is not part of ﬁctional situations. The given analysis has the following merits. The effects of the preterit are kept within the scope of the imagination component. This is in line with the observations in Section 4.1, according to which the preterit is a local morphological category that does not bear on the global question of whether a narrative is ﬁctional or not. In particular, the analysis thus also complies with temporal readings of the preterit within the ﬁction. In addition, it inspires a promising take on the effects of present tense within ﬁction. Recall from Section 4.1 that the use of an atemporal present in a narrative does not suspend its ﬁction, which is arguably a major obstacle to the idea that the atemporal preterit marks ﬁction. By contrast, the imagination-based approach to the atemporal preterit suggests a plausible analysis of the atemporal present as well. It simply instructs an interpreter to imagine the relevant topic situations not from a distance, but in a way as if she were part of them. In other words, the interpreter is invited to experience the situations that are described from their inside. This complies with the common intuition that the use of present tense in narratives creates the impression of immediate perception. 5 Conclusion This paper has been concerned with the distinction between narrator-creating and narrator-neutral ﬁctional narratives from a linguistic perspective. As a ﬁrst step, I have argued against the proposal by Eckardt (2015), according to which ﬁctional narratives always involve a narrating situation with a narrator, while the impression of narrator-neutral narration can be traced back to a lack of knowledge about this sit- uation. Three problems have been presented. For one, it remains unclear why a lack of knowledge about the narrator should advance the impression of its nonexistence. Furthermore, the narrating instance is potentially abstract and thus at odds with the role of an ordinary speaker within the story. Finally, the preterit in German can receive an atemporal interpretation along the lines of the epic preterit as introduced by Hamburger (31977), which avoids the logical need for a narrating time and thus a narrator within the ﬁction. As a second step, I have proposed an imagination-based account of narrator- neutral narration. This alternative account combines ideas from the Institutional Theory of Fiction as defended by Walton (1990) and Köppe/Stühring (2011) with the formal tools of Attitude Description Theory as developed by Maier (2017). Correspondingly, ﬁctional narratives are conceived of as invitations to an update of the imagination component of the mental state of an interpreter. The distinc- Narration Without Narrating 63 tion between narrator-creating and narrator-neutral narration is captured by optional existential binding of a narrating situation and a narrator within this imagination component. tion between narrator-creating and narrator-neutral narration is captured by optional existential binding of a narrating situation and a narrator within this imagination component. As a third step, I have tackled the grammatically crucial follow-up question of which role the preterit in ﬁctional narratives plays for the proposed account. I have conﬁrmed the general idea that the preterit in German contributes a distance feature that can be resolved either temporally or atemporally. However, in contrast to the suggestion by traditional German linguistics, the atemporal reading of the preterit cannot be treated as a grammatical marker for ﬁction. Speciﬁcally, as shown by a detailed comparison between the epic preterit on the one hand and fake tense as known from counterfactuals on the other, ﬁctionality should be considered a global text feature that does not interact in any reliable semantic way with local mor- phology. 5 Conclusion Finally, I have proposed an alternative imagination-based approach to the contribution of the epic preterit. In lieu of marking the ﬁction, it marks the way in which the contents of a ﬁctional narrative should be imagined, namely, it instructs interpreters to imagine the situations the narrative is about from the perspective of a distant observer. Acknowledgements This research was supported by the Deutsche Forschungsgemeinschaft (project A1 of the CRC 833 ›The Construction of Meaning‹, ID 75650358). I very much thank the anonymous reviewer, the editors of LiLi, and Claudia Maienborn for their helpful comments on earlier versions of the present paper. Parts of this work were presented at »Event Semantics 19« in Berlin (November, 2019). I thank the audience for their valuable feedback. Funding Open Access funding enabled and organized by Projekt DEAL. Funding Open Access funding enabled and organized by Projekt DEAL. 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 in- cluded in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. 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https://openalex.org/W3159736346	https://iris.unige.it/bitstream/11567/1046858/1/Monteleone%20et%20al%2c%20antioxidants-10-00691.pdf	English	null	PKCα Inhibition as a Strategy to Sensitize Neuroblastoma Stem Cells to Etoposide by Stimulating Ferroptosis	Antioxidants	2,021	cc-by	18,160	6 Department of Molecular Medicine, University of Padua, 35122 Padua, Italy; antonella.roveri@unipd.it (A.R.); fulvio.ursini@unipd.it (F.U.) Citation: Monteleone, L.; Speciale, A.; Valenti, G.E.; Traverso, N.; Ravera, S.; Garbarino, O.; Leardi, R.; Farinini, E.; Roveri, A.; Ursini, F.; et al. PKCα Inhibition as a Strategy to Sensitize Neuroblastoma Stem Cells to Etoposide by Stimulating Ferroptosis. Antioxidants 2021, 10, 691. https://doi.org/ 10.3390/antiox10050691 Academic Editor: José M. Matés Received: 29 March 2021 Accepted: 24 April 2021 Published: 28 April 2021 Citation: Monteleone, L.; Speciale, A.; Valenti, G.E.; Traverso, N.; Ravera, S.; Garbarino, O.; Leardi, R.; Farinini, E.; Roveri, A.; Ursini, F.; et al. PKCα Inhibition as a Strategy to Sensitize Neuroblastoma Stem Cells to Etoposide by Stimulating Ferroptosis. Antioxidants 2021, 10, 691. https://doi.org/ 10.3390/antiox10050691 Academic Editor: José M. Matés Received: 29 March 2021 Accepted: 24 April 2021 Published: 28 April 2021 Citation: Monteleone, L.; Speciale, A.; Valenti, G.E.; Traverso, N.; Ravera, S.; Garbarino, O.; Leardi, R.; Farinini, E.; Roveri, A.; Ursini, F.; et al. PKCα Inhibition as a Strategy to Sensitize Neuroblastoma Stem Cells to Etoposide by Stimulating Ferroptosis. Antioxidants 2021, 10, 691. https://doi.org/ 10.3390/antiox10050691 Academic Editor: José M Matés 7 Laboratory of Clinical and Experimental Immunology, Integrated Department of Services and Laboratories, IRCCS Istituto G. Gaslini, 35122 Genoa, Italy y * Correspondence: barbara.marengo@unige.it (B.M.); cinzia.domenicotti@unige.it (C.D.); Tel.: +39-010-3538831 (B.M.); +39-010-3538830 (C.D.) Tel.: +39-010-3538831 (B.M.); +39-010-3538830 (C.D.) † These authors contributed equally to this work. † These authors contributed equally to this work. Abstract: Cancer stem cells (CSCs) are a limited cell population inside a tumor bulk characterized by high levels of glutathione (GSH), the most important antioxidant thiol of which cysteine is the limiting amino acid for GSH biosynthesis. In fact, CSCs over-express xCT, a cystine transporter stabilized on cell membrane through interaction with CD44, a stemness marker whose expression is modulated by protein kinase Cα (PKCα). Since many chemotherapeutic drugs, such as Etoposide, exert their cytotoxic action by increasing reactive oxygen species (ROS) production, the presence of high antioxidant defenses confers to CSCs a crucial role in chemoresistance. In this study, Etoposide- sensitive and -resistant neuroblastoma CSCs were chronically treated with Etoposide, given alone or in combination with Sulfasalazine (SSZ) or with an inhibitor of PKCα (C2-4), which target xCT directly or indirectly, respectively. Both combined approaches are able to sensitize CSCs to Etoposide by decreasing intracellular GSH levels, inducing a metabolic switch from OXPHOS to aerobic glycolysis, down-regulating glutathione-peroxidase-4 activity and stimulating lipid peroxidation, thus leading to ferroptosis. Our results suggest, for the ﬁrst time, that PKCα inhibition inducing ferroptosis might be a useful strategy with which to ﬁght CSC chemoresistance. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Keywords: cancer stem cells; chemoresistance; glutathione; lipid peroxidation; ZEB-1; GPX4; ferroptosis Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Article PKCα Inhibition as a Strategy to Sensitize Neuroblastoma Stem Cells to Etoposide by Stimulating Ferroptosis Lorenzo Monteleone 1,†, Andrea Speciale 2,† , Giulia Elda Valenti 1 , Nicola Traverso 1, Silvia Ravera 3 , Ombretta Garbarino 4, Riccardo Leardi 5, Emanuele Farinini 5, Antonella Roveri 6 , Fulvio Ursini 6, Claudia Cantoni 1,7 , Maria Adelaide Pronzato 1, Umberto Maria Marinari 1, Barbara Marengo 1,,† and Cinzia Domenicotti 1,,† 1 General Pathology Section, Department of Experimental Medicine, University of Genoa, 16126 Genoa, Italy; lorenzo.monteleone@edu.unige.it (L.M.); giuliaelda.valenti@edu.unige.it (G.E.V.); nicola.traverso@unige.it (N.T.); claudia.cantoni@unige.it (C.C.); maidep@unige.it (M.A.P.); umm@unige.it (U.M.M.) 1 General Pathology Section, Department of Experimental Medicine, University of Genoa, 16126 Genoa, Italy; lorenzo.monteleone@edu.unige.it (L.M.); giuliaelda.valenti@edu.unige.it (G.E.V.); nicola.traverso@unige.it (N.T.); claudia.cantoni@unige.it (C.C.); maidep@unige.it (M.A.P.); umm@unige.it (U.M.M.) g ( ) 2 Mutagenesis and Cancer Prevention Unit, IRCCS Ospedale Policlinico San Martino, 16132 Genoa, Italy; andrea.speciale@hsanmartino.it 2 Mutagenesis and Cancer Prevention Unit, IRCCS Ospedale Policlinico San Martino, 16132 Genoa, Italy; andrea.speciale@hsanmartino.it 2 Mutagenesis and Cancer Prevention Unit, IRCCS Ospedale Policlinico San Martino, 16132 Genoa, Italy; andrea.speciale@hsanmartino.it 3 Human Anatomy Section, Department of Experimental Medicine, University of Genoa, 16126 Genoa, Italy; silvia.ravera@unige.it 4 Department of Cardiovascular Medicine, IRCCS Humanitas Clinical and Research Center, Rozzano, 20089 Milan, Italy; ombretta.garbarino@humanitasresearch.it 5 Department of Pharmacy, University of Genoa, 16126 Genoa, Italy; riclea@difar.unige.it (R.L.); farinini@difar.unige.it (E.F.) antioxidants antioxidants antioxidants antioxidants 1. Introduction Neuroblastoma (NB), a pediatric cancer originating from the neural crest cells, ac- counts for about 6% of all cancers in children [1]. The development of the neural crest is correlated with the epithelial–mesenchymal transition (EMT) [2,3], a phenomenon that is strictly related to MYCN ampliﬁcation [4], a negative prognostic factor that characterizes advanced tumor stages and aggressive progression of NB [5,6]. https://www.mdpi.com/journal/antioxidants Antioxidants 2021, 10, 691. https://doi.org/10.3390/antiox10050691 2 of 22 Antioxidants 2021, 10, 691 Several studies have reported that the genes involved in the EMT process are also implicated in regulating cancer stem cell (CSC) generation and propagation [7]. CSCs are a limited cell population found in different tumors, including NB [8], and are involved in various stages of the tumorigenic process and in cancer response to therapy [9–11]. In this regard, it has been demonstrated that CSCs, having physiologically low levels of reactive oxygen species (ROS) and high antioxidant defenses in comparison to cancer cells, are strongly involved in chemoresistance [12]. The maintenance of these low ROS levels is due to the ability of CSCs to synthesize high amounts of glutathione (GSH) by over-expressing xCT, a membrane transporter able to modulate the intracellular levels of cysteine, the limiting amino acid involved in GSH synthesis [13]. Moreover, it has also been found that CSCs express high levels of CD44, a well-known stemness marker [14] that is able to induce xCT membrane stabilization [15] and whose expression is modulated by protein kinase C α (PKCα) [16,17]. Based on these considerations, anti-tumor research should be focused on discovering innovative approaches able to limit GSH availability in cancer cells and, in particular, in CSCs. In fact, it has been shown that L-Buthionine-sulfoximine (BSO), a GSH-depleting agent, sensitizes NB cells to chemotherapeutic drugs [18–21] and CSCs to radiotherapy [13], by inducing ROS over-production. However, the clinical use of BSO is not actually feasible due to the secondary toxic effects of this compound, in addition to those resulting from the use of traditional radiation and drug therapies [22]. Therefore, in order to investigate new nontoxic therapeutic approaches, both Etoposide- sensitive and Etoposide-resistant NB-derived CSCs were long-term treated with Etopo- side, alone or in combination with sulfasalazine (SSZ), or with an inhibitor of the PKCα- dependent pathway (C2-4, a peptide inhibitor), which can target xCT directly orindirectly, respectively. 2.1. Chemicals Etoposide was purchased from Calbiochem (Merk KGaA, Darmstadt, Germany) and xCT inhibitor sulfasalazine (SSZ) and PKCα peptide inhibitor (C2-4) from Cayman Chemical (Ann Arbor, MI, USA). The stock solutions of these compounds were prepared using dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) as a solvent. 1. Introduction The herein results demonstrate that both combinations of Etoposide with SSZ or C2-4 efﬁciently counteract the propagation of Etoposide-resistant CSCs by preventing EMT transition and by triggering ferroptosis via down-regulation of GPX4 activity. 2.4. Immunoﬂuorescence and Cytoﬂuorimetric Analysis In order to investigate the expression of CD44, Oct3/4, CD44v9, xCT, and vimentin (used as a control of cell permeabilization), cells were analyzed by ﬂow cytometry. In detail, both untreated CSC populations were collected, centrifuged (117 rcf × 8 min), and then re-suspended in the TrypLE ™Express Enzyme 1X solution (Invitrogen, Carlsbad, CA, USA). After monitoring CSC disaggregation by microscope analysis, cells were diluted in PBS (137 mM NaCl, 2.6 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4). For intracellular staining, prior to incubation with primary mAbs, cells were permeabilized and ﬁxed for 30 min at 4 ◦C with the BD Cytoﬁx/Cytoperm ™kit (BD Biosciences, Franklin Lakes, NJ, USA). After this step, CSCs were re-suspended in 1% PBS/Saponin solution (Sigma-Aldrich, Milano, Italy) and then centrifuged (365 rcf × 10 min). Subsequently, CSCs were incubated for 30 min at 4 ◦C with primary antibodies: rabbit polyclonal anti-CD44 (Cosmo Bio, Tokio, Japan), rat monoclonal anti-Oct3/4 (EM92, PE-linked; eBioscience, San Diego, CA, USA), rat monoclonal anti-CD44v9 (RV3, Cosmo Bio), rabbit polyclonal anti-xCT (H-121, Santa Cruz Biotechnology, Dallas, TX, USA), and mouse monoclonal anti-vimentin (V9, Santa Cruz Biotechnology). gy Labelled cells were then washed with 2% FBS solution in PBS and incubated for 30 min at 4 ◦C with the appropriate isotype-matched secondary antibodies: goat anti-rabbit- IgG-PE (Southern Biotech, Birmingham, AL, USA), goat anti-rat-IgG-FITC (Sigma-Aldrich), goat anti-mouse-IgG-PE (Southern Biotech). Finally, all samples were ﬁxed with PBS/1% formaldehyde and analyzed with the FACScalibur ﬂow cytometer (BD Biosciences). Data were analyzed by the CellQuest software (BD Biosciences). 2.2. Cancer Stem Cell (Csc) Generation To select neurospheres (3D cultures), ﬂoating parental cells derived from 2D cul- tures of HTLA-230 and HTLA-ER [23] cells were harvested, centrifuged (117 rcf × 8 min), seeded (16 × 104) and grown in DMEM-F12 Knock-out (Life Technologies, Carlsbad, CA, USA) containing 1% penicillin/streptomycin (Euroclone S.p.A., Pavia, Italy), 2% B27 (Life Technologies), 40 ng/mL basal growth factor for ﬁbroblasts (bFGF) (R&D Systems, Inc., Minneapolis, MN, USA) and 20 ng/mL epidermal growth factor (EGF) (Life Technolo- gies) [24]. It is important to outline that the expression of stem cell markers was evaluated (see Section 2.4) in order to verify the staminality and, only after this characterization, were the neurospheres renamed as CSCs. The HTLA-CSCs and ER-CSCs were split once a week to favor their propagation and, at each passage, the culture medium consisted of 50% fresh medium and 50% of the medium in which the cells were grown [24]. In order to analyze CSC propagation, at any split, the CSCs were collected, centrifuged, mechanically dissociated in PBS and then counted using a Burker chamber (Marienfeld, Germany). g y Parental HTLA-230 and HTLA-ER cells were maintained in RPMI 1640 medium (Eu- roclone) containing 10% fetal bovine serum (FBS; Euroclone), 2 mM glutamine (Euroclone), 1% penicillin/streptomycin (Euroclone), 1% sodium pyruvate and 2% amino acid solu- tion (Sigma-Aldrich, St. Louis, MO, USA). All cell lines were periodically tested with Antioxidants 2021, 10, 691 3 of 22 semi-quantitative PCR for the evaluation of any mycoplasma contamination (Mycoplasma Reagent Set; Euroclone). semi-quantitative PCR for the evaluation of any mycoplasma contamination (Mycoplasma Reagent Set; Euroclone). 2.3. Treatments Both CSC populations were treated once a week with 1 mM BSO or three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. The treatments were protracted for 6 weeks and stock solutions (1 mM C2-4, 50 mM SSZ and 50 mM Etoposide) were prepared by dissolving each drug in DMSO. In order to demonstrate that the doses of DMSO used to dissolve the compounds did not affect the subsequent analyses, pilot studies were performed in parallel with the experiments. 2.10. MDA Production Malondialdehyde (MDA) levels were analyzed by thiobarbituric acid assay (TBARS) as previously reported [23,27]. Brieﬂy, 50 µg of total proteins, derived from each sample, were dissolved in 300 µL of Milli-Q water and added to 600 µL of TBARS solution containing 15% trichloroacetic acid (TCA) and 26 mM thiobarbituric acid (TBA) in 0.25 N HCl. This mixture was incubated for 40 min at 100 ◦C and then centrifuged (20,800 rcf × 2 min). Subsequently, the supernatant was collected and analyzed on the spectrophotometer using a wavelength of 532 nm. In order to calculate MDA concentration, the absorbance of each sample was compared with that obtained from a standard curve evaluated with known concentrations of MDA (0.75, 1, and 2 µM). 2.5. HPLC Analysis of Intracellular Glutathione Levels Intracellular levels of reduced glutathione (GSH) and oxidized glutathione (GSSG) were assessed by high performance liquid chromatography (HPLC) using the methods reported by Reed for total GSH [25] and Asensi for GSSG quantiﬁcation [26]. Untreated and treated CSCs (1 × 106 cells) were collected and centrifuged (117 rcf × 8 min). Then, the pellets were washed in PBS, precipitated with 10% perchloric acid (PCA) and the thiol groups were blocked with iodacetic acid (alkaline pH 8–9, 10 min, room temperature, in the dark). Subsequently, the analytes were converted to 2,4-dinitrophenyl derivatives by incubating the samples with 1% 1-Fluoro-2,4-dinitrobenzene (FDNB) at 4 ◦C in the dark overnight. The quantitative determination of the derivatized analytes was carried out by HPLC equipped with an NH2 Spherisorb column and a UV detector set at 365 nm with a ﬂow rate of 1.25 mL/min. The mobile phase was maintained at 80% solution A (80% methanol in water) and 20% solution B (0.5 M sodium acetate in 64% methanol in water) for 5 min, followed by a 10-min-linear gradient to 1% A and 99% B. The chromatography was performed with gradient elution. g p y p g In order to evaluate GSSG, the samples were incubated with 20 mM N-ethyl-maleimide (NEM) in PCA and, after precipitation and alkalization of the sample (alkaline pH 8–9, 10 min, room temperature, in the dark), derivatization was carried out by adding 1% FDNB. Antioxidants 2021, 10, 691 4 of 22 In order to allow the elution of GSSG, the mobile phase was kept at 99% solution B for a further 15 min. In order to allow the elution of GSSG, the mobile phase was kept at 99% solution B for a further 15 min. The data obtained was normalized by the intracellular amount of protein and ex- pressed as µEq/mg of protein. 2.9. Lactate Release The concentration of lactic acid released by CSCs in the culture medium was analyzed by spectrophotometric analysis as previously reported [23]. The assay buffer contained 100 mM Tris/HCl (pH 8), 5 mM NAD+ and 1 IU/mL of lactate dehydrogenase. Samples were analyzed before and after the addition of 4 µg of puriﬁed lactate dehydrogenase. 2.6. ATP Synthesis The ATP content was measured using the luciferin/luciferase ATP bioluminescence assay kit CLSII (Roche, Basel, Switzerland) on a Luminometer (Triathler, Bioscan, Washing- ton, DC, USA) [23]. ATP standard solutions (Roche, Basel, Switzerland) in the concentration range of 10−10–10−7 M were used for calibration. Assay was carried out at 37 ◦C over 2 min by measuring formed ATP from added ADP. Untreated and treated CSCs were incubated for 10 min with the assay buffer (10 mM Tris-HCl pH 7.4, 50 mM KCl, 1 mM EGTA, 2 mM EDTA, 5 mM KH2PO4, 2 mM MgCl2, 0.6 mM Ouabain, 0.040 mg/mL Ampi- cillin, 0.2 mM di(adenosine-5′) penta-phosphate, 0.2 mM and 5 mM pyruvate plus 2.5 mM malate). Afterwards, ATP synthesis was induced by the addition of 0.3 mM ADP. 2.8. Glucose Consumption Glucose consumption was assessed by measuring its concentration using a double beam spectrophotometer (UNICAM UV2, Analytical S.n.c., Langhirano, PR, Italy), by the hexokinase (HK) and glucose 6 phosphate dehydrogenase (G6PD) coupling system, following the reduction of NADP at 340 nm. [23]. The buffer assay contained 100 mM Tris HCl, pH 7.4, 2 mM ATP, 10 mM NADP, 2 mM MgCl2, 2 IU of HK and 2 IU of G6PD. The reaction was started after the addition of 5 µL of CSC medium. 2.7. Oxygen Consumption Rate (OCR) In order to measure the respiratory activity, 2 × 105 cells were analyzed by using an amperometric electrode for O2 placed in an isolated chamber and stirring maintained at 37 ◦C, as previously reported [23]. Untreated and treated CSCs were collected and centrifuged (117 rcf × 8 min). Then, the pellets were suspended in the assay buffer (137 mM NaCl, 5 mM KH2PO4, 5 mM KCl, 0.5 mM EDTA, 3 mM MgCl2 and 25 mM Tris– HCl, pH 7.4), and permeabilized with 0.3% digitonin for 10 min. Then, the sample was transferred to the chamber; so as to measure the maximum respiration rate, 5 mM pyruvate plus 2.5 mM malate were added. Detection of ROS levels was evaluated by incubating CSCs with 5µM 2′-7′ dichlorofluorescein- diacetate (DCFH-DA; Sigma-Aldrich) as previously reported [23]. 2.13. GPX4 Activity GPX4 activity was evaluated by standard methods [29]. In detail, cell pellets were re-suspended in 0.75 mL lysis buffer (0.1 M Tris-HCl, 0.25 M sucrose, protease inhibitors, pH 7.5) and then sonicated and used in the test (0.1–0.2 mL of sample per test). Samples were mixed with the assay buffer (0.1 M Tris-HCl pH 7.8, 5 mM EDTA, 5 mM GSH, 0.1% (v/v) Triton X-100, 0.16 mM NADPH and 0.6 IU/mL Glutathione Reductase (GR)) and incubated for 5 min at 25 ◦C. Then the baseline was recorded at 340 nm for about 1 min and ﬁnally the enzymatic activity was started by adding phosphatidylcholine hydroperoxide (0.020 mM). The quantiﬁcation of the activity was done on the basis of the net speed with which the absorbance decreases after the addition of the substrate (net speed = speed after the addition of the substrate −baseline speed). 2.15. Statistical Analysis Results were expressed as mean ± SEM from at least four independent experiments. The statistical signiﬁcance of parametric differences among the sets of experimental data was evaluated by one-way ANOVA and Dunnett’s test for multiple comparisons. 2.12. Western Blot Analyses Immunoblots were carried out according to standard methods [28] using rabbit an- tibody anti-N-Cadherin (D4R1H), anti-ZEB-1 (D80D3), anti-Vimentin (D21H3), anti-β- Catenin (D10A8), anti-Claudin-1 (D5H1D) (Cell Signaling Technology Inc., Danvers, MA, USA Upstate, Lake Placid, NY, USA) and anti-GPX4 (Abcam, Cambridge, UK). Anti-rabbit secondary antibodies coupled with horseradish peroxidase (Cell signaling Technologies) were utilized. 2.14. Principal Component Analyses (PCA) PCA is a data display method for multivariate data. Given a data set in which each sample is described by n variables, PCA aims to ﬁnd new directions, linear combinations of the original ones [30]. The ﬁrst component (PC1) corresponds to the direction explaining the maximum variance, while PC2 is the direction, orthogonal to PC1, explaining the maximum variance not explained by PC1, and so on. The result of such a transformation is that a limited number of components is sufﬁcient to explain the relevant part of the information. The loadings are the coefﬁcients of the linear combinations corresponding to the PCs. By plotting them in a loading plot it is possible to understand the relationships among the variables in the multivariate space. On the other side, the score plot (the scores being the coordinates of the samples in the new space deﬁned by the PCs) allows visualization of the location of the samples in the space described by the PCs, hence making possible to check similarities and differ- ences among the samples. The elaborations and the plots were carried out by using CAT software [31]. 2.11. ROS Production Detection of ROS levels was evaluated by incubating CSCs with 5µM 2′-7′ dichlorofluorescein- diacetate (DCFH-DA; Sigma-Aldrich) as previously reported [23]. 5 of 22 Antioxidants 2021, 10, 691 2.12. Western Blot Analyses 3. Results and Discussion 3.1. Neurospheres, Isolated from Parental HTLA-230 and HTLA-ER Cells, Are Cancer Stem Cells (CSCs) and GSH Plays a Crucial Role in Their Generation and Propagation In order to verify if the ﬂoating neurospheres, isolated from HTLA-230 and HTLA-ER cells [23], were formed by stem cells, the expression of CD44 and Oct-3/4, known mark- ers of stemness, was analyzed. As shown in Figure 1a, isolated neurospheres expressed both CD44 and Oct-3/4, demonstrating that both cell populations have staminality char- acteristics and, consequently, have been referred to as HTLA- and ER-cancer stem cells (HTLA-CSCs and ER-CSCs). 6 of 22 Antioxidants 2021, 10, 691 (HTLA-CSCs and ER-CSCs). Figure 1. Flow cytometric analysis of CD44, Oct3/4 (a) and of CD44v9 and xCT (c) expression in untreated HTLA-CSCs and ER-CSCs. Cells were permeabilized, stained with mAbs to the indi- cated molecules, and analyzed by flow cytometry. Grey profiles indicate cells stained with the Figure 1. Flow cytometric analysis of CD44, Oct3/4 (a) and of CD44v9 and xCT (c) expression in untreated HTLA-CSCs and ER-CSCs. Cells were permeabilized, stained with mAbs to the indicated molecules, and analyzed by ﬂow cytometry. Grey proﬁles indicate cells stained with the different mAbs, while white proﬁles correspond to isotype control. One representative experiment of ﬁve is shown. (b) Evaluation of cell number in HTLA-CSCs and ER-CSCs chronically treated with 1mM BSO. Both CSC populations were treated (once a week for six weeks) with 1 mM BSO and, at any split, disaggregated CSCs were counted using a Burker chamber as described in Materials and Methods. ** p < 0.01 vs. time 0; ◦◦p < 0.01 vs. untreated HTLA-CSCs; §§ p < 0.01 vs. untreated ER-CSCs. Figure 1. Flow cytometric analysis of CD44, Oct3/4 (a) and of CD44v9 and xCT (c) expression in untreated HTLA-CSCs and ER-CSCs. Cells were permeabilized, stained with mAbs to the indi- cated molecules, and analyzed by flow cytometry. Grey profiles indicate cells stained with the Figure 1. Flow cytometric analysis of CD44, Oct3/4 (a) and of CD44v9 and xCT (c) expression in untreated HTLA-CSCs and ER-CSCs. Cells were permeabilized, stained with mAbs to the indicated molecules, and analyzed by ﬂow cytometry. Grey proﬁles indicate cells stained with the different mAbs, while white proﬁles correspond to isotype control. One representative experiment of ﬁve is shown. (b) Evaluation of cell number in HTLA-CSCs and ER-CSCs chronically treated with 1mM BSO. 3.2. Etoposide Prevents the Formation of HTLA-CSCs after 3 Weeks of Treatment While It Completely Counteracts the Formation of ER-CSCs when It Is Combined with SSZ or C2-4 for 6 Weeks 3.2. Etoposide Prevents the Formation of HTLA-CSCs after 3 Weeks of Treatment While It Completely Counteracts the Formation of ER-CSCs when It Is Combined with SSZ or C2-4 for 6 Weeks By analyzing HTLA-CSCs, it was observed that Etoposide reduced the propagation of the formed CSCs by 35% after only one week of exposure and totally prevented CSC formation after three weeks (Figure 2a). Moreover, similar effects were obtained in CSCs co-treated with either Etoposide and SSZ (Figure 2a, left panel) or Etoposide and C2-4 (Figure 2a, right panel). However, treatments with SSZ (Figure 2a, left panel) or C2-4 alone (Figure 2a, right panel) did not signiﬁcantly alter this parameter during the analyzed time points. ER REVIEW 8 of 23 Figure 2. Evaluation of cell number in HTLA-CSCs (a) and ER-CSCs (b) treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. The treatments were protracted for six weeks. In order to evaluate CSC propagation, at any split, disaggregated CSCs were counted using a Burker chamber as described in Materials and Methods. °° p < 0.01 vs. untreated HTLA-CSCs (Ctr); * p < 0.05 vs. untreated ER-CSCs (Ctr); ## p < 0.01 vs. Etoposide-treated ER-CSCs. Together these results demonstrate that ER-CSCs maintain their resistance to Etopo- Figure 2. Evaluation of cell number in HTLA-CSCs (a) and ER-CSCs (b) treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. The treatments were protracted for six weeks. In order to evaluate CSC propagation, at any split, disaggregated CSCs were counted using a Burker chamber as described in Materials and Methods. ◦◦p < 0.01 vs. untreated HTLA-CSCs (Ctr); * p < 0.05 vs. untreated ER-CSCs (Ctr); ## p < 0.01 vs. Etoposide-treated ER-CSCs. Figure 2. Evaluation of cell number in HTLA-CSCs (a) and ER-CSCs (b) treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. The treatments were protracted for six weeks. In order to evaluate CSC propagation, at any split, disaggregated CSCs were counted using a Burker chamber as described in Materials and Methods. °° p < 0.01 vs. 3. Results and Discussion Both CSC populations were treated (once a week for six weeks) with 1 mM BSO and, at any split, disaggregated CSCs were counted using a Burker chamber as described in Materials and Methods. ** p < 0.01 vs. time 0; ◦◦p < 0.01 vs. untreated HTLA-CSCs; §§ p < 0.01 vs. untreated ER-CSCs. Since the presence of CSCs [12] and glutathione (GSH) play a crucial role in chemore- sistance [23], both CSC populations were chronically treated (once a week for 6 weeks) with 1 mM BSO in order to investigate whether a relationship between GSH levels and stemness exists. As shown in Figure 1b, BSO markedly counteracted the formation of HTLA-CSCs after two weeks of treatment, while a similar effect was observed in ER-CSCs after ﬁve weeks. These results demonstrate that GSH plays a crucial role in the formation and maintenance of CSCs and suggest that its depletion could be employed to increase sensitivity of CSCs to therapeutic approaches. p pp However, although BSO has been found to counteract CSC formation, both in vitro and in vivo [32–34], and has been included in NB clinical trials [35], its therapeutic use is limited by its short half-life and by its non-selective depleting effect on neoplastic cells [22]. 7 of 22 Antioxidants 2021, 10, 691 In order to overcome this limitation, an effective strategy could be the indirect modulation of GSH levels by acting on GSH-related targets. In this context, it has been reported that CD44, a known stemness membrane marker [17,36], and, in particular, its variant 9 (CD44v9) contributes to the stabilization of xCT, an anti-port protein involved in the uptake of cystine, an amino acid that is essential for GSH biosynthesis. Considering that the expression of CD44 is modulated by protein kinase C (PKC)α [17,36], an opportunity would be to reduce the CD44 expression by inhibiting this kinase. As shown in Figure 1c, both CSC populations expressed CD44v9 and xCT, thus justifying the use of C2-4, a PKCα inhibitor, or of sulphasalazine (SSZ), a xCT inhibitor, in the reported studies. 3.2. Etoposide Prevents the Formation of HTLA-CSCs after 3 Weeks of Treatment While It Completely Counteracts the Formation of ER-CSCs when It Is Combined with SSZ or C2-4 for 6 Weeks untreated HTLA-CSCs (Ctr); * p < 0.05 vs. untreated ER-CSCs (Ctr); ## p < 0.01 vs. Etoposide-treated ER-CSCs. Figure 2. Evaluation of cell number in HTLA-CSCs (a) and ER-CSCs (b) treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. The treatments were protracted for six weeks. In order to evaluate CSC propagation, at any split, disaggregated CSCs were counted using a Burker chamber as described in Materials and Methods. ◦◦p < 0.01 vs. untreated HTLA-CSCs (Ctr); * p < 0.05 vs. untreated ER-CSCs (Ctr); ## p < 0.01 vs. Etoposide-treated ER-CSCs. Antioxidants 2021, 10, 691 8 of 22 8 of 22 In contrast, the same treatments did not induce any signiﬁcant alteration in the forma- tion/propagation of ER-CSCs until two weeks of exposure (Figure 2b). In fact, Etoposide treatment reduced CSC propagation by 38% at four weeks, and this effect remained un- changed until six weeks of treatment (Figure 2b). By analyzing the results obtained in co-treated CSCs, it was observed that Etoposide combined with SSZ or Etoposide combined with C 2-4 reduced the ER-CSC propagation by 30% and 55%, respectively, after three weeks and totally prevent the formation of CSCs after six weeks (Figure 2b). Moreover, as shown in Figure 2b left panel, the results obtained in ER-CSCs, treated with SSZ alone for six weeks, were comparable to those found in CSCs exposed to only Etoposide, whereas the treatment with C2-4 did not affect CSC formation and propagation (Figure 2b, right panel). p p g g g p Since the formation of HTLA-CSCs was totally inhibited after three weeks of treat- ments and that of ER-CSCs after six weeks, the following analyses were performed after two and ﬁve weeks respectively. Together these results demonstrate that ER-CSCs maintain their resistance to Etoposide and show that ER-CSC generation is totally inhibited by co-treatments with SSZ or C2-4. Moreover, the data obtained conﬁrm that the inhibition of xCT is a useful strategy for enhancing the effect of chemotherapy [37–42] and, although the use of SSZ is limited due to its low bioavailability, its employment has been recently considered to treat high risk NB [43]. 3.2. Etoposide Prevents the Formation of HTLA-CSCs after 3 Weeks of Treatment While It Completely Counteracts the Formation of ER-CSCs when It Is Combined with SSZ or C2-4 for 6 Weeks In addition, the results obtained in C2-4-co-treated cells suggest that the inhibition of PKCα could be a novel way to sensitize CSCs to therapy by sustaining GSH depletion. In this regard, several clinical trials (NCT00042679; NCT00003236; NCT00017407) have been carried out using Aprinocarsen, an anti-sense oligonucleotide that targets PKCα mRNA, even if, unfortunately, the results obtained are not encouraging [44]. However, a large number of studies have reported the efﬁcacy of PKCα inhibitors in sensitizing cancer cells to chemotherapeutic drugs [45–48]. 3.3. Etoposide Induces a Marked Depletion of GSH in HTLA-CSCs after Two Weeks While the Same Effect Is Not Observed in ER-CSCs until Five Weeks of Treatment Therefore, based on these ﬁndings, the metabolic proﬁle of untreated and treated CSCs was analyzed. 80% reduction in GSH was observed in HTLA-CSCs with respect to untreated CSCs (Fig- ure 3a). At the same time, both co-treatment conditions (C2-4 + Etoposide or SSZ + Etopo- side), exerted similar effects to those detected in Etoposide-treated CSCs (Figure 3a), while C2-4 or SSZ alone did not significantly alter the GSH amount (Figure 3a). Notably, the same two-week treatments in ER-CSCs did not induce any significant changes in GSH levels (Figure 3b). Figure 3. Glutathione (GSH) levels in both CSC populations treated for two (a,b) or five weeks (c). HTLA-CSCs and ER- CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. GSH concentrations were determined by HPLC analysis and expressed as percentages of the control value. * p < 0.05 vs. untreated CSCs (Ctr); ** p < 0.01 vs. untreated CSCs (Ctr). After five weeks, untreated HTLA-CSCs and ER-CSCs displayed a different GSH Figure 3. Glutathione (GSH) levels in both CSC populations treated for two (a,b) or ﬁve weeks (c). HTLA-CSCs and ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. GSH concentrations were determined by HPLC analysis and expressed as percentages of the control value. * p < 0.05 vs. untreated CSCs (Ctr); ** p < 0.01 vs. untreated CSCs (Ctr). chemoresistant cancer cells [23,51,52] and CSCs undergo a metabolic rewiring, stimulating OXPHOS with a major ATP production [53–56]. Therefore, based on these ﬁndings, the metabolic proﬁle of untreated and treated CSCs was analyzed. side), exerted similar effects to those detected in Etoposide-treated CSCs (Figure 3a), while C2-4 or SSZ alone did not significantly alter the GSH amount (Figure 3a). Notably, the same two-week treatments in ER-CSCs did not induce any significant changes in GSH levels (Figure 3b). Figure 3. Glutathione (GSH) levels in both CSC populations treated for two (a,b) or five weeks (c). HTLA-CSCs and ER- CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. 3.3. Etoposide Induces a Marked Depletion of GSH in HTLA-CSCs after Two Weeks While the Same Effect Is Not Observed in ER-CSCs until Five Weeks of Treatment 3.3. Etoposide Induces a Marked Depletion of GSH in HTLA-CSCs after Two Weeks While the Same Effect Is Not Observed in ER-CSCs until Five Weeks of Treatment Untreated HTLA-CSCs and ER-CSCs displayed comparable GSH levels (about 17.07 µEq/g and 17.37 µEq/g proteins, respectively). After two weeks of Etoposide exposure, an 80% reduction in GSH was observed in HTLA-CSCs with respect to untreated CSCs (Figure 3a). At the same time, both co-treatment conditions (C2-4 + Etoposide or SSZ + Etoposide), exerted similar effects to those detected in Etoposide-treated CSCs (Figure 3a), while C2-4 or SSZ alone did not signiﬁcantly alter the GSH amount (Figure 3a). Notably, the same two-week treatments in ER-CSCs did not induce any signiﬁcant changes in GSH levels (Figure 3b). After ﬁve weeks, untreated HTLA-CSCs and ER-CSCs displayed a different GSH con- tent of 4.7 µEq/g and 8.99 µE/g protein, respectively. Since the ﬁve-week-treatment with Etoposide, alone or in combination, totally counteracted HTLA-CSC formation (Figure 2a), it was not possible to analyze GSH. As shown in Figure 3c, Etoposide decreased GSH of ER-CSCs by 60% in comparison to untreated CSCs, and co-treatments, with C2-4 or SSZ, which did not modify the GSH-depleting effect of Etoposide. The amount of GSSG, the oxidized form of GSH, was below the detection limits in both CSC populations after two weeks while it was depleted by 60–65% in ER-CSCs after the ﬁve-week-treatment with Etoposide, alone or in combination with C2-4 or SSZ (data not shown). These results demonstrate that ﬁve-week treatment with Etoposide markedly reduces GSH levels in ER-CSCs and that this effect is only due to the action of Etoposide, given that a similar loss of GSH levels is observed in co-treated CSCs and neither C2-4 nor SSZ, alone or combined with Etoposide, modulate GSH levels. This data demonstrates that the survival of Etoposide-resistant CSCs is not totally dependent on GSH since, even though Etoposide exerts a severe GSH-depleting action, it does not totally counteract CSC generation, suggesting that other factors might contribute to the maintenance of cancer stem cell survival. In this regard, it has been widely demon- strated that cancer cells usually depend on glycolysis for energy production [49,50] while 9 of 22 C (Fi Antioxidants 2021, 10, 691 9 of 22 C (Fi Antioxidants 2021, 10, 691 9 of 22 chemoresistant cancer cells [23,51,52] and CSCs undergo a metabolic rewiring, stimulating OXPHOS with a major ATP production [53–56]. 3.3. Etoposide Induces a Marked Depletion of GSH in HTLA-CSCs after Two Weeks While the Same Effect Is Not Observed in ER-CSCs until Five Weeks of Treatment GSH concentrations were determined by HPLC analysis and expressed as percentages of the control value. * p < 0.05 vs. untreated CSCs (Ctr); ** p < 0.01 vs. untreated CSCs (Ctr). Figure 3. Glutathione (GSH) levels in both CSC populations treated for two (a,b) or ﬁve weeks (c). HTLA-CSCs and ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. GSH concentrations were determined by HPLC analysis and expressed as percentages of the control value. * p < 0.05 vs. untreated CSCs (Ctr); ** p < 0.01 vs. untreated CSCs (Ctr). p y content of 4.7 µEq/g and 8.99 µE/g protein, respectively. Since the five-week-tr with Etoposide, alone or in combination, totally counteracted HTLA-CSC formati 3.4. ER-CSCs Maintain an Efﬁcient OXPHOS Metabolism Which Is Impaired Only by Co-Treatments HTLA- CSCs and ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. Results were reported as nmol ATP/min/106 cells. Bar graph summarizes quantitative data of means ± S.E.M. of three independent experiments. * p < 0.05 vs. untreated CSCs (Ctr); ** p < 0.01 vs. untreated CSCs (Ctr); ## p < 0 01 vs Etoposide-treated CSCs Figure 4. ATP synthesis in both CSC populations treated for two (a,b) or ﬁve weeks (c). HTLA-CSCs and ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. Results were reported as nmol ATP/min/106 cells. Bar graph summarizes quantitative data of means ± S.E.M. of three independent experiments. * p < 0.05 vs. untreated CSCs (Ctr); ** p < 0.01 vs. untreated CSCs (Ctr); ## p < 0.01 vs. Etoposide-treated CSCs. After ﬁve weeks, untreated CSC populations had comparable levels of ATP (about 10 nmol/min/1 × 106 cells). Since ﬁve weeks of treatment with Etoposide, alone or in combination, totally counteracted HTLA-CSC formation (Figure 2a), the evaluation of ATP synthesis was not possible while in ER-CSCs, ATP decreased by 10% after Etoposide and by 35% by the co-treatments (Figure 4c). Moreover, in HTLA-CSCs, two weeks of Etoposide treatment reduced the oxygen consumption rate (OCR) by 75% in respect of untreated CSCs (Figure 5a) and similar results were obtained in co-treated ones (Figure 5a). At the same time point, in ER-CSCs, the OCR was not modiﬁed either by Etoposide or co-treatments and it was increased by 15% and 35% only by C2-4 or SSZ respectively (Figure 5b). Since the ﬁve-week-treatment with Etoposide, alone or in combination, totally counteracted HTLA-CSC formation (Figure 2a), OCR analysis was not possible. Notably, in ER-CSCs, none of the treatments signiﬁcantly altered the OCR (Figure 5c). To verify the efﬁciency of OXPHOS, the P/O value was calculated. In detail, in both CSC populations, the P/O ratio was 2.3 ± 0.2, which is comparable to the physiological level of 2.5 as reported by Hinkle [57] and interestingly, only SSZ, administered alone or in combination with Etoposide for two or ﬁve weeks, led to a P/O value of 1.8 ± 0.15. p y content of 4.7 µEq/g and 8.99 µE/g protein, respectively. Since the five-week-tr with Etoposide, alone or in combination, totally counteracted HTLA-CSC formati 3.4. ER-CSCs Maintain an Efﬁcient OXPHOS Metabolism Which Is Impaired Only by Co-Treatments p y content of 4.7 µEq/g and 8.99 µE/g protein, respectively. Since the five-week-tr with Etoposide, alone or in combination, totally counteracted HTLA-CSC formati 3.4. ER-CSCs Maintain an Efﬁcient OXPHOS Metabolism Which Is Impaired Only by Co-Treatments ure 2a), it was not possible to analyze GSH. As shown in Figure 3c, Etoposide decreased GSH of ER-CSCs by 60% in comparison to untreated CSCs, and co-treatments, with C2-4 or SSZ, which did not modify the GSH-depleting effect of Etoposide. As shown in Figure 4a, ATP synthesis, detected in untreated HTLA-CSCs, after two weeks, was doubled in respect of that of ER-CSCs. In HTLA-CSCs, two weeks of Etoposide, alone or in combination with C2-4 or SSZ, reduced ATP production by 70% in respect of untreated CSCs (Figure 4a). Notably, in ER-CSCs, two weeks of Etoposide was ineffective and only co-treatment of Etoposide with SSZ reduced ATP synthesis by 25% compared to both untreated and Etoposide treated CSCs (Figure 4b). 10 of 22 10 of 22 Antioxidants 2021, 10, 691cr been found to increase therapy sensitivity of cancer stem cells [60,61]. Figure 4. ATP synthesis in both CSC populations treated for two (a,b) or five weeks (c). HTLA- CSCs and ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. Results were reported as nmol ATP/min/106 cells. Bar graph summarizes quantitative data of means ± S.E.M. of three independent experiments. * p < 0.05 vs. untreated CSCs (Ctr); ** p < 0.01 vs. untreated CSCs (Ctr); ## p < 0.01 vs. Etoposide-treated CSCs. Figure 4. ATP synthesis in both CSC populations treated for two (a,b) or ﬁve weeks (c). HTLA-CSCs and ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. Results were reported as nmol ATP/min/106 cells. Bar graph summarizes quantitative data of means ± S.E.M. of three independent experiments. * p < 0.05 vs. untreated CSCs (Ctr); p < 0.01 vs. untreated CSCs (Ctr); ## p < 0.01 vs. Etoposide-treated CSCs. py y Figure 4. ATP synthesis in both CSC populations treated for two (a,b) or five weeks (c). p y content of 4.7 µEq/g and 8.99 µE/g protein, respectively. Since the five-week-tr with Etoposide, alone or in combination, totally counteracted HTLA-CSC formati 3.4. ER-CSCs Maintain an Efﬁcient OXPHOS Metabolism Which Is Impaired Only by Co-Treatments HTLA-CSCs and ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. Results were reported as nmol O/min/106 cells. Bar graph summarizes quantitative data of means ± S.E.M. of three independent experiments. p < 0.01 vs. untreated CSCs (Ctr); ## p < 0.01 vs. Etoposide-treated CSCs. umption rate (OCR) in both CSC populations treated for two (a,b) or five weeks (c). HTLA-CSCs and ER-CSCs es a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in 4 or SSZ. Results were reported as nmol O/min/106 cells. Bar graph summarizes quantitative data of means ± ndent experiments. * p < 0.05 vs. untreated CSCs (Ctr); ** p < 0.01 vs. untreated CSCs (Ctr); ## p < 0.01 vs. Etopo- nsumption in both CSC populations treated for two (a,b) or five weeks (c). HTLA-CSCs and ER-CSCs were week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in onsumption in both CSC populations treated for two (a,b) or five weeks (c). HTLA-CSCs and ER-CSCs were a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in Figure 6. Glucose consumption in both CSC populations treated for two (a,b) or ﬁve weeks (c). HTLA-CSCs and ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. Results were reported as mM glucose/106 cells. Bar graph summarizes quantitative data of means ± S.E.M. of three independent experiments. * p < 0.05 vs. untreated CSCs (Ctr); ** p < 0.01 vs. untreated CSCs (Ctr); # p < 0.05 vs. Etoposide-treated CSCs; ## p < 0.01 vs. Etoposide-treated CSCs. CSC populations treated for two (a,b) or five wee C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, nsumption in both CSC populations treated for two (a,b) or five weeks (c). HTLA-CSCs and ER-CSCs were week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in Figure 6. p y content of 4.7 µEq/g and 8.99 µE/g protein, respectively. Since the five-week-tr with Etoposide, alone or in combination, totally counteracted HTLA-CSC formati 3.4. ER-CSCs Maintain an Efﬁcient OXPHOS Metabolism Which Is Impaired Only by Co-Treatments In order to complete the analysis of cell metabolism, glucose consumption and lactate release were evaluated. As shown in Figure 6a, untreated HTLA-CSCs consumed about 40% more glucose than untreated ER-CSCs. In HTLA-CSCs, two-week-Etoposide exposure increased glucose consumption by 60% in respect of untreated CSCs and the co-treatments did not modify the Etoposide-induced effect (Figure 6a). In ER-CSCs, treatments with either Etoposide, C2-4 or SSZ increased glucose consumption by 30% as compared to 11 of 22 11 of 22 Antioxidants 2021, 10, 691 untreated CSCs, and the co-treatments with C2-4 or SSZ contributed to stimulating this parameter by a further 60% and 25%, respectively (Figure 6b). OR PEER REVIEW 12 of 23 untreated CSCs, and the co-treatments with C2-4 or SSZ contributed to stimulating this parameter by a further 60% and 25%, respectively (Figure 6b). R PEER REVIEW 12 of 23 untreated CSCs, and the co-treatments with C2-4 or SSZ contributed to stimulating this parameter by a further 60% and 25%, respectively (Figure 6b). OR PEER REVIEW 12 of 23 umption rate (OCR) in both CSC populations treated for two (a,b) or five weeks (c). HTLA-CSCs and ER-CSCs es a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in 4 or SSZ. Results were reported as nmol O/min/106 cells. Bar graph summarizes quantitative data of means ± ndent experiments. * p < 0.05 vs. untreated CSCs (Ctr); p < 0.01 vs. untreated CSCs (Ctr); ## p < 0.01 vs. Etopo- Figure 5. Oxygen consumption rate (OCR) in both CSC populations treated for two (a,b) or ﬁve weeks (c). HTLA-CSCs and ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. Results were reported as nmol O/min/106 cells. Bar graph summarizes quantitative data of means ± S.E.M. of three independent experiments. p < 0.01 vs. untreated CSCs (Ctr); ## p < 0.01 vs. Etoposide-treated CSCs. FOR PEER REVIEW 12 of 23 nsumption rate (OCR) in both CSC populations treated for two (a,b) or five weeks (c). HTLA-CSCs and ER-CSCs mes a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in -4 or SSZ. Results were reported as nmol O/min/106 cells. p y content of 4.7 µEq/g and 8.99 µE/g protein, respectively. Since the five-week-tr with Etoposide, alone or in combination, totally counteracted HTLA-CSC formati 3.4. ER-CSCs Maintain an Efﬁcient OXPHOS Metabolism Which Is Impaired Only by Co-Treatments Bar graph summarizes quantitative data of means ± endent experiments. * p < 0.05 vs. untreated CSCs (Ctr); ** p < 0.01 vs. untreated CSCs (Ctr); ## p < 0.01 vs. Etopo- FOR PEER REVIEW umption rate (OCR) in both CSC populations treated for two (a,b) or five weeks (c). HTLA-CSCs and ER-CSCs s a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in or SSZ. Results were reported as nmol O/min/106 cells. Bar graph summarizes quantitative data of means ± dent experiments. * p < 0.05 vs. untreated CSCs (Ctr); p < 0.01 vs. untreated CSCs (Ctr); ## p < 0.01 vs. Etopo- Figure 5. Oxygen consumption rate (OCR) in both CSC populations treated for two (a,b) or ﬁve weeks (c). HTLA-CSCs and ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. Results were reported as nmol O/min/106 cells. Bar graph summarizes quantitative data of means ± S.E.M. of three independent experiments. p < 0.01 vs. untreated CSCs (Ctr); ## p < 0.01 vs. Etoposide-treated CSCs. umption rate (OCR) in both CSC populations treated for two (a,b) or five weeks (c). HTLA-CSCs and ER-CSCs es a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in 4 or SSZ. Results were reported as nmol O/min/106 cells. Bar graph summarizes quantitative data of means ± ndent experiments. * p < 0.05 vs. untreated CSCs (Ctr); ** p < 0.01 vs. untreated CSCs (Ctr); ## p < 0.01 vs. Etopo- umption rate (OCR) in both CSC populations treated for two (a,b) or five weeks (c). HTLA-CSCs and ER-CSCs s a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in or SSZ. Results were reported as nmol O/min/106 cells. Bar graph summarizes quantitative data of means ± dent experiments. * p < 0.05 vs. untreated CSCs (Ctr); p < 0.01 vs. untreated CSCs (Ctr); ## p < 0.01 vs. Etopo- Figure 5. Oxygen consumption rate (OCR) in both CSC populations treated for two (a,b) or ﬁve weeks (c). p y content of 4.7 µEq/g and 8.99 µE/g protein, respectively. Since the five-week-tr with Etoposide, alone or in combination, totally counteracted HTLA-CSC formati 3.4. ER-CSCs Maintain an Efﬁcient OXPHOS Metabolism Which Is Impaired Only by Co-Treatments p < 0.01 vs. untreated CSCs (Ctr); # p < 0.05 vs. Etoposide-treated CSCs; ## p < 0.01 vs. Etoposide-treated CSCs. Figure 7. Lactate release in both CSC populations treated for two (a,b) or ﬁve weeks (c). HTLA-CSCs and ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. Results were reported as mM lactate/106 cells. Bar graph summarizes quantitative data of means ± S.E.M. of three independent experiments. p < 0.01 vs. untreated CSCs (Ctr); # p < 0.05 vs. Etoposide-treated CSCs; ## p < 0.01 vs. Etoposide-treated CSCs. Figure 7. Lactate release in both CSC populations treated for two (a,b) or five weeks (c). HTLA- CSCs and ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. Results were reported as mM lactate/106 cells. Bar graph summarizes quantitative data of means ± S.E.M. of three independent experiments. p < 0.01 vs. untreated CSCs (Ctr); # p < 0.05 vs. Etoposide-treated CSCs; ## p < 0.01 vs. Etoposide-treated CSCs. Figure 7. Lactate release in both CSC populations treated for two (a,b) or ﬁve weeks (c). HTLA-CSCs and ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. Results were reported as mM lactate/106 cells. Bar graph summarizes quantitative data of means ± S.E.M. of three independent experiments. ** p < 0.01 vs. untreated CSCs (Ctr); # p < 0.05 vs. Etoposide-treated CSCs; ## p < 0.01 vs. Etoposide-treated CSCs. eatments Are Able to Induce Lipid Peroxidation in ER-CSCs rder to better characterize cell damage, the production of malondialdehyde a marker of membrane lipid peroxidation, was evaluated in both CSC popula- observed in Figure 8a, two-week-treatments of HTLA-CSCs with Etoposide or e increased MDA levels by 100% and the co-treatment with SSZ increased this r by a further 30% (Figure 8a). p y content of 4.7 µEq/g and 8.99 µE/g protein, respectively. Since the five-week-tr with Etoposide, alone or in combination, totally counteracted HTLA-CSC formati 3.4. ER-CSCs Maintain an Efﬁcient OXPHOS Metabolism Which Is Impaired Only by Co-Treatments Glucose consumption in both CSC populations treated for two (a,b) or ﬁve weeks (c). HTLA-CSCs and ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. Results were reported as mM glucose/106 cells. Bar graph summarizes quantitative data of means ± S.E.M. of three independent experiments. * p < 0.05 vs. untreated CSCs (Ctr); p < 0.01 vs. untreated CSCs (Ctr); # p < 0.05 vs. Etoposide-treated CSCs; ## p < 0.01 vs. Etoposide-treated CSCs. 12 of 22 Antioxidants 2021, 10, 691 12 of 22 Since ﬁve-week-treatment with Etoposide, alone or in combination, totally counter- acted HTLA-CSC formation (Figure 2a), glucose consumption analysis was not possible. Noteworthy, in ER-CSCs, the trend observed after ﬁve weeks was similar to that observed after two weeks and, also in this case, the co-treatments stimulated the Etoposide-induced effect on glucose consumption (Figure 6c). As expected, in line with the highest level of glucose consumption observed in un- treated HTLA-CSCs, an increase of 50% in the lactate release was detected in compari- son with untreated ER-CSCs (Figure 7a,b). After two weeks, the Etoposide exposure of HTLA-CSCs stimulated the lactate release by 80% in respect of untreated CSCs, and the co-treatments did not modify the Etoposide-induced effect (Figure 7a). In ER-CSCs, a simi- lar result was obtained only in the presence of C2-4 or SSZ, given alone or in combination with Etoposide, while Etoposide per se did not induce any effect (Figure 7b). Interestingly, in ER-CSCs, after ﬁve weeks, the co-treatments with C2-4 or SSZ stimulated lactate release by 30% in respect of both untreated and Etoposide-treated ER-CSCs (Figure 7c). 13 of 23 e reported as mM glucose/106 cells. Bar graph summarizes quantitative data of means ± p < 0.05 vs. untreated CSCs (Ctr); p < 0.01 vs. untreated CSCs (Ctr); # p < 0.05 vs. Etopo- eated CSCs. Figure 7. Lactate release in both CSC populations treated for two (a,b) or five weeks (c). HTLA- CSCs and ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. Results were reported as mM lactate/106 cells. Bar graph summarizes quantitative data of means ± S.E.M. of three independent experiments. p y content of 4.7 µEq/g and 8.99 µE/g protein, respectively. Since the five-week-tr with Etoposide, alone or in combination, totally counteracted HTLA-CSC formati 3.4. ER-CSCs Maintain an Efﬁcient OXPHOS Metabolism Which Is Impaired Only by Co-Treatments MDA production in both CSC populations treated for two (a,b) or ﬁve weeks (c). HTLA-CSCs and ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. Results were reported as µM/mg protein. Bar graph summarizes quantitative data of means ± S.E.M. of three independent experiments. p < 0.01 vs. untreated CSCs (Ctr); # p < 0.05 vs. Etoposide-treated CSCs; ## p < 0.01 vs. Etoposide-treated CSCs. Considering that mitochondrial oxidative phosphorylation can generate ROS, th parameter was evaluated in both CSC populations. As shown in Figure 9a, after two-week-exposure to Etoposide, alone or in combinati populations. In agreement with these ﬁndings, OXPHOS inhibitors (e.g., metformin) have been found to increase therapy sensitivity of cancer stem cells [60,61]. populations. In agreement with these ﬁndings, OXPHOS inhibitors (e.g., metformin) have been found to increase therapy sensitivity of cancer stem cells [60,61]. 3.5. Co-Treatments Are Able to Induce Lipid Peroxidation in ER-CSCs In order to better characterize cell damage, the production of malondialdehyde (MDA), a marker of membrane lipid peroxidation, was evaluated in both CSC populations. As observed in Figure 8a, two-week-treatments of HTLA-CSCs with Etoposide or SSZ alone increased MDA levels by 100% and the co-treatment with SSZ increased this parameter by a further 30% (Figure 8a). Notably, in ER-CSCs, only two-week-exposure to SSZ, alone or in combination with Etoposide, induced an 85% increase in MDA production (Figure 8b). After ﬁve weeks, in ER-CSCs, all treatments maintained MDA at similar levels to those observed after two weeks. In addition, a 35% increase in MDA levels was found in C2-4-co- treated ER-CSCs (Figure 8c). REVIEW 14 of 23 treated ER-CSCs (Figure 8c). Figure 8. MDA production in both CSC populations treated for two (a,b) or five weeks (c). HTLA- CSCs and ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. Results were reported as µM/mg protein. Bar graph summarizes quantitative data of means ± S.E.M. of three independent experiments. p < 0.01 vs. untreated CSCs (Ctr); # p < 0.05 vs. Etoposide-treated CSCs; ## p < 0.01 vs. Etoposide-treated CSCs. Figure 8. MDA production in both CSC populations treated for two (a,b) or ﬁve weeks (c). HTLA-CSCs and ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. Results were reported as µM/mg protein. Bar graph summarizes quantitative data of means ± S.E.M. of three independent experiments. p < 0.01 vs. untreated CSCs (Ctr); # p < 0.05 vs. Etoposide-treated CSCs; ## p < 0.01 vs. Etoposide-treated CSCs. C id i th t it h d i l id ti h h l ti t ROS thi Figure 8. MDA production in both CSC populations treated for two (a,b) or five weeks (c). HTL CSCs and ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. Results we reported as µM/mg protein. Bar graph summarizes quantitative data of means ± S.E.M. of three independent experiments. p < 0.01 vs. p y content of 4.7 µEq/g and 8.99 µE/g protein, respectively. Since the five-week-tr with Etoposide, alone or in combination, totally counteracted HTLA-CSC formati 3.4. ER-CSCs Maintain an Efﬁcient OXPHOS Metabolism Which Is Impaired Only by Co-Treatments Notably, in ER-CSCs, only two-week-exposure to e or in combination with Etoposide, induced an 85% increase in MDA produc- Taken together, the metabolic ﬁndings indicate that, although both untreated CSC populations have an efﬁcient OXPHOS metabolism, HTLA-CSCs, following Etoposide ex- posure, activate glycolysis while ER-CSCs maintain their metabolic adaptation and are able to choose glycolytic metabolism after ﬁve-week-co-treatments with C2-4 or SSZ. This data, besides conﬁrming the propensity of CSCs to obtain energy by stimulating OXPHOS [58], also demonstrates that this metabolic rewiring is reversible [59] and that, in having a crucial role in CSC survival, it could be targeted in order to eradicate chemoresistant CSC Antioxidants 2021, 10, 691 13 of 22 13 of 22 populations. In agreement with these ﬁndings, OXPHOS inhibitors (e.g., metformin) ha been found to increase therapy sensitivity of cancer stem cells [60,61]. 3.5. Co-Treatments Are Able to Induce Lipid Peroxidation in ER-CSCs In order to better characterize cell damage, the production of malondialdehyde (MD a marker of membrane lipid peroxidation, was evaluated in both CSC populations. observed in Figure 8a, two-week-treatments of HTLA-CSCs with Etoposide or SSZ alo increased MDA levels by 100% and the co-treatment with SSZ increased this parameter a further 30% (Figure 8a). Notably, in ER-CSCs, only two-week-exposure to SSZ, alone in combination with Etoposide, induced an 85% increase in MDA production (Figure 8 After ﬁve weeks, in ER-CSCs, all treatments maintained MDA at similar levels to tho observed after two weeks. In addition, a 35% increase in MDA levels was found in C2-4- treated ER-CSCs (Figure 8c). ntioxidants 2021, 10, x FOR PEER REVIEW 14 o Figure 8. MDA production in both CSC populations treated for two (a,b) or five weeks (c). HTL CSCs and ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. Results we reported as µM/mg protein. Bar graph summarizes quantitative data of means ± S.E.M. of three independent experiments. p < 0.01 vs. untreated CSCs (Ctr); # p < 0.05 vs. Etoposide-treated CSCs; ## p < 0.01 vs. Etoposide-treated CSCs. Altogether these results demonstrate that although Etoposide alone significantly creased the level of GSH, GSSG amount was not enhanced and, in parallel, ROS and MD production did not increase In this context we cannot exclude that other antioxidant s Figure 8. 3.5. Co-Treatments Are Able to Induce Lipid Peroxidation in ER-CSCs HTLA- CSCs and ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. Results were expressed as percentages of the control value. Bar graph summarizes quantitative data of means ± S.E.M. of three independent experiments. p < 0.01 vs. untreated CSCs (Ctr). Figure 9. ROS production in both CSC populations treated for two (a,b) or ﬁve weeks (c). HTLA-CSCs and ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. Results were expressed as percentages of the control value. Bar graph summarizes quantitative data of means ± S.E.M. of three independent experiments. p < 0.01 vs. untreated CSCs (Ctr). Figure 9. ROS production in both CSC populations treated for two (a,b) or five weeks (c). HTLA- CSC d ER CSC t t d th ti k ith 0 1 M C2 4 ith 5 M SSZ ith 1 25 Figure 9. ROS production in both CSC populations treated for two (a,b) or ﬁve weeks (c). HTLA-CSCs and ER-CSCs were Figure 9. ROS production in both CSC populations treated for two (a,b) or five weeks (c). HTLA- CSCs and ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. Results were expressed as percentages of the control value. Bar graph summarizes quantitative data of means ± S.E.M. of three independent experiments. p < 0.01 vs. untreated CSCs (Ctr). Figure 9. ROS production in both CSC populations treated for two (a,b) or ﬁve weeks (c). HTLA-CSCs and ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. Results were expressed as percentages of the control value. Bar graph summarizes quantitative data of means ± S.E.M. of three independent experiments. p < 0.01 vs. untreated CSCs (Ctr). 3.5. Co-Treatments Are Able to Induce Lipid Peroxidation in ER-CSCs untreated CSCs (Ctr); # p < 0.05 vs. Etoposide-treated CSCs; ## p < 0.01 vs. Etoposide-treated CSCs. Figure 8. MDA production in both CSC populations treated for two (a,b) or ﬁve weeks (c). HTLA-CSCs and ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. Results were reported as µM/mg protein. Bar graph summarizes quantitative data of means ± S.E.M. of three independent experiments. p < 0.01 vs. untreated CSCs (Ctr); # p < 0.05 vs. Etoposide-treated CSCs; ## p < 0.01 vs. Etoposide-treated CSCs. Altogether these results demonstrate that although Etoposide alone significantly de- creased the level of GSH, GSSG amount was not enhanced and, in parallel, ROS and MDA Considering that mitochondrial oxidative phosphorylation can generate ROS, this parameter was evaluated in both CSC populations. Altogether these results demonstrate that although Etoposide alone significantly de- creased the level of GSH GSSG amount was not enhanced and in parallel ROS and MDA Considering that mitochondrial oxidative phosphorylation can generate ROS, this parameter was evaluated in both CSC populations. creased the level of GSH, GSSG amount was not enhanced and, in parallel, ROS and MDA production did not increase. In this context, we cannot exclude that other antioxidant sys- tems might contribute to the maintenance of CSC oxidative status [23,62] and in support p p p As shown in Figure 9a, after two-week-exposure to Etoposide, alone or in combination with C2-4 or SSZ, no changes in ROS production were observed in HTLA-CSCs in respect Antioxidants 2021, 10, 691 14 of 22 14 of 22 of untreated CSCs. Instead, two-week treatment of ER-CSCs with Etoposide, alone or combined with C2-4 or SSZ, induced a 60% reduction of ROS levels in respect of untreated CSCs (Figure 9b). After ﬁve weeks of all treatments, no changes in ROS production were detected in ER-CSCs (Figure 9c). 15 of 23 of untreated CSCs. Instead, two-week treatment of ER-CSCs with Etoposide, alone or combined with C2-4 or SSZ, induced a 60% reduction of ROS levels in respect of untreated CSCs (Figure 9b). After ﬁve weeks of all treatments, no changes in ROS production were detected in ER-CSCs (Figure 9c). 15 of 23 Figure 9. ROS production in both CSC populations treated for two (a,b) or five weeks (c). 3.5. Co-Treatments Are Able to Induce Lipid Peroxidation in ER-CSCs tments Are Able to Induce Down-Regulation of GPX4 Activity and of ZEB-1 dering that xCT inhibition by SSZ or erastin has been demonstrated to induce de production and ferroptosis, a form of non-apoptotic cell death consequent GSH and of Glutathione peroxidase 4 (GPX4) activity [64,65], the role of this s investigated. wn in Figure 10, after five weeks, in untreated ER-CSCs, the activity of GPX4 d by 20% in respect of that observed after two weeks. Two-week-exposure to Altogether these results demonstrate that although Etoposide alone signiﬁcantly de- creased the level of GSH, GSSG amount was not enhanced and, in parallel, ROS and MDA production did not increase. In this context, we cannot exclude that other antiox- idant systems might contribute to the maintenance of CSC oxidative status [23,62] and in support of these ﬁndings, it has been recently found that the ability of breast CSCs to maintain low ROS production confers cancer stemness and drug resistance [63]. Interest- ingly, the co-treatments which are able to activate glycolysis, reduce GSH levels without altering cell redox state and induce lipid peroxidation are also effective in counteracting ER-CSC propagation. alone or in combination with SSZ, reduced the activity of GP 4 by 30% com at analyzed in untreated CSCs. Instead, two-week co-treatments with C2-4 fur- sed GPX4 activity by 38% in respect of Etoposide alone and by 55% in compar- 3.6. Co-Treatments Are Able to Induce Down-Regulation of GPX4 Activity and of ZEB-1 Expression y y p p y p control. Moreover, after five weeks, Etoposide alone or in combination with ased GPX4 activity by 40% in respect of untreated CSCs, while SSZ co-treat- her inhibited the activity by 60% in comparison to Etoposide-treated CSCs and espect of the control (Figure 10). Considering that xCT inhibition by SSZ or erastin has been demonstrated to induce lipid peroxide production and ferroptosis, a form of non-apoptotic cell death consequent to a loss of GSH and of Glutathione peroxidase 4 (GPX4) activity [64,65], the role of this enzyme was investigated. 15 of 22 15 of 22 Antioxidants 2021, 10, 691 As shown in Figure 10, after ﬁve weeks, in untreated ER-CSCs, the activity of GPX4 was reduced by 20% in respect of that observed after two weeks. Two-week-exposure to Etoposide, alone or in combination with SSZ, reduced the activity of GPX4 by 30% compared to that analyzed in untreated CSCs. Instead, two-week co-treatments with C2-4 further decreased GPX4 activity by 38% in respect of Etoposide alone and by 55% in comparison to the control. Moreover, after ﬁve weeks, Etoposide alone or in combination with C2-4 decreased GPX4 activity by 40% in respect of untreated CSCs, while SSZ co- treatments further inhibited the activity by 60% in comparison to Etoposide-treated CSCs and by 75% in respect of the control (Figure 10). REVIEW 16 of 23 role in supporting CSC generation [69]. In fact, ZEB-1 has been found to have a pleiotropic role in controlling lipid metabolism, growth, proliferation and death of CSCs [68,70]. Figure 10. GPX4 activity in ER-CSC populations treated for two or five weeks. ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, adminis- tered once a week alone or in combination with C2-4 or SSZ. Results were reported as nmol/min/mg protein. Bar graph summarizes quantitative data of means ± S.E.M. of three inde- pendent experiments. p < 0.01 vs. untreated ER-CSCs (Ctr); °° p < 0.01 vs. Etoposide-treated ER- CSCs. Figure 10. GPX4 activity in ER-CSC populations treated for two or ﬁve weeks. ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. Results were reported as nmol/min/mg protein. Bar graph summarizes quantitative data of means ± S.E.M. alone or in combination with SSZ, reduced the activity of GP 4 by 30% com at analyzed in untreated CSCs. Instead, two-week co-treatments with C2-4 fur- sed GPX4 activity by 38% in respect of Etoposide alone and by 55% in compar- 3.6. Co-Treatments Are Able to Induce Down-Regulation of GPX4 Activity and of ZEB-1 Expression of three independent experiments. p < 0.01 vs. untreated ER-CSCs (Ctr); ◦◦p < 0.01 vs. Etoposide-treated ER-CSCs. Figure 10. GPX4 activity in ER-CSC populations treated for two or five weeks. ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, adminis- tered once a week alone or in combination with C2-4 or SSZ. Results were reported as nmol/min/mg protein. Bar graph summarizes quantitative data of means ± S.E.M. of three inde- pendent experiments. p < 0.01 vs. untreated ER-CSCs (Ctr); °° p < 0.01 vs. Etoposide-treated ER- CSCs. Figure 10. GPX4 activity in ER-CSC populations treated for two or ﬁve weeks. ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. Results were reported as nmol/min/mg protein. Bar graph summarizes quantitative data of means ± S.E.M. of three independent experiments. p < 0.01 vs. untreated ER-CSCs (Ctr); ◦◦p < 0.01 vs. Etoposide-treated ER-CSCs. Based on these considerations and since Etoposide, alone or in combination with C2- 4 or SSZ, was able to totally prevent ER-CSC formation, the subsequent analyses were focused on the role played by EMT in drug resistance. As shown in Figure 11, both untreated HTLA- and ER-CSCs expressed N-Cadherin and ZEB-1, two proteins favoring the EMT process. Two-week-treatments of HTLA-CSCs fully counteracted the expression of these two proteins while, interestingly, two-week- Etoposide treatment of ER-CSCs reduced the expression of N-Cadherin and ZEB-1 by 40% and 70% respectively in comparison to untreated CSCs (Figure 11a b) After five weeks Since GPX4 plays a crucial role in the suppression of lipid peroxidation by stimulating a GSH-dependent reduction of phospholipid hydroperoxides [64,66], GPX4 inhibitors could be employed as anticancer drugs capable of inducing ferroptosis [67]. Interestingly, it has been reported that chemoresistant cancer cells, that are preserved from ferroptosis through GPX4 activation, expressed high levels of ZEB-1 [68]. ZEB-1 is a protein involved in the epithelial to mesenchymal transition (EMT) process, which plays a fundamental role in supporting CSC generation [69]. In fact, ZEB-1 has been found to have a pleiotropic role in controlling lipid metabolism, growth, proliferation and death of CSCs [68,70]. and 70%, respectively, in comparison to untreated CSCs (Figure 11a,b). alone or in combination with SSZ, reduced the activity of GP 4 by 30% com at analyzed in untreated CSCs. Instead, two-week co-treatments with C2-4 fur- sed GPX4 activity by 38% in respect of Etoposide alone and by 55% in compar- 3.6. Co-Treatments Are Able to Induce Down-Regulation of GPX4 Activity and of ZEB-1 Expression After five weeks, the combination of Etoposide with C2-4 reduced the expression of both proteins by 50% and co-treatment with SSZ decreased ZEB-1 levels by 90% in respect of untreated CSCs (Fi 11 b) g p g p Based on these considerations and since Etoposide, alone or in combination with C2-4 or SSZ, was able to totally prevent ER-CSC formation, the subsequent analyses were focused on the role played by EMT in drug resistance. (Figure 11a,b). Changes in the expression of N-Cadherin and ZEB-1 were not accompanied by sig- nificant alterations in the expression of Vimentin and β-catenin, another two EMT-related proteins (Figure 11c). In addition, as shown in Figure 11d, only treated HTLA-CSCs ex- pressed Claudin, a protein inhibiting the EMT process. Therefore, it is conceivable that co-treatments of Etoposide with SSZ/C2-4 are able to counteract the ability of ER-CSCs to generate spheres and to maintain the EMT phenotype. This data, although in vitro, supports the results obtained in early clinical trials demon- h h d f d/ h E T f l As shown in Figure 11, both untreated HTLA- and ER-CSCs expressed N-Cadherin and ZEB-1, two proteins favoring the EMT process. Two-week-treatments of HTLA-CSCs fully counteracted the expression of these two proteins while, interestingly, two-week- Etoposide treatment of ER-CSCs reduced the expression of N-Cadherin and ZEB-1 by 40% and 70%, respectively, in comparison to untreated CSCs (Figure 11a,b). After ﬁve weeks, the combination of Etoposide with C2-4 reduced the expression of both proteins by 50% and co-treatment with SSZ decreased ZEB-1 levels by 90% in respect of untreated CSCs (Figure 11a,b). 16 of 22 r, it has SCs [78] 16 of 22 r, it has SCs [78] Antioxidants 2021, 10, 691 Protein levels of N-Cadherin (a), ZEB-1 (b), Vimentin (c), β-Catenin (c) and Claudin (d) in both CSC populatio ive weeks. HTLA-CSCs and ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or wit administered once a week alone or in combination with C2-4 or SSZ. Immunoblots shown are representativ nt experiments. To ensure normalized protein content all filters were stained with Red Ponceau. Bar graph su 11. Protein levels of N-Cadherin (a), ZEB-1 (b), Vimentin (c), β-Catenin (c) and Claudin (d) in both CSC populat d for two or ﬁve weeks. alone or in combination with SSZ, reduced the activity of GP 4 by 30% com at analyzed in untreated CSCs. Instead, two-week co-treatments with C2-4 fur- sed GPX4 activity by 38% in respect of Etoposide alone and by 55% in compar- 3.6. Co-Treatments Are Able to Induce Down-Regulation of GPX4 Activity and of ZEB-1 Expression HTLA-CSCs and ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. Immunoblots sh presentative of three independent experiments. To ensure normalized protein content all ﬁlters were stained onceau. Bar graph summarizes quantitative data of means ± S.E.M. of three independent experiments. p < treated CSCs (Ctr); * p < 0.001 vs. untreated CSCs (Ctr); ◦◦p < 0.01 vs. Etoposide-treated CSCs; ◦◦◦p < 0.00 ide-treated CSCs. Protein levels of N-Cadherin (a), ZEB-1 (b), Vimentin (c), β-Catenin (c) and Claudin (d) in both CSC populat ive weeks. HTLA-CSCs and ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or w administered once a week alone or in combination with C2-4 or SSZ. Immunoblots shown are representat nt experiments. To ensure normalized protein content all filters were stained with Red Ponceau. Bar graph s 11. Protein levels of N-Cadherin (a), ZEB-1 (b), Vimentin (c), β-Catenin (c) and Claudin (d) in both CSC popul d for two or ﬁve weeks. HTLA-CSCs and ER-CSCs were treated three times a week with 0.1 µM C2-4, with with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. Immunoblots s presentative of three independent experiments. To ensure normalized protein content all ﬁlters were stained onceau. Bar graph summarizes quantitative data of means ± S.E.M. of three independent experiments. p treated CSCs (Ctr); * p < 0.001 vs. untreated CSCs (Ctr); ◦◦p < 0.01 vs. Etoposide-treated CSCs; ◦◦◦p < 0.0 side treated CSCs ure 11. Protein levels of N-Cadherin (a), ZEB-1 (b), Vimentin (c), β-Catenin (c) and Claudin (d) in both CSC populations trea two or five weeks. HTLA-CSCs and ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 poside, administered once a week alone or in combination with C2-4 or SSZ. Immunoblots shown are representative of th ependent experiments. To ensure normalized protein content all filters were stained with Red Ponceau. Bar graph summar Figure 11. Protein levels of N-Cadherin (a), ZEB-1 (b), Vimentin (c), β-Catenin (c) and Claudin (d) in both CSC populations treated for two or ﬁve weeks. alone or in combination with SSZ, reduced the activity of GP 4 by 30% com at analyzed in untreated CSCs. Instead, two-week co-treatments with C2-4 fur- sed GPX4 activity by 38% in respect of Etoposide alone and by 55% in compar- 3.6. Co-Treatments Are Able to Induce Down-Regulation of GPX4 Activity and of ZEB-1 Expression HTLA-CSCs and ER-CSCs were treated three times a week with 0.1 µM C2-4, with 5 µM SSZ or with 1.25 µM Etoposide, administered once a week alone or in combination with C2-4 or SSZ. Immunoblots shown are representative of three independent experiments. To ensure normalized protein content all ﬁlters were stained with Red Ponceau. Bar graph summarizes quantitative data of means ± S.E.M. of three independent experiments. p < 0.01 vs. untreated CSCs (Ctr); * p < 0.001 vs. untreated CSCs (Ctr); ◦◦p < 0.01 vs. Etoposide-treated CSCs; ◦◦◦p < 0.001 vs. Etoposide-treated CSCs. Antioxidants 2021, 10, 691 17 of 22 17 of 22 Changes in the expression of N-Cadherin and ZEB-1 were not accompanied by sig- niﬁcant alterations in the expression of Vimentin and β-catenin, another two EMT-related proteins (Figure 11c). In addition, as shown in Figure 11d, only treated HTLA-CSCs expressed Claudin, a protein inhibiting the EMT process. Therefore, it is conceivable that co-treatments of Etoposide with SSZ/C2-4 are able to counteract the ability of ER-CSCs to generate spheres and to maintain the EMT phe- notype. This data, although in vitro, supports the results obtained in early clinical trials demonstrating that the disruption of cancer stemness and/or the EMT process is a useful approach in ﬁghting tumor recurrence [71,72]. pp g g Furthermore, in ER-CSCs, which become sensitive to the drug, GPX4 activity is reduced and is accompanied by ZEB-1 down-regulation leading to ferroptotic death [73,74]. The results obtained in co-treated CSCs conﬁrm the ferroptotic-inducer action of SSZ [75] and suggest that C2-4, the PKCα inhibitor, is able to trigger ferroptosis of ER-CSCs. The relationship between PKCα and ferroptosis has been previously suggested in dopamin- ergic cells [76] but, to our knowledge, this is the ﬁrst time it has been demonstrated that the anti-PKCα peptide can sensitize CSCs to chemotherapy by inhibiting GPX4 and inducing ferroptosis. PKCα activation has been implicated in promoting cancer progression [18,77] and in the formation and survival of cancer stem cells [78] Moreover, it has been demon- strated that the pharmacologic inhibition of PKCα can target breast CSCs [78] and override ZEB1-induced chemoresistance in hepatocarcinoma [79]. 3.7. Chemoresistance of ER-CSCs Is Characterized by An Efﬁcient OXPHOS Metabolism, High GSH Levels, ZEB-1 Up-Regulation and GPX4 Activation 3.7. Chemoresistance of ER-CSCs Is Characterized by An Efﬁcient OXPHOS Metabolism, High GSH Levels, ZEB-1 Up-Regulation and GPX4 Activation 3.7. alone or in combination with SSZ, reduced the activity of GP 4 by 30% com at analyzed in untreated CSCs. Instead, two-week co-treatments with C2-4 fur- sed GPX4 activity by 38% in respect of Etoposide alone and by 55% in compar- 3.6. Co-Treatments Are Able to Induce Down-Regulation of GPX4 Activity and of ZEB-1 Expression 18 of 22 19 of 23 Antioxidants 2021, 10, 691 Antioxidants 2021 10 x FO 12. Loading (a) and score (b) plots. (a) The variables which sustain chemoresistance (green rectangle: ATP, G have negative loadings on PC1 values, while the variables that are able to induce chemosensitivity (red rectan , lactate release and glucose consumption) have positive loadings on PC1; variables N-CAD, OCR and Vim ha s on PC2 (b) In the score plot, the black-colored samples were treated for two weeks, while the red-colored on weeks. ure 12. Loading (a) and score (b) plots. (a) The variables which sustain chemoresistance (green rectangle: A O, ZEB-1, GPX4) have negative loadings on PC1 values, while the variables that are able to induce chemosensi tangle: MDA production, lactate release and glucose consumption) have positive loadings on PC1; variable CR and Vim have instead high loadings on PC2 (b) In the score plot, the black-colored samples were treate eks, while the red-colored ones were treated for ﬁve weeks. gure 12. Loading (a) and score (b) plots. (a) The variables which sustain chemoresistance (green rectangle: ATP, GSH, P/O, ZE PX4) have negative loadings on PC1 values, while the variables that are able to induce chemosensitivity (red rectangle: MDA p uction, lactate release and glucose consumption) have positive loadings on PC1; variables N-CAD, OCR and Vim have instead h adings on PC2 (b) In the score plot, the black-colored samples were treated for two weeks, while the red-colored ones were trea r five weeks. Figure 12. Loading (a) and score (b) plots. (a) The variables which sustain chemoresistance (green rectangle: ATP, GSH, P/O, ZEB-1, GPX4) have negative loadings on PC1 values, while the variables that are able to induce chemosensitivity (red rectangle: MDA production, lactate release and glucose consumption) have positive loadings on PC1; variables N-CAD, OCR and Vim have instead high loadings on PC2 (b) In the score plot, the black-colored samples were treated for two weeks, while the red-colored ones were treated for ﬁve weeks. gure 12. Loading (a) and score (b) plots. alone or in combination with SSZ, reduced the activity of GP 4 by 30% com at analyzed in untreated CSCs. Instead, two-week co-treatments with C2-4 fur- sed GPX4 activity by 38% in respect of Etoposide alone and by 55% in compar- 3.6. Co-Treatments Are Able to Induce Down-Regulation of GPX4 Activity and of ZEB-1 Expression Chemoresistance of ER-CSCs Is Characterized by An Efﬁcient OXPHOS Metabolism, High GSH Levels, ZEB-1 Up-Regulation and GPX4 Activation In order to identify which variables are more determinant in maintaining cancer cell survival and, consequently, responsible for chemoresistance, Principal Component Analysis (PCA) was carried out by collecting all the analyzed metabolic, biochemical and morphological variables. The ﬁrst two components explain 45.2% and 19.5% of the variance (64.6% in total). The loading plot (Figure 12a) shows the correlation between the following variables: P/O ratio, ATP production, GSH and ZEB-1 expression and GPX4 activity. They are all characterized by negative loadings on PC1 (grouped in the green rectangle) and are all related since they allow the propagation of ER-CSCs. On the other side of the plot (positive loadings on PC1) another group of correlated variables can be detected (red rectangle): lactate release, glucose consumption and MDA production. They reduce ER- CSC propagation, and are responsible for their susceptibility to the co-treatments. The two groups of correlated variables are negatively correlated, since they are opposite compared to the origin (red cross in the plot). A third group of correlated variables can be detected, made by N-cad, OCR and Vim. These variables, having high loadings on PC2, are uncorrelated with the other ones. As expected, the score plot (Figure 12b) shows that the two-week and ﬁve-week controls are close to each other, having negative PC1 scores. Therefore, they are characterized by the high values of the variables grouped into the green rectangle and the low values of the variables grouped into the red rectangle in the loading plot. The effect of the three different treatments and of the different times, together with the metabolic modiﬁcations involved, can be estimated by taking into account the distance and the direction of the different points in the score plot, compared to the controls. First of all, it can be seen that for all of them the effect after ﬁve weeks is larger than the effect after two weeks. It can also be seen that the treatment with Etoposide by itself is much less efﬁcient than the other two treatments in which Etoposide is combined with another drug. This treatment has almost no effect after two weeks, while after ﬁve weeks it leads to a more positive score on PC1, this meaning a reduction of the “green” variables and an increase of the “red” ones. alone or in combination with SSZ, reduced the activity of GP 4 by 30% com at analyzed in untreated CSCs. Instead, two-week co-treatments with C2-4 fur- sed GPX4 activity by 38% in respect of Etoposide alone and by 55% in compar- 3.6. Co-Treatments Are Able to Induce Down-Regulation of GPX4 Activity and of ZEB-1 Expression (a) The variables which sustain chemoresistance (green rectangle: ATP, GSH, P/O, ZE X4) have negative loadings on PC1 values, while the variables that are able to induce chemosensitivity (red rectangle: MDA ction, lactate release and glucose consumption) have positive loadings on PC1; variables N-CAD, OCR and Vim have instead h dings on PC2 (b) In the score plot, the black-colored samples were treated for two weeks, while the red-colored ones were tre five weeks. Figure 12. Loading (a) and score (b) plots. (a) The variables which sustain chemoresistance (green rectangle: ATP, GSH, P/O, ZEB-1, GPX4) have negative loadings on PC1 values, while the variables that are able to induce chemosensitivity (red rectangle: MDA production, lactate release and glucose consumption) have positive loadings on PC1; variables N-CAD, OCR and Vim have instead high loadings on PC2 (b) In the score plot, the black-colored samples were treated for two weeks, while the red-colored ones were treated for ﬁve weeks. ure 12. Loading (a) and score (b) plots. (a) The variables which sustain chemoresistance (green rectangle: ATP, GSH, P/O, ZE X4) have negative loadings on PC1 values, while the variables that are able to induce chemosensitivity (red rectangle: MDA ction, lactate release and glucose consumption) have positive loadings on PC1; variables N-CAD, OCR and Vim have instead h dings on PC2 (b) In the score plot, the black-colored samples were treated for two weeks, while the red-colored ones were tre five weeks. Figure 12. Loading (a) and score (b) plots. (a) The variables which sustain chemoresistance (green rectangle: ATP, GSH, P/O, ZEB-1, GPX4) have negative loadings on PC1 values, while the variables that are able to induce chemosensitivity (red rectangle: MDA production, lactate release and glucose consumption) have positive loadings on PC1; variables N-CAD, OCR and Vim have instead high loadings on PC2 (b) In the score plot, the black-colored samples were treated for two weeks, while the red-colored ones were treated for ﬁve weeks. Antioxidants 2021, 10, 691 19 of 22 19 of 22 The co-treatment with SSZ has a much greater effect, already relevant after two weeks (corresponding to the effect after ﬁve weeks of Etoposide by itself). Since the direction is the same as the one already observed with Etoposide, it can be concluded that SSZ just ampliﬁes the effect of Etoposide. alone or in combination with SSZ, reduced the activity of GP 4 by 30% com at analyzed in untreated CSCs. Instead, two-week co-treatments with C2-4 fur- sed GPX4 activity by 38% in respect of Etoposide alone and by 55% in compar- 3.6. Co-Treatments Are Able to Induce Down-Regulation of GPX4 Activity and of ZEB-1 Expression Instead, when looking at the co-treatment with C2-4 it can be seen that a strong effect is also present already at two weeks, but the trajectory followed is quite different. In this case an increase along PC1 together with a decrease along PC2 is observed, corresponding to a decrease of variables N-Cad, OCR and Vim. It can therefore be concluded that C2-4 not only ampliﬁes the effect of Etoposide, but also produces different metabolic variations. Collectively, these results further underline that CSC chemoresistance can be the result of a complex cellular adaptation that involves several metabolic and biochemical pathways and, therefore, it is necessary “to attack the enemy” simultaneously from several fronts in order to bypass this adaptation. 4. Conclusions Even if the mechanism underlying CSC chemoresistance has not yet been fully clari- ﬁed, the results herein reported suggest that drug refractoriness is maintained as long as CSCs are able to keep a ﬁne balance between correlated molecular actors. In fact, the main- tenance of an oxidative metabolism, high levels of GSH and the expression of ZEB-1 concur to allow the survival of resistant CSCs. Therefore, an approach able to down-regulate these molecular targets and to induce ferroptosis may be the winning strategy to counteract drug resistance. In this context, the results herein reported suggest that PKCα might be a new pharmacological target able to ﬁght chemoresistance of cancer stem cells by preventing drug-induced metabolic adaptation and triggering ferroptotic death. Author Contributions: Conceptualization, C.D. and B.M.; methodology, B.M., N.T., L.M., A.S., A.R. and F.U.; formal analysis, C.D., B.M., R.L., E.F.; software, R.L. and E.F.; validation, C.D., B.M.; investigation, B.M., L.M., A.S., N.T., C.C., S.R., O.G., G.E.V. and A.R.; data curation, C.D. and B.M.; writing-original draft, B.M.; writing-review and editing, C.D., B.M., N.T., M.A.P. and U.M.M.; supervision, C.D. and B.M. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by University of Genoa. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: We would like to thank Giuseppe Catalano (DIMES-University of Genoa) for his technical assistance and Suzanne Patten for her language editing. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 6. Seeger, R.C.; Brodeur, G.M.; Sather, H.; Dalton, A.; Siegel, S.E.; Wong, K.Y.; Hammond, D. Association of multiple copies of the N-myc oncogene with rapid progression of neuroblastomas. N. Engl. J. Med. 1985, 313, 1111–1116. [CrossRef] 4. Maris, J.M.; Hogarty, M.D.; Bagatell, R.; Cohn, S.L. Neuroblastoma. Lancet 2007, 369, 2106–2120. [CrossRef] 5. Brodeur, G.M.; Seeger, R.C.; Schwab, M.; Varmus, H.E.; Bishop, J.M. Ampliﬁcation of N-myc in untreated human neuroblastomas correlates with advanced disease stage. Science 1984, 224, 1121–1124. [CrossRef] [PubMed] References Evolution of the cancer stem cell model. Cell Stem Cell 2014, 14, 275–291. [CrossRef] 12. Nagano, O.; Okazaki, S.; Saya, H. 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https://openalex.org/W2947552323	https://www.e3s-conferences.org/articles/e3sconf/pdf/2019/23/e3sconf_form2018_04061.pdf	English	null	Experience in designing and operating the buildings located on subsiding and heaving soils of the Minusinsk Hollows	E3S web of conferences	2,019	cc-by	4,320	1. Introduction The Minusinsk Hollows are located between the mountains: Kuznetskiy Alatau on the west, the spurs of the Western Sayan on the South, the ridges of the Eastern Sayan on the east and the foothills of the Eastern Sayan and Kuznetsk Alatau on the north. The Yenisei, Abakan, and Tuba rivers play a significant role in the formation of the modern relief of the South Minusinsk Hollows. So do the Syda and Erba rivers for the formation of the Mid- Minusinsk Hollow and the Bely and Cherny Iyus and Chulym rivers – for the North Minusinsk Hollow [1, 2]. Experience in designing and operating the buildings located on subsiding and heaving soils of the Minusinsk Hollows Dmitry Strelnikov* and Oleg Khalimov Dmitry Strelnikov and Oleg Khalimov Khakas Technical Institute – SFU branch, Komarova 15, Abakan 655016 Russian Federation Khakas Technical Institute – SFU branch, Komarova 15, Abakan 655016 Russian Federation Abstract. The article presents the experience of design, expert research within the period of construction and operation of the buildings located on subsiding soils. It describes the experiment in conducting the organized soaking of a mounted house made of reinforced-concrete slabs and claydite-concrete panels. It shows the absence of danger of frost heaving while soaking the subsiding soils. There are tables demonstrating the expert studies during the inspections of deformable buildings and long- term observations over their behavior in freezing and thawing depending on the groundwater level and pressures under the bottom of the foundations. As a result of the analysis of long-term studies it has been established that the building load contributes to the reduction of frost heaving for sandy soils (fine and dust sands). In case of clayey soils, in contrast to the views on this problem emerged at the end of the 20th century, it is impossible to extinguish the migration process using pressure. If the migration flow slows down under the foundation bottom, it occurs due to the higher rate of soil freezing arising from the high thermal conductivity of the foundation body and the formation of vertical ice lenses on the borders of tension zones. The most important thing showed herein is that the thawing process in these lenses provokes the process of stability loss, where the soil protrudes from the foundation bottom. The long-term studies of frost heaving in a severe continental climate of the Minusinsk hollows enabled the development of the most effective direction of the anti-heave stabilization. E3S Web of Conferences 97, 04061 (2019) FORM-2019 E3S Web of Conferences 97, 04061 (2019) FORM-2019 https://doi.org/10.1051/e3sconf/20199704061 E3S Web of Conferences 97, 04061 (2019) FORM-2019 https://doi.org/10.1051/e3sconf/20199704061 The climate of the region is severe continental with warm summers and cold winters. The average annual temperature varies from - 0.5 to + 2.0 ° C. The temperature in the intermontain basins is lower as compared to that of the surrounding mountain slopes, since the cold air flows down the slopes and stagnates in the basins. In this regard, the low temperatures and the shallow snow cover contribute to the deep freezing of the soils. However, these are not only low temperatures which change its structure, but also a high wind [3,4]. The origin of the soils prone to subsidence, as a rule, is wind erosion. The capacity of the subsiding soils varies from 2 to 20 meters. So, for example, in the most developing cities of Abakan and Chernogorsk, the subsiding depth does not exceed two meters. However, in Chernogorsk there are areas with fault lines, filled with aeolian deposits to the depth of 18 meters. In the eastern part of the Minusinsk Hollow, in the Minusinsk, Idrinsky, Karatuzsky, Shushensky districts, the depth of the subsidence varies from 8 to 15 meters. The scheme is shown on Figure 1 [5]. Fig. 1. The location map of loess rocks in the southern part of the Krasnoyarsk territory * Corresponding author: Lacky_traine@mail.ru © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). E3S Web of Conferences 97, 04061 (2019) FORM-2019 Fig. 1. The location map of loess rocks in the southern part of the Krasnoyarsk territory The relevance of the topic is determined by the needs of the foundations design practice, where the fear of these specific soils leads designers to the use of driven piles. However, such costly solutions can be eliminated by applying anti-heave and anti-subsiding stabilization. But its implementation requires a continuous improvement of both research technology (from models to production experiments) and the promotion of innovation into design practice using the geotechnical consulting at all stages of the building life cycle. 2 2 https://doi.org/10.1051/e3sconf/20199704061 E3S Web of Conferences 97, 04061 (2019) FORM-2019 E3S Web of Conferences 97, 04061 (2019) E3S Web of Conferences 97, 04061 (2019) FORM-2019 https://doi.org/10.1051/e3sconf/20199704061 The deformations from soaking under the load from the structures of a dwelling house without the imposed load (from people, equipment, materials used in the functional process) were homogeneous. The existing cracks in the concrete-enveloped joints of the panels did not open. The drawbacks were that the porch dropped one step down and the zero mark occurred in relation to the planned mark. Thirty-six years of operation of these houses have shown the working capacity of the foundation soils. There have been no deformations reducing the reliability of the supporting structures. However, this method of organized or regulated soaking has not yet been adopted either in Krasnoyarsk or in the south of the Krasnoyarsk Territory. At the same time, the entrepreneurs, who do not want to bury money in the foundations, are considering more rational options for building foundations on the subsiding soils. p g g While conducting a survey in the Shushensky district, the subsiding soils were found and then investigated and the results obtained are presented in Table 1. Table 1. Calculation of the type of soil conditions for subsidence Depth of soil sampl- ing Value of relative subsidence under Consis- tency of water- deposited soil t/m3 Natural pressure, mPa Relative subsidence under natural pressure, εsl Strength of layer Subsi- dence Initial subsidence pressure, Psl 0,200 0,300 2,0 0,039 0,048 1,89 0,038 0,012 2,00 0,024 0,033 3,0 0,039 0,047 1,87 0,057 0,012 1,00 0,012 0,045 4,0 0,039 0,050 1,88 0,076 0,018 1,00 0,018 0,033 5,0 0,035 0,046 1,88 0,095 0,018 1,00 0,018 0,043 6,0 0,032 0,040 1,87 0,114 0,018 1,00 0,018 0,056 7,0 0,020 0,020 1,89 0,133 0,012 1,00 0,012 0.091 8,0 0,026 0,040 1,89 0,152 0,022 1,00 0,022 0,050 9,0 0,029 0,029 1,91 0,171 0,020 1,00 0,020 0,067 10,0 0,024 0,029 1,93 0,190 0,021 1,00 0,021 0,059 11,0 0,024 0,018 1,93 0,209 0,017 1,00 0,017 0,091 12,0 0,026 0,027 1,95 0,229 0,024 1,00 0,024 0,082 13,0 0,015 0,009 1,94 0,248 0,011 1,00 0,011 0,192 14,0 0,010 0,012 1,96 0,268 0,012 1,00 0,012 0,221 15,0 0,013 0,006 1,98 0,288 0,009 1,00 0,000 0,215 16,0 0,017 0,008 1,97 0,308 0,013 1,00 0,013 0,179 17,0 0,011 0,006 1,98 0,328 0,010 1,00 0,010 0,248 18,0 0,005 0,007 1,98 0,348 0,008 1,00 0,000 0,419 19,0 0,004 0,005 1,99 0,368 0,006 1,00 0,000 0,550 21,0 0,004 0,005 1,98 0,408 0,007 2,00 0,000 0,550 Table 1. 2. Example of improving the design of buildings located on subsiding soils Khakas Technical Institute has experience in the design, construction and operation of single-storey buildings made of reinforced concrete slabs and claydite-concrete panels in the village Solontsy of the city of Krasnoyarsk. The subsidence column in this village varies from 5 to 12 meters. The value of subsidence of gravity load while soaking according to calculations in the lower, most powerful layer of subsiding soils reaches up to 15 cm. In accordance with the design solution, it was necessary to load 12-gauge piles with making pilot holes (Figure 2). Fig. 2. Original version proposed by the design institute Fig. 2. Original version proposed by the design institute However, the customer agreed to the proposal of Khakas Technical Institute and implemented an organized soaking method, which reduced the time and cost of construction (Figure 3). Fig. 3. Implemented version of construction with method of soils soaking Fig. 3. Implemented version of construction with method of soils soaking The houses were built according to the following technology: erection of strip foundations on the natural base, installation of basement panels, floors above the basement, walls of the first floor, floors above the first floor and a roof. After that, the base soils were soaked by pouring water into the basement. The infiltration occurred due to the infiltration of water without drilling the pilot holes and filling the sand into them, as later was required by the standards for carrying out the procedure of organized soaking. All the works were carried out in 1982. The deformations of the unfinished objects were as follows: at the house located down the slope - 152mm, at the house located forty meters up the slope - 127mm. 3 3 E3S Web of Conferences 97, 04061 (2019) FORM-2019 Fig. 4. Chart of freezing and ground water level in winter Fig. 4. Chart of freezing and ground water level in winter This chart shows the lowering of the surface water at the end of autumn and throughout winter. The main reasons in this case are snow precipitations which do not lead to the filling of soil interstices with water, and freezing of the surface [9]. Depending on the temperature of the outside air, there is a change in the speed of freezing of the foundation soil [10]. E3S Web of Conferences 97, 04061 (2019) FORM-2019 Calculation of the type of soil conditions for subsidence Thus, the test showed that the soil subsidence of gravity load was 0.252 m or 25.20 cm. The soaking soil refers to type II [6].However, the customer decides to abandon costly decisions on pile-sinking. He assumes the risk of an emergency condition with possible soaking. And it is quite possible that these hopes are real. Moreover, in the event of possible deformations, the progressive designer will prescribe a system of geotechnical monitoring of the controlled parameters. However, the basis for the state expert examination of project documentation should be a regulatory document, that is territorial construction standards. These standards exist in Moscow and in the Leningrad Region. The nearest regulatory documents to the south of the Krasnoyarsk Territory are in the Tomsk Region. And if the “small” Khakassia cannot allow the development of such standards, then the vast Krasnoyarsk Territory should develop such standards and Khakassia’s geotechnicians can contribute to the results of geotechnical monitoring at the facilities located in the Republic and in the South of the Krasnoyarsk Territory. 4 4 4 E3S Web of Conferences 97, 04061 (2019) FORM-2019 https://doi.org/10.1051/e3sconf/20199704061 2.1 Analysis of the work of experimental objects located on the heaving soi 1- Possible trends depending on the speed of soil freezing; 2 – trend of ground water level; 3 – overlap by the freezing point of the position of the ground water level; 4 – distance from freezing point to ground water level. 1- Possible trends depending on the speed of soil freezing; 2 trend of ground water level; 3 – overlap by the freezing point of the position of the ground water level; 4 – distance from freezing point to ground water level. 2.1 Analysis of the work of experimental objects located on the heaving soi However, the greatest danger in the Republic of Khakassia is not the subsiding soils, not the fear of them among the designers, but the danger from the frost heaving of the soils, which especially arises during the construction period. This fear leads designers to eliminate this danger by sinking piles at those points where it is possible to use foundations on the natural base. One of the reasons why designers are afraid of frost heaving is that the criteria for heaving assessment are not objective. So, three levels for the analysis of soils heaving, presented in the landmark work of Nevzorov, are of undoubted interest [7,8]. However, for most designers, the main document is a report on engineering and geological surveys, where almost all the soils with possible autumn soaking are regarded as heaving, though laboratory experiments do not show this. y p The years of experience in expert study of the causes for the occurrence of an emergency condition of buildings and structures show that in the conditions of the Minusinsk Hollows the frost heaving especially during construction is more dangerous than subsidence. Therefore, if the soil has moved from the subsiding to the non-subsiding type in the result of preliminary or organized soaking, then according to the experts of the survey organizations and designers, it will become a heaving one. However, this is not what occurs in practice. The frost heaving reliably develops when there is a recharge from ground waters. Of course, with increased natural humidity, when the index of liquidity exceeds zero mark, the process of migration and frost heaving is underway. But the coefficient of water accumulation in this case (without recharge from ground waters) is much lower (Figure 4). 1- Possible trends depending on the speed of soil freezing; 2 – trend of ground water level; 3 – overlap by the freezing point of the position of the ground water level; 4 – distance from freezing point to ground water level. 1- Possible trends depending on the speed of soil freezing; 2 – trend of ground water level; 1- Possible trends depending on the speed of soil freezing; 2 – trend of ground water level; 3 – overlap by the freezing point of the position of the ground water level; 4 – distance from freezing point to ground water level. 2.2 Research methodology of frost heaving and cryogenic structures The laboratory studies of cryogenic textures in the conditions of frost heaving were carried out in the portable ground trays and in the stationary ground tray. It is established that the most effective cryogenic migration occurs under the slow freezing in the conditions of high negative temperatures (not lower than -3 ° C). To study the frost heaving and the formation of a cryogenic texture at the border of the soils both treated with KCl and non-treated, the experiments were carried out according to the following procedure: a layer of 100 mm sand soil was placed on a gravel drainage pad; the treated KCl soil was tightly placed through the polyethylene film, which was then removed; the tray was taken out to the open space at negative temperatures for four days; after freezing the body of frozen soil was opened and sawn at negative temperatures to expose the cryogenic texture [11-12]. 5 5 5 https://doi.org/10.1051/e3sconf/20199704061 E3S Web of Conferences 97, 04061 (2019) E3S Web of Conferences 97, 04061 (2019) FORM-2019 Figure 5 shows one of the typical schemes for the formation of cryogenic textures on the contact of treated and non-treated soils. Fig. 5. A cryogenic texture in a layer contact area obtained in 1980 s. Fig. 5. A cryogenic texture in a layer contact area obtained in 1980 s. The treated soil (on the left) has a massive cryogenic texture. The non-treated one has a stratified texture with horizontal ice lenses. At the point of contact there are vertical and inclined ice lenses. When discussing the results with Professor I.A. Tyutyunov we came to the conclusion that the vertical ice lenses were formed due to the advance freezing [13] of the treated soil and the formation of the freezing front of the non-treated soil at the phase boundary in the horizontal direction. A similar texture can be formed at the boundary of the tense zones below the bottom of the foundation. Fig. 6. A plot of moisture redistribution (coefficient of water accumulation) at frost penetration in different conditions. Fig. 6. A plot of moisture redistribution (coefficient of water accumulation) at frost penetration in different conditions. Fig. 6. A plot of moisture redistribution (coefficient of water accumulation) at frost penetration in different conditions. Below is the information on the conditions of soils freezing at the facilities according to Figure 6. 2.2 Research methodology of frost heaving and cryogenic structures Below is the information on the conditions of soils freezing at the facilities according to Figure 6. g 1) In the freezer with a constant negative temperature; g 1) In the freezer with a constant negative temperature; 2) In a soil hood in the open air; 3) In the stationary soil pan during natural freezing and artificial thawing in the period of negative temperatures; g p 4) Under the foundation base in Vilners house, Minusinsk; 5) Under the foundation base in 27, Vyatkina str. in Abakan during freezing from the basement; 6) In a field site during freezing below the groundwater level; ) g g g 7) A site of geo-engineering investigations for a residential house, 2, Katernaya str. in 7) A site of geo-engineering investigations for a residential house, 2, Katernaya str. in Abakan; clayey sand lies at 0,4-0,8 m from the ground surface, silty sand – at the depth of 0,8-1,8 m underlain by gravel, the groundwater level is 3 m. p y g g The study of the effects of frost heaving on the deformations of buildings has been conducted for a long time. The list of observed objects is presented in table 2. The study of the effects of frost heaving on the deformations of buildings has been conducted for a long time. The list of observed objects is presented in table 2. 6 6 https://doi.org/10.1051/e3sconf/20199704061 E3S Web of Conferences 97, 04061 (2019) FORM-2019 Table 2. Results of expert investigations of coil deformed by frost heave under building and structures. Location/floor Construction scheme Soil Groundwater level from designed/from basement floor Stress/soil parameters State Technical College – 59, Abakan / 2 floor, basement Frame; foundation posts 0,85 x 0,85 Gravel with silty – clayey filling 3,5/1,0 0,55/partly collapsed Kindergaten, Zeleny / 2 floors Walls Clayey sand 4.5/1,5 0,20/more than 10 mm Prikholmye Secondary General Education School No.4, settlement Prikholmye/ 2 floor, basement Walls Loam 3,0 0,20/up to 12 mm Askiz, residential house, Khakasia/ 1 floor Longitudinal bearing walls; strip foundations Clayey sand 1,7 0/up to 8 mm District hospital Shira/ 2 floor Longitudinal bearing walls; strip foundations Clayey sand 5/4,0 0,20/up to 8 mm Supply depot, 18, Itygina str., Abakan / 1 floor Walls Clayey sand 3,5/3,0 0,20/up to 3 mm 27, Vyatkina str. 3. Conclusion The presented materials, the analysis of emergency facilities show that even in excessively - heaving soils, the danger of impact of cryogenic processes on the foundations can be excluded. So, the eastern part of the GPTU 59 facility has maintained a healthy state. In the case of presence of reinforcement grid in the concrete floors, the breaking of concrete in the floors could be avoided. Under the concrete floor it was possible to make a reliable thermal insulation, but when the collapse occurred in 1983, the builders did not have the ability to carry out conservation of objects with expensive thermal insulation solutions during the winter construction period. At the present time, such insulation is necessary not only as an anti-abrasion stabilization, but also as a thermal protection technology while mounting the heated floors. The use of pile foundations for both subsiding and heaving soils is not the most rational solution. If the negative friction affects the piles in the subsiding soils, then the tangential forces of frost heaving also negatively affect their bearing capacity in the heaving soils.Thus, in practical terms, these soils are united by the designers' fear of their poor knowledge of the work with the soils and the possible inaccuracy of engineering and geological survey materials. The method of organized soaking, implemented in the village Solontsy, as well as methods of anti-heave stabilization, shows the relevance of rejecting expensive driven piles. 2.2 Research methodology of frost heaving and cryogenic structures It is worth noting that the deformation of the frost heaving [17-19] in the village Priholmye is lower than that described above, but one conclusion can be made: it is impossible to suppress the frost heaving of the soil on the excessively heaving soils of the Minusinsk Hollows by loading and pressing the soil under the bottom of the foundations. years. The similar emergency condition occurred in the village Priholmye [16]. There, during the construction period, the pressure under the bottom did not exceed 0.1 mPa, however, the foundation during the thawing period inclined more than in the above described example. However, during the period of operation, the soils stabilized and this object is still in use. It is worth noting that the deformation of the frost heaving [17-19] in the village Priholmye is lower than that described above, but one conclusion can be made: it is impossible to suppress the frost heaving of the soil on the excessively heaving soils of the Minusinsk Hollows by loading and pressing the soil under the bottom of the foundations. 2.2 Research methodology of frost heaving and cryogenic structures Abakan/ 2 floors, basement Walls Silty sand 4,5/2,5 0,10/more than 10 mm Vilner House, Minusinsk/ 3 floor, basement Walls Silty sand 4.5/1,5 0,20/up to 5 mm Heated parking, Shira, Lineynaya str./ 1 floor Frame Sandy clay IL=0,5-0,6 5,5/5,5 0,22/ up to 5 mm in concrete floors Table 2. Results of expert investigations of coil deformed by frost heave under building and structures. The analysis of the materials presented in the table shows that the pressures under the bottom of foundation of the collapsed part of the GPTU-59 building [14] reached 0.55 mPa, but this did not lead to a decrease in the migration process [15], the soil received additional moisture while and lost stability during thawing. It is necessary to pay attention to the fact that in the second half of the building with the concrete floors in the basement, the loss of stability - protrusion of soil from the bottom of foundation was substantially lower. This is due to the fact that the beginning of the process of soil protrusion was stopped by a concrete floor, which resisted the movement of the soil being squeezed out from below. As a result, the concrete floor was raised to a height of 65 mm. Between the fragments of the concrete floor there appeared a crack of up to one hundred millimeters, where one could fit a hand to a depth of up to 350 mm. The kindergarten in the village Zelenoe was also in an emergency condition during the construction period, the pressure under the bottom did not exceed 0.2 mPa, but the deformations in the walls, particularly the crack opening exceeded 10 mm. The basement wall blocks received a distinct inclination. The customer agreed to fill the basement with soil. This block - section has been currently in operation for more than 30 7 E3S Web of Conferences 97, 04061 (2019) FORM-2019 https://doi.org/10.1051/e3sconf/20199704061 years. The similar emergency condition occurred in the village Priholmye [16]. There, during the construction period, the pressure under the bottom did not exceed 0.1 mPa, however, the foundation during the thawing period inclined more than in the above described example. However, during the period of operation, the soils stabilized and this object is still in use. References 1. L. Kochurova, V. Eliseev. Human ecology. Ecological situation in Khakassia. 15, 10 (2011). 2. A. Gerasimova. The climate of Abakan, Leningrad Gidrometeoizdat, 42 (1985) 3. A. Budikova, N. Otepbergen. Problems of science. Engineering and geological studies of loess subsidence deposits. 43, 4 (2018). 4. A. Bogomolov, Yu Olyanskii, E. Shchekochikhina, I. Kuzmenko. Bulletin of Perm National Research Polytechnic University. Construction and architecture. Features of deformation behavior, slow subsidence of loess soils in the foundations of engineering structures in the man-made flooding. 3, 34 (2016). 5. Yu. Kozakov, N. Bulankin, G. Shishkanov, V. Korol. Specific features of the use of piles in Eastern Siberia. 268 p. (1992). 6. Rules and regulations 22. 13330.2016. Foundations of buildings and structures. Updated edition of Construction rules and regulations 2.02.01-83*. p. 6.1.13 (2017) 7. A. Nevzorov. Foundations on seasonally freezing soils. 67 (2000). 8. H. Rathmayer. Frost in geotechnical engineering: Int. Symp. 1,2, 34 (1989 9. S. Kudryavtsev. Scientific reports. Effect of migration moisture on the frost heaving process of seasonally freezing soils. 233 (2003). 8 https://doi.org/10.1051/e3sconf/20199704061 E3S Web of Conferences 97, 04061 (2019) FORM-2019 10. S. Kudryavtsev. Modern technologies. System analysis. Modeling. Numerical simulation of the process of frost heaving and thawing depending on the speed of soil freezing. 2, 105 (2012). 10. S. Kudryavtsev. Modern technologies. System analysis. Modeling. Numerical simulation of the process of frost heaving and thawing depending on the speed of soil freezing. 2, 105 (2012). 11. O. Khalimov. The Method of Pysical – Chemical Anti – Heave Stabilization of Soils during Construction in case of High Ground Water Level. Synopsis of PhD Thesis. 23 (1989). 12. S. Kudriavtsev. Freezing and defrosting soil. Series «Progress modern geotechniks». 116 (2014). 13. I. Tyutyunov, O. Khalimov. Anti-shock stabilization in the fight against subsidence of thawing soils, Proceedings of the institute. 118 (1986). 14. O. Khalimov. Soil – Struc. Inter. Underg. Struct. and Reta. Wal. Methods of Research on Frost Heave of Soil and Foundation – Freezing Soil Bulk Interaction in Terms of Seasonal Deep Frost Penetration including Areas of Pressure Migration. 1, 133 (2014). 15. V. Puskov. Thesis research. Force impacts of frost heaving of soils on the foundations of structures and methods for their calculation. 1, 65 (1995). 16. O. Khalimov, D. Strelnikov. : AIP Conference Proceedings. Geotechnical consulting at the stages of design and full repair: A case study of village school in Minusinsk region, Russia. 1899, (2017). 17. References T. L. Wang, Z. R. Yue, T. C. Sun, and J. C. Hua, Sciences in Cold and Arid Regions, 7, 407–413 (2015). 18. A. Sato and T. Yamanashi, Proceedings of the ISSMGE Technical Committee 207 Int. Conf. on Geotech. Eng. 4, 366–373 (2015). 19. A. J. J. Pauderham, Development of Cities and Geotechnical Construction, 11, 211–221 (2007) 9
https://openalex.org/W2548505476	http://journals.iucr.org/x/issues/2016/11/00/su4077/su4077.pdf	English	null	1-Benzyl-3′-[(1<i>H</i>-indol-3-yl)carbonyl]-1′-methyl-2-oxo-4′-(pyridin-3-yl)spiro[indoline-3,2′-pyrrolidine]-3′-carbonitrile	IUCrData	2,016	cc-by	4,523	data reports data reports ISSN 2414-3146 ISSN 2414-3146 P. Seethalakshmia and C. Palanivelb,a* Structure description Structure description Spiro-pyrrolidine derivatives are unique tetracyclic 5-HT(2A) receptor antagonists (Obniska et al., 2003; Peddi et al., 2004). These derivatives possess anticancer (Zapf et al., 2011) and anti-inﬂuenza virus (Stylianakis et al., 2003) activities. Highly functionalized pyrrolidines have attracted much interest in recent years as they constitute the main structural element of many natural and synthetic pharmacologically active compounds (Waldmann, 1995). Optically active pyrrolidines have been used as intermediates, chiral ligands or auxiliaries in controlled asymmetric synthesis (Suzuki et al., 1994; Huryn et al., 1991). In view of their importance and in continuation of our work on the crystal structure analysis of spiro-pyrrolidine derivatives, we report herein on the synthesis and crystal structure of a new spiro-pyrrolidine derivative. The molecular structure of the title compound is illustrated in Fig. 1. The ﬁve- membered ring (N2/C6–C9) in the pyrrolidine moiety adopts an envelope conformation, with atom N2 as the ﬂap atom [puckering parameters: q2 = 0.410 (2) A˚ and ’2 = 181.7 (3)], and the pyridine ring (N1/C1–C5) exhibits a slightly twisted conformation [puckering parameters: q2 = 0.093 (2) A˚ and 2 = 49.9 (2)]. The sum of angles at atom P. Seethalakshmia and C. Palanivelb,a* aDepartment of Chemistry, Jawahar Science College, Cuddalore, Neyveli 607 803, India, and bPG & Research Department of Chemistry, Government Arts College, Chidambaram, Tamilnadu, India. Correspondence e-mail: palanivelchem@gmail.com Received 12 September 2016 Accepted 20 October 2016 Received 12 September 2016 Accepted 20 October 2016 In the title compound, C34H27N5O2, the central pyrrolidine ring adopts an envelope conformation, with the N atom as the ﬂap. The mean planes of the two indoline ring systems are inclined to the mean plane of the central pyrrolidine ring by 86.26 (9) and 69.30 (9), respectively. The dihedral angle between the benzene and pyridine rings is 75.09 (11). In the crystal, molecules are linked by N—H N and C—H N hydrogen bonds, forming sheets parallel to the ab plane. In the title compound, C34H27N5O2, the central pyrrolidine ring adopts an envelope conformation, with the N atom as the ﬂap. The mean planes of the two indoline ring systems are inclined to the mean plane of the central pyrrolidine ring by 86.26 (9) and 69.30 (9), respectively. The dihedral angle between the benzene and pyridine rings is 75.09 (11). In the crystal, molecules are linked by N—H N and C—H N hydrogen bonds, forming sheets parallel to the ab plane. Edited by H. Stoeckli-Evans, University of Neuchaˆtel, Switzerland Keywords: crystal structure; spiro-pyrrolidine derivatives; N—H N hydrogen bonds; C— H N hydrogen bonds. CCDC reference: 1510867 Structural data: full structural data are available from iucrdata.iucr.org Structural data: full structural data are available from iucrdata.iucr.org 1 of 3 http://dx.doi.org/10.1107/S2414314616016862 1 of 3 IUCrData (2016). 1, x161686 IUCrData (2016). 1, x161686 IUCrData (2016). 1, x161686 http://dx.doi.org/10.1107/S2414314616016862 1 data reports Table 1 Hydrogen-bond geometry (A˚ , ). D—H A D—H H A D A D—H A N5—H5 N1i 0.86 1.96 2.812 (2) 170 C19—H19A N4ii 0.97 2.46 3.370 (3) 157 Symmetry codes: (i) x þ 1; y þ 1 2; z þ 1 2; (ii) x 1 2; y; z þ 1 2. Table 2 Experimental details. Crystal data Chemical formula C34H27N5O2 Mr 537.60 Crystal system, space group Orthorhombic, Pbca Temperature (K) 296 a, b, c (A˚ ) 14.7421 (6), 17.5573 (6), 21.4675 (9) V (A˚ 3) 5556.5 (4) Z 8 Radiation type Mo K (mm1) 0.08 Crystal size (mm) 0.25 0.16 0.12 Data collection Diffractometer Bruker APEXII CCD No. of measured, independent and observed [I > 2(I)] reﬂections 22838, 4884, 3251 Rint 0.031 (sin /)max (A˚ 1) 0.595 Reﬁnement R[F 2 > 2(F 2)], wR(F 2), S 0.040, 0.095, 0.97 No. of reﬂections 4884 No. of parameters 372 H-atom treatment H-atom parameters constrained max, min (e A˚ 3) 0.13, 0.17 Computer programs: APEX2 and SAINT (Bruker, 2014), SHELXS97 and SHELXTL (Sheldrick, 2008), SHELXL2014 (Sheldrick, 2015), Mercury (Macrae et al., 2008) and PLATON (Spek, 2009). Figure 1 The molecular structure of the title compound, showing the atom labelling and 20% probability displacement ellipsoids. Figure 1 Figure 1 The molecular structure of the title compound, showing the atom labelling and 20% probability displacement ellipsoids. The molecular structure of the title compound, showing the atom labelling and 20% probability displacement ellipsoids. N2 [328.4 (15)] is in accordance with sp2-hybiridization, and the sum of angles at atom N3 [313.6 (14)] is in accordance with sp3-hybridization. The dihedral angle between the benzene (C20–C25) and pyridine (N1/C1–C5) rings is 75.09 (11). N2 [328.4 (15)] is in accordance with sp2-hybiridization, and the sum of angles at atom N3 [313.6 (14)] is in accordance with sp3-hybridization. The dihedral angle between the benzene (C20–C25) and pyridine (N1/C1–C5) rings is 75.09 (11). In the crystal, molecules are linked by N—H N and C— H N hydrogen bonds, forming sheets parallel to the ab plane (Table 1 and Fig. 2). Computer programs: APEX2 and SAINT (Bruker, 2014), SHELXS97 and SHELXTL (Sheldrick, 2008), SHELXL2014 (Sheldrick, 2015), Mercury (Macrae et al., 2008) and PLATON (Spek, 2009). Refinement Crystal data, data collection and structure reﬁnement details are summarized in Table 2. Synthesis and crystallization N-Benzylisatin, (1) (0.3 mmol), was mixed with sarcosine, (2) (0.3 mmol), and (E)-2-[(1H-indol-3-yl)carbonyl]-3-(pyridin-3- yl)acrylonitrile in ethanol (10 ml) in a round-bottomed ﬂask. The reaction mixture was heated at 358 K for 3 h. After cooling to ambient temperature, the reaction mixture was ﬁltered to afford the pure title product as a white solid (yield 92%). The ﬁltrate was left to evaporate slowly and after 48 h yellow crystals of the title compound were obtained. Figure 2 The crystal packing of the title compound, viewed along the c axis. Hydrogen bonds are shown as dashed lines (see Table 1) and, for clarity, only H atoms H5 and H19A have been included. Synthesis and crystallization Acknowledgements PS and CP thank the Department of Chemistry, IIT, Chennai, India, for the X-ray diffraction data collection. Figure 2 References Huryn, D. M., Trost, B. M. & Fleming, I. (1991). Curr. Org. Synth. 1, 64–74. Macrae, C. F., Bruno, I. J., Chisholm, J. A., Edgington, P. R., McCabe, P., Pidcock, E., Rodriguez-Monge, L., Taylor, R., van de Streek, J. & Wood, P. A. (2008). J. Appl. Cryst. 41, 466–470. Obniska, J., Pawlowski, M., Kolaczkowski, M., Czopek, A., Duszyn’ska, B., Klodzin’ska, A., Tatarczyn’ska, E. & Chojnacka- Wo’jcik, E. (2003). Pol. J. Pharmacol. 55, 553–557. Peddi, S., Roth, B. L., Glennon, R. A. & Westkaemper, R. B. (2004). Bioorg. Med. Chem. Lett. 14, 2279–2283. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Sheldrick, G. M. (2015). Acta Cryst. C71, 3–8. Seethalakshmi and Palanivel C34H27N5O2 3 of 3 Stylianakis, I., Kolocouris, A., Kolocouris, N., Fytas, G., Foscolos, G. B., Padalko, E., Neyts, J. & De Clercq, E. (2003). Bioorg. Med. Chem. Lett. 13, 1699–1703. Spek, A. L. (2009). Acta Cryst. D65, 148–155. Huryn, D. M., Trost, B. M. & Fleming, I. (1991). Curr. Org. Synth. 1, 64–74. full crystallographic data IUCrData (2016). 1, x161686 [https://doi.org/10.1107/S2414314616016862] Crystal data C34H27N5O2 Mr = 537.60 Orthorhombic, Pbca a = 14.7421 (6) Å b = 17.5573 (6) Å c = 21.4675 (9) Å V = 5556.5 (4) Å3 Z = 8 F(000) = 2256 Dx = 1.285 Mg m−3 Mo Kα radiation, λ = 0.71073 Å Cell parameters from 5384 reflections θ = 2.3–24.1° µ = 0.08 mm−1 T = 296 K Rectangular, yellow 0.25 × 0.16 × 0.12 mm Data collection Bruker APEXII CCD diffractometer φ and ω scans 22838 measured reflections 4884 independent reflections 3251 reflections with I > 2σ(I) Rint = 0.031 θmax = 25.0°, θmin = 1.9° h = −14→17 k = −20→20 l = −18→25 H-atom parameters constrained w = 1/[σ2(Fo2) + (0.0237P)2 + 2.8668P] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max < 0.001 Δρmax = 0.13 e Å−3 Δρmin = −0.17 e Å−3 Extinction correction: SHELXL2014 (Sheldrick 2015), Fc=kFc[1+0.001xFc2λ3/sin(2θ)]-1/4 Extinction coefficient: 0.00063 (9) References g The crystal packing of the title compound, viewed along the c axis. Hydrogen bonds are shown as dashed lines (see Table 1) and, for clarity, only H atoms H5 and H19A have been included. g The crystal packing of the title compound, viewed along the c axis. Hydrogen bonds are shown as dashed lines (see Table 1) and, for clarity, only H atoms H5 and H19A have been included. Bruker (2014). APEX2 and SAINT. Bruker AXS Inc., Madison, Wisconsin, USA. 2 of 3 Seethalakshmi and Palanivel C34H27N5O2 IUCrData (2016). 1, x161686 IUCrData (2016). 1, x161686 data reports Spek, A. L. (2009). Acta Cryst. D65, 148–155. Stylianakis, I., Kolocouris, A., Kolocouris, N., Fytas, G., Foscolos, G. B., Padalko, E., Neyts, J. & De Clercq, E. (2003). Bioorg. Med. Chem. Lett. 13, 1699–1703. Suzuki, H., Aoyagi, S. & Kibayashi, C. (1994). Tetrahedron Lett. 35, 6119–6122. Waldmann, H. (1995). Synlett, pp. 133–141. Zapf, C. W., Bloom, J. D., Li, Z., Dushin, R. G., Nittoli, T., Otteng, M., Nikitenko, A., Golas, J. M., Liu, H., Lucas, J., Boschelli, F., Vogan, E., Olland, A., Johnson, M. & Levin, J. I. (2011). Bioorg. Med. Chem. Lett. 21, 4602–4607. IUCrData (2016). 1, x161686 data reports data report full crystallographic data IUCrData (2016). 1, x161686 [https://doi.org/10.1107/S2414314616016862] 1-Benzyl-3′-[(1H-indol-3-yl)carbonyl]-1′-methyl-2-oxo-4′-(pyridin-3-yl)spiro- [indoline-3,2′-pyrrolidine]-3′-carbonitrile P. Seethalakshmi and C. Palanivel 1-Benzyl-3′-[(1H-indol-3-yl)carbonyl]-1′-methyl-2-oxo-4′-(pyridin-3-yl)spiro[indoline-3,2′-pyrrolidine]-3′- carbonitrile Crystal data C34H27N5O2 Mr = 537.60 Orthorhombic, Pbca a = 14.7421 (6) Å b = 17.5573 (6) Å c = 21.4675 (9) Å V = 5556.5 (4) Å3 Z = 8 F(000) = 2256 Dx = 1.285 Mg m−3 Mo Kα radiation, λ = 0.71073 Å Cell parameters from 5384 reflections θ = 2.3–24.1° µ = 0.08 mm−1 T = 296 K Rectangular, yellow 0.25 × 0.16 × 0.12 mm Data collection Bruker APEXII CCD diffractometer φ and ω scans 22838 measured reflections 4884 independent reflections 3251 reflections with I > 2σ(I) Rint = 0.031 θmax = 25.0°, θmin = 1.9° h = −14→17 k = −20→20 l = −18→25 Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.040 wR(F2) = 0.095 S = 0.97 4884 reflections 372 parameters 0 restraints Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained w = 1/[σ2(Fo2) + (0.0237P)2 + 2.8668P] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max < 0.001 Δρmax = 0.13 e Å−3 Δρmin = −0.17 e Å−3 Extinction correction: SHELXL2014 (Sheldrick 2015), Fc=kFc[1+0.001xFc2λ3/sin(2θ)]-1/4 Extinction coefficient: 0.00063 (9) Special details Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes. IUCrData (2016). Special details 1, x161686 data-1 data reports Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq C1 0.63234 (14) −0.06447 (11) 0.42023 (9) 0.0517 (5) H1 0.6404 −0.0377 0.4572 0.062* C2 0.70167 (15) −0.10825 (13) 0.39656 (11) 0.0627 (6) H2 0.7568 −0.1116 0.4174 0.075* C3 0.68837 (16) −0.14676 (12) 0.34183 (11) 0.0644 (6) H3 0.7358 −0.1758 0.3259 0.077* C4 0.54388 (15) −0.10303 (11) 0.33427 (10) 0.0524 (5) H4 0.4889 −0.1024 0.3131 0.063* C5 0.55064 (13) −0.06054 (10) 0.38871 (9) 0.0438 (5) C6 0.47250 (13) −0.01328 (10) 0.41220 (9) 0.0445 (5) H6 0.4907 0.0063 0.4531 0.053* C7 0.38438 (14) −0.05742 (11) 0.42225 (10) 0.0537 (5) H7A 0.3763 −0.0960 0.3904 0.064* H7B 0.3837 −0.0817 0.4628 0.064* C8 0.33670 (12) 0.04828 (10) 0.36441 (9) 0.0419 (5) C9 0.44388 (13) 0.05810 (10) 0.37192 (8) 0.0405 (4) C10 0.31830 (13) 0.01138 (11) 0.29978 (10) 0.0458 (5) C11 0.24722 (13) 0.12705 (11) 0.29981 (9) 0.0470 (5) C12 0.19527 (15) 0.18821 (12) 0.28185 (11) 0.0637 (6) H12 0.1732 0.1926 0.2414 0.076* C13 0.17724 (17) 0.24293 (13) 0.32648 (13) 0.0788 (8) H13 0.1433 0.2855 0.3155 0.095* C14 0.20803 (18) 0.23604 (13) 0.38641 (12) 0.0757 (7) H14 0.1939 0.2734 0.4155 0.091* C15 0.26003 (15) 0.17389 (11) 0.40409 (10) 0.0593 (6) H15 0.2804 0.1689 0.4449 0.071* C16 0.28089 (13) 0.11976 (10) 0.35996 (9) 0.0455 (5) C17 0.22168 (15) −0.02969 (13) 0.41757 (12) 0.0748 (7) H17A 0.1792 0.0112 0.4119 0.112* H17B 0.2097 −0.0547 0.4565 0.112* H17C 0.2155 −0.0656 0.3841 0.112* C18 0.48887 (13) 0.05771 (10) 0.31095 (10) 0.0426 (5) C19 0.24710 (15) 0.04824 (13) 0.19947 (10) 0.0622 (6) H19A 0.1814 0.0489 0.1969 0.075* H19B 0.2673 −0.0025 0.1883 0.075* C20 0.28439 (15) 0.10381 (12) 0.15286 (9) 0.0522 (5) C21 0.22805 (17) 0.13774 (15) 0.10973 (11) 0.0762 (7) H21 0.1660 0.1283 0.1113 0.091* C22 0.2618 (2) 0.18511 (17) 0.06460 (13) 0.0908 (9) H22 0.2226 0.2070 0.0357 0.109* C23 0.3517 (2) 0.20027 (16) 0.06171 (13) 0.0888 (9) H23 0.3743 0.2321 0.0308 0.107* C24 0.40917 (18) 0.16848 (16) 0.10455 (12) 0.0805 (8) H24 0.4708 0.1797 0.1033 0.097* C25 0.37569 (15) 0.11979 (13) 0.14957 (10) 0.0620 (6) Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) omic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) IUCrData (2016). Special details 1, x161686 data-2 data-2 data reports data reports H25 0.4153 0.0975 0.1780 0.074 C26 0.47020 (13) 0.13396 (10) 0.40690 (9) 0.0453 (5) C27 0.47548 (13) 0.20507 (10) 0.37413 (9) 0.0455 (5) C28 0.44590 (14) 0.22198 (11) 0.31459 (10) 0.0528 (5) H28 0.4239 0.1863 0.2863 0.063* C29 0.48880 (14) 0.33263 (11) 0.35534 (10) 0.0531 (5) C30 0.50841 (16) 0.40925 (12) 0.36446 (13) 0.0700 (7) H30 0.4986 0.4452 0.3334 0.084* C31 0.54281 (18) 0.42924 (13) 0.42120 (14) 0.0776 (8) H31 0.5563 0.4801 0.4291 0.093* C32 0.55800 (16) 0.37571 (13) 0.46725 (12) 0.0710 (7) H32 0.5813 0.3914 0.5054 0.085* C33 0.53938 (14) 0.29933 (12) 0.45804 (10) 0.0565 (6) H33 0.5506 0.2637 0.4892 0.068* C34 0.50338 (13) 0.27721 (10) 0.40092 (9) 0.0465 (5) N1 0.61087 (13) −0.14454 (9) 0.31048 (8) 0.0588 (5) N2 0.31418 (11) 0.00086 (9) 0.41796 (8) 0.0509 (4) N3 0.27357 (10) 0.06328 (9) 0.26387 (7) 0.0477 (4) N4 0.52502 (12) 0.05610 (10) 0.26381 (8) 0.0572 (5) N5 0.45319 (12) 0.29669 (9) 0.30315 (8) 0.0587 (5) H5 0.4382 0.3189 0.2690 0.070* O1 0.34090 (10) −0.05210 (7) 0.28292 (7) 0.0599 (4) O2 0.48569 (11) 0.12999 (7) 0.46262 (7) 0.0646 (4) Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 C1 0.0643 (14) 0.0493 (12) 0.0416 (12) −0.0037 (11) −0.0093 (11) 0.0065 (10) C2 0.0557 (14) 0.0682 (14) 0.0643 (16) 0.0042 (12) −0.0136 (12) 0.0109 (13) C3 0.0679 (16) 0.0581 (13) 0.0671 (16) 0.0158 (12) −0.0047 (13) 0.0013 (13) C4 0.0580 (13) 0.0468 (11) 0.0525 (14) 0.0043 (10) −0.0129 (11) −0.0027 (11) C5 0.0566 (13) 0.0371 (10) 0.0377 (11) −0.0020 (9) −0.0059 (10) 0.0053 (9) C6 0.0592 (13) 0.0388 (10) 0.0354 (11) −0.0025 (9) −0.0034 (10) 0.0058 (9) C7 0.0684 (14) 0.0415 (11) 0.0512 (13) −0.0009 (10) 0.0045 (11) 0.0087 (10) C8 0.0483 (11) 0.0384 (10) 0.0388 (11) −0.0013 (8) 0.0022 (9) −0.0019 (9) C9 0.0496 (11) 0.0392 (10) 0.0326 (11) −0.0036 (9) −0.0018 (9) 0.0026 (9) C10 0.0423 (11) 0.0433 (11) 0.0516 (13) −0.0045 (9) −0.0008 (10) −0.0034 (10) C11 0.0439 (11) 0.0456 (11) 0.0515 (13) 0.0002 (9) 0.0001 (10) −0.0020 (10) C12 0.0667 (15) 0.0608 (13) 0.0638 (15) 0.0106 (12) −0.0107 (12) −0.0001 (12) C13 0.0919 (19) 0.0590 (14) 0.086 (2) 0.0292 (14) −0.0058 (16) −0.0034 (15) C14 0.100 (2) 0.0584 (14) 0.0689 (18) 0.0263 (14) 0.0033 (15) −0.0124 (13) C15 0.0755 (15) 0.0527 (12) 0.0495 (13) 0.0070 (11) 0.0058 (12) −0.0051 (11) C16 0.0474 (12) 0.0430 (10) 0.0460 (12) −0.0008 (9) 0.0037 (10) −0.0013 (10) C17 0.0643 (16) 0.0686 (15) 0.0914 (19) −0.0096 (12) 0.0192 (14) 0.0141 (14) C18 0.0445 (11) 0.0431 (11) 0.0401 (12) −0.0027 (9) −0.0077 (10) 0.0038 (10) C19 0.0595 (14) 0.0734 (15) 0.0535 (14) −0.0075 (12) −0.0152 (12) −0.0114 (12) C20 0.0577 (14) 0.0597 (12) 0.0393 (12) 0.0074 (11) −0.0110 (11) −0.0101 (11) C21 0.0650 (16) 0.105 (2) 0.0589 (16) 0.0134 (15) −0.0171 (14) −0.0009 (16) H25 0.4153 0.0975 0.1780 0.074* C26 0.47020 (13) 0.13396 (10) 0.40690 (9) 0.0453 (5) C27 0.47548 (13) 0.20507 (10) 0.37413 (9) 0.0455 (5) C28 0.44590 (14) 0.22198 (11) 0.31459 (10) 0.0528 (5) H28 0.4239 0.1863 0.2863 0.063* C29 0.48880 (14) 0.33263 (11) 0.35534 (10) 0.0531 (5) C30 0.50841 (16) 0.40925 (12) 0.36446 (13) 0.0700 (7) H30 0.4986 0.4452 0.3334 0.084* C31 0.54281 (18) 0.42924 (13) 0.42120 (14) 0.0776 (8) H31 0.5563 0.4801 0.4291 0.093* C32 0.55800 (16) 0.37571 (13) 0.46725 (12) 0.0710 (7) H32 0.5813 0.3914 0.5054 0.085* C33 0.53938 (14) 0.29933 (12) 0.45804 (10) 0.0565 (6) H33 0.5506 0.2637 0.4892 0.068* C34 0.50338 (13) 0.27721 (10) 0.40092 (9) 0.0465 (5) N1 0.61087 (13) −0.14454 (9) 0.31048 (8) 0.0588 (5) N2 0.31418 (11) 0.00086 (9) 0.41796 (8) 0.0509 (4) N3 0.27357 (10) 0.06328 (9) 0.26387 (7) 0.0477 (4) N4 0.52502 (12) 0.05610 (10) 0.26381 (8) 0.0572 (5) N5 0.45319 (12) 0.29669 (9) 0.30315 (8) 0.0587 (5) H5 0.4382 0.3189 0.2690 0.070* O1 0.34090 (10) −0.05210 (7) 0.28292 (7) 0.0599 (4) O2 0.48569 (11) 0.12999 (7) 0.46262 (7) 0.0646 (4) Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 C1 0.0643 (14) 0.0493 (12) 0.0416 (12) −0.0037 (11) −0.0093 (11) 0.0065 (10) C2 0.0557 (14) 0.0682 (14) 0.0643 (16) 0.0042 (12) −0.0136 (12) 0.0109 (13) C3 0.0679 (16) 0.0581 (13) 0.0671 (16) 0.0158 (12) −0.0047 (13) 0.0013 (13) C4 0.0580 (13) 0.0468 (11) 0.0525 (14) 0.0043 (10) −0.0129 (11) −0.0027 (11) C5 0.0566 (13) 0.0371 (10) 0.0377 (11) −0.0020 (9) −0.0059 (10) 0.0053 (9) C6 0.0592 (13) 0.0388 (10) 0.0354 (11) −0.0025 (9) −0.0034 (10) 0.0058 (9) C7 0.0684 (14) 0.0415 (11) 0.0512 (13) −0.0009 (10) 0.0045 (11) 0.0087 (10) C8 0.0483 (11) 0.0384 (10) 0.0388 (11) −0.0013 (8) 0.0022 (9) −0.0019 (9) C9 0.0496 (11) 0.0392 (10) 0.0326 (11) −0.0036 (9) −0.0018 (9) 0.0026 (9) C10 0.0423 (11) 0.0433 (11) 0.0516 (13) −0.0045 (9) −0.0008 (10) −0.0034 (10) C11 0.0439 (11) 0.0456 (11) 0.0515 (13) 0.0002 (9) 0.0001 (10) −0.0020 (10) C12 0.0667 (15) 0.0608 (13) 0.0638 (15) 0.0106 (12) −0.0107 (12) −0.0001 (12) C13 0.0919 (19) 0.0590 (14) 0.086 (2) 0.0292 (14) −0.0058 (16) −0.0034 (15) C14 0.100 (2) 0.0584 (14) 0.0689 (18) 0.0263 (14) 0.0033 (15) −0.0124 (13) C15 0.0755 (15) 0.0527 (12) 0.0495 (13) 0.0070 (11) 0.0058 (12) −0.0051 (11) C16 0.0474 (12) 0.0430 (10) 0.0460 (12) −0.0008 (9) 0.0037 (10) −0.0013 (10) C17 0.0643 (16) 0.0686 (15) 0.0914 (19) −0.0096 (12) 0.0192 (14) 0.0141 (14) C18 0.0445 (11) 0.0431 (11) 0.0401 (12) −0.0027 (9) −0.0077 (10) 0.0038 (10) C19 0.0595 (14) 0.0734 (15) 0.0535 (14) −0.0075 (12) −0.0152 (12) −0.0114 (12) C20 0.0577 (14) 0.0597 (12) 0.0393 (12) 0.0074 (11) −0.0110 (11) −0.0101 (11) C21 0.0650 (16) 0.105 (2) 0.0589 (16) 0.0134 (15) −0.0171 (14) −0.0009 (16) Atomic displacement parameters (Å2) IUCrData (2016). Special details 1, x161686 data-3 data reports data reports data-4 IUCrData (2016). Special details 1, x161686 C22 0.099 (2) 0.113 (2) 0.0605 (18) 0.0353 (19) −0.0144 (17) 0.0123 (17) C23 0.112 (3) 0.095 (2) 0.0593 (18) 0.0244 (19) 0.0108 (17) 0.0166 (15) C24 0.0728 (17) 0.103 (2) 0.0654 (17) 0.0064 (15) 0.0134 (15) 0.0091 (16) C25 0.0571 (15) 0.0769 (15) 0.0520 (14) 0.0146 (12) −0.0048 (12) 0.0003 (13) C26 0.0529 (12) 0.0434 (11) 0.0397 (12) −0.0050 (9) −0.0050 (10) 0.0033 (10) C27 0.0533 (13) 0.0407 (10) 0.0426 (12) −0.0071 (9) −0.0053 (10) 0.0067 (10) C28 0.0608 (13) 0.0461 (11) 0.0516 (14) −0.0110 (10) −0.0063 (11) 0.0084 (10) C29 0.0521 (13) 0.0463 (11) 0.0611 (14) −0.0071 (10) 0.0001 (11) 0.0064 (11) C30 0.0755 (17) 0.0464 (13) 0.0882 (19) −0.0106 (11) 0.0065 (15) 0.0124 (13) C31 0.0872 (19) 0.0482 (13) 0.097 (2) −0.0185 (13) 0.0103 (17) −0.0056 (15) C32 0.0763 (17) 0.0647 (15) 0.0719 (17) −0.0191 (13) 0.0038 (14) −0.0151 (14) C33 0.0570 (13) 0.0539 (12) 0.0585 (14) −0.0078 (10) −0.0008 (11) −0.0026 (11) C34 0.0448 (11) 0.0435 (11) 0.0512 (13) −0.0059 (9) −0.0001 (10) 0.0024 (10) N1 0.0668 (12) 0.0492 (10) 0.0603 (12) 0.0118 (9) −0.0086 (10) −0.0058 (9) N2 0.0547 (11) 0.0461 (9) 0.0518 (11) −0.0049 (8) 0.0098 (9) 0.0080 (8) N3 0.0474 (10) 0.0515 (9) 0.0440 (10) −0.0014 (8) −0.0062 (8) −0.0062 (8) N4 0.0557 (11) 0.0711 (12) 0.0447 (11) 0.0006 (9) 0.0005 (9) 0.0045 (10) N5 0.0698 (12) 0.0487 (10) 0.0575 (12) −0.0070 (9) −0.0063 (10) 0.0211 (9) O1 0.0665 (10) 0.0466 (8) 0.0666 (10) 0.0015 (7) −0.0079 (8) −0.0144 (7) O2 0.1075 (13) 0.0464 (8) 0.0399 (9) −0.0101 (8) −0.0152 (9) 0.0022 (7) Geometric parameters (Å, º) C1—C2 1.376 (3) C17—H17A 0.9600 C1—C5 1.383 (3) C17—H17B 0.9600 C1—H1 0.9300 C17—H17C 0.9600 C2—C3 1.370 (3) C18—N4 1.144 (2) C2—H2 0.9300 C19—N3 1.460 (2) C3—N1 1.327 (3) C19—C20 1.502 (3) C3—H3 0.9300 C19—H19A 0.9700 C4—N1 1.329 (2) C19—H19B 0.9700 C4—C5 1.390 (3) C20—C25 1.377 (3) C4—H4 0.9300 C20—C21 1.379 (3) C5—C6 1.507 (3) C21—C22 1.370 (4) C6—C7 1.528 (3) C21—H21 0.9300 C6—C9 1.580 (2) C22—C23 1.353 (4) C6—H6 0.9800 C22—H22 0.9300 C7—N2 1.458 (2) C23—C24 1.369 (3) C7—H7A 0.9700 C23—H23 0.9300 C7—H7B 0.9700 C24—C25 1.381 (3) C8—N2 1.458 (2) C24—H24 0.9300 C8—C16 1.504 (2) C25—H25 0.9300 C8—C10 1.555 (3) C26—O2 1.220 (2) C8—C9 1.598 (3) C26—C27 1.435 (2) C9—C18 1.467 (3) C27—C28 1.383 (3) C9—C26 1.578 (2) C27—C34 1.451 (2) C10—O1 1.218 (2) C28—N5 1.339 (2) C10—N3 1.364 (2) C28—H28 0.9300 IUCrData (2016). Special details 1, x161686 data-4 data reports 1.374 (3) C29—C30 1.390 (3) 1.389 (3) C29—N5 1.389 (3) 1.414 (2) C29—C34 1.397 (3) 1.383 (3) C30—C31 1.365 (3) 0.9300 C30—H30 0.9300 1.370 (3) C31—C32 1.382 (3) 0.9300 C31—H31 0.9300 1.387 (3) C32—C33 1.383 (3) 0.9300 C32—H32 0.9300 1.377 (3) C33—C34 1.391 (3) 0.9300 C33—H33 0.9300 1.465 (3) N5—H5 0.8600 119.6 (2) H17A—C17—H17C 109.5 120.2 H17B—C17—H17C 109.5 120.2 N4—C18—C9 178.5 (2) 119.1 (2) N3—C19—C20 114.55 (17) 120.5 N3—C19—H19A 108.6 120.5 C20—C19—H19A 108.6 123.0 (2) N3—C19—H19B 108.6 118.5 C20—C19—H19B 108.6 118.5 H19A—C19—H19B 107.6 124.34 (19) C25—C20—C21 117.8 (2) 117.8 C25—C20—C19 121.6 (2) 117.8 C21—C20—C19 120.5 (2) 116.54 (19) C22—C21—C20 121.2 (3) 121.97 (18) C22—C21—H21 119.4 121.49 (18) C20—C21—H21 119.4 114.71 (15) C23—C22—C21 120.5 (3) 117.24 (15) C23—C22—H22 119.8 104.63 (15) C21—C22—H22 119.8 106.5 C22—C23—C24 119.7 (3) 106.5 C22—C23—H23 120.2 106.5 C24—C23—H23 120.2 103.80 (15) C23—C24—C25 120.1 (3) 111.0 C23—C24—H24 120.0 111.0 C25—C24—H24 120.0 111.0 C20—C25—C24 120.8 (2) 111.0 C20—C25—H25 119.6 109.0 C24—C25—H25 119.6 113.72 (15) O2—C26—C27 121.35 (17) 115.22 (15) O2—C26—C9 117.69 (16) 101.27 (15) C27—C26—C9 120.97 (17) 101.96 (14) C28—C27—C26 128.52 (18) 117.23 (15) C28—C27—C34 105.57 (16) 107.90 (15) C26—C27—C34 125.49 (18) 108.50 (15) N5—C28—C27 110.76 (18) 111.34 (15) N5—C28—H28 124.6 C11—C12 1.374 (3) C29—C30 1.390 (3) C11—C16 1.389 (3) C29—N5 1.389 (3) C11—N3 1.414 (2) C29—C34 1.397 (3) C12—C13 1.383 (3) C30—C31 1.365 (3) C12—H12 0.9300 C30—H30 0.9300 C13—C14 1.370 (3) C31—C32 1.382 (3) C13—H13 0.9300 C31—H31 0.9300 C14—C15 1.387 (3) C32—C33 1.383 (3) C14—H14 0.9300 C32—H32 0.9300 C15—C16 1.377 (3) C33—C34 1.391 (3) C15—H15 0.9300 C33—H33 0.9300 C17—N2 1.465 (3) N5—H5 0.8600 C2—C1—C5 119.6 (2) H17A—C17—H17C 109.5 C2—C1—H1 120.2 H17B—C17—H17C 109.5 C5—C1—H1 120.2 N4—C18—C9 178.5 (2) C3—C2—C1 119.1 (2) N3—C19—C20 114.55 (17) C3—C2—H2 120.5 N3—C19—H19A 108.6 C1—C2—H2 120.5 C20—C19—H19A 108.6 N1—C3—C2 123.0 (2) N3—C19—H19B 108.6 N1—C3—H3 118.5 C20—C19—H19B 108.6 C2—C3—H3 118.5 H19A—C19—H19B 107.6 N1—C4—C5 124.34 (19) C25—C20—C21 117.8 (2) N1—C4—H4 117.8 C25—C20—C19 121.6 (2) C5—C4—H4 117.8 C21—C20—C19 120.5 (2) C1—C5—C4 116.54 (19) C22—C21—C20 121.2 (3) C1—C5—C6 121.97 (18) C22—C21—H21 119.4 C4—C5—C6 121.49 (18) C20—C21—H21 119.4 C5—C6—C7 114.71 (15) C23—C22—C21 120.5 (3) C5—C6—C9 117.24 (15) C23—C22—H22 119.8 C7—C6—C9 104.63 (15) C21—C22—H22 119.8 C5—C6—H6 106.5 C22—C23—C24 119.7 (3) C7—C6—H6 106.5 C22—C23—H23 120.2 C9—C6—H6 106.5 C24—C23—H23 120.2 N2—C7—C6 103.80 (15) C23—C24—C25 120.1 (3) N2—C7—H7A 111.0 C23—C24—H24 120.0 C6—C7—H7A 111.0 C25—C24—H24 120.0 N2—C7—H7B 111.0 C20—C25—C24 120.8 (2) C6—C7—H7B 111.0 C20—C25—H25 119.6 H7A—C7—H7B 109.0 C24—C25—H25 119.6 N2—C8—C16 113.72 (15) O2—C26—C27 121.35 (17) N2—C8—C10 115.22 (15) O2—C26—C9 117.69 (16) C16—C8—C10 101.27 (15) C27—C26—C9 120.97 (17) N2—C8—C9 101.96 (14) C28—C27—C26 128.52 (18) C16—C8—C9 117.23 (15) C28—C27—C34 105.57 (16) C10—C8—C9 107.90 (15) C26—C27—C34 125.49 (18) C18—C9—C26 108.50 (15) N5—C28—C27 110.76 (18) C18—C9—C6 111.34 (15) N5—C28—H28 124.6 data-5 data-5 IUCrData (2016). Special details 1, x161686 data reports C26—C9—C6 110.09 (14) C27—C28—H28 124.6 C18—C9—C8 110.89 (15) C30—C29—N5 129.2 (2) C26—C9—C8 112.48 (15) C30—C29—C34 122.9 (2) C6—C9—C8 103.51 (14) N5—C29—C34 107.86 (17) O1—C10—N3 125.13 (19) C31—C30—C29 116.8 (2) O1—C10—C8 126.77 (18) C31—C30—H30 121.6 N3—C10—C8 108.09 (16) C29—C30—H30 121.6 C12—C11—C16 122.13 (19) C30—C31—C32 121.6 (2) C12—C11—N3 128.23 (19) C30—C31—H31 119.2 C16—C11—N3 109.63 (16) C32—C31—H31 119.2 C11—C12—C13 117.1 (2) C33—C32—C31 121.7 (2) C11—C12—H12 121.4 C33—C32—H32 119.2 C13—C12—H12 121.4 C31—C32—H32 119.2 C14—C13—C12 121.7 (2) C32—C33—C34 118.2 (2) C14—C13—H13 119.1 C32—C33—H33 120.9 C12—C13—H13 119.1 C34—C33—H33 120.9 C13—C14—C15 120.7 (2) C33—C34—C29 118.79 (18) C13—C14—H14 119.7 C33—C34—C27 134.53 (18) C15—C14—H14 119.7 C29—C34—C27 106.67 (18) C16—C15—C14 118.6 (2) C3—N1—C4 117.45 (19) C16—C15—H15 120.7 C8—N2—C7 106.80 (15) C14—C15—H15 120.7 C8—N2—C17 114.61 (17) C15—C16—C11 119.75 (18) C7—N2—C17 113.82 (16) C15—C16—C8 130.88 (19) C10—N3—C11 110.69 (16) C11—C16—C8 109.36 (16) C10—N3—C19 122.93 (17) N2—C17—H17A 109.5 C11—N3—C19 125.88 (17) N2—C17—H17B 109.5 C28—N5—C29 109.12 (17) H17A—C17—H17B 109.5 C28—N5—H5 125.4 N2—C17—H17C 109.5 C29—N5—H5 125.4 C5—C1—C2—C3 0.4 (3) C21—C22—C23—C24 −0.5 (4) C1—C2—C3—N1 −0.7 (3) C22—C23—C24—C25 1.5 (4) C2—C1—C5—C4 0.8 (3) C21—C20—C25—C24 0.2 (3) C2—C1—C5—C6 −179.71 (17) C19—C20—C25—C24 177.1 (2) N1—C4—C5—C1 −1.9 (3) C23—C24—C25—C20 −1.4 (4) N1—C4—C5—C6 178.56 (18) C18—C9—C26—O2 −139.91 (19) C1—C5—C6—C7 −123.4 (2) C6—C9—C26—O2 −17.8 (2) C4—C5—C6—C7 56.0 (2) C8—C9—C26—O2 97.0 (2) C1—C5—C6—C9 113.2 (2) C18—C9—C26—C27 40.2 (2) C4—C5—C6—C9 −67.3 (2) C6—C9—C26—C27 162.28 (17) C5—C6—C7—N2 −156.36 (16) C8—C9—C26—C27 −82.8 (2) C9—C6—C7—N2 −26.51 (19) O2—C26—C27—C28 −168.1 (2) C5—C6—C9—C18 10.9 (2) C9—C26—C27—C28 11.8 (3) C7—C6—C9—C18 −117.49 (17) O2—C26—C27—C34 3.4 (3) C5—C6—C9—C26 −109.52 (18) C9—C26—C27—C34 −176.69 (18) C7—C6—C9—C26 122.14 (17) C26—C27—C28—N5 172.8 (2) C5—C6—C9—C8 130.04 (16) C34—C27—C28—N5 −0.1 (2) C7—C6—C9—C8 1.70 (18) N5—C29—C30—C31 179.6 (2) IUCrData (2016). Special details 1, x161686 data-6 data reports N2—C8—C9—C18 142.99 (15) C34—C29—C30—C31 −0.4 (3) C16—C8—C9—C18 −92.16 (19) C29—C30—C31—C32 0.4 (4) C10—C8—C9—C18 21.2 (2) C30—C31—C32—C33 0.2 (4) N2—C8—C9—C26 −95.31 (16) C31—C32—C33—C34 −0.8 (4) C16—C8—C9—C26 29.5 (2) C32—C33—C34—C29 0.8 (3) C10—C8—C9—C26 142.95 (15) C32—C33—C34—C27 −179.9 (2) N2—C8—C9—C6 23.50 (17) C30—C29—C34—C33 −0.2 (3) C16—C8—C9—C6 148.35 (16) N5—C29—C34—C33 179.80 (18) C10—C8—C9—C6 −98.24 (16) C30—C29—C34—C27 −179.7 (2) N2—C8—C10—O1 −48.4 (3) N5—C29—C34—C27 0.3 (2) C16—C8—C10—O1 −171.61 (19) C28—C27—C34—C33 −179.5 (2) C9—C8—C10—O1 64.7 (2) C26—C27—C34—C33 7.4 (4) N2—C8—C10—N3 132.87 (16) C28—C27—C34—C29 −0.2 (2) C16—C8—C10—N3 9.69 (19) C26—C27—C34—C29 −173.29 (19) C9—C8—C10—N3 −113.99 (16) C2—C3—N1—C4 −0.4 (3) C16—C11—C12—C13 −0.2 (3) C5—C4—N1—C3 1.7 (3) N3—C11—C12—C13 178.6 (2) C16—C8—N2—C7 −169.48 (16) C11—C12—C13—C14 −1.3 (4) C10—C8—N2—C7 74.2 (2) C12—C13—C14—C15 1.1 (4) C9—C8—N2—C7 −42.32 (18) C13—C14—C15—C16 0.7 (4) C16—C8—N2—C17 63.5 (2) C14—C15—C16—C11 −2.2 (3) C10—C8—N2—C17 −52.8 (2) C14—C15—C16—C8 178.4 (2) C9—C8—N2—C17 −169.36 (16) C12—C11—C16—C15 1.9 (3) C6—C7—N2—C8 44.46 (19) N3—C11—C16—C15 −177.05 (17) C6—C7—N2—C17 171.97 (17) C12—C11—C16—C8 −178.56 (18) O1—C10—N3—C11 172.32 (19) N3—C11—C16—C8 2.4 (2) C8—C10—N3—C11 −9.0 (2) N2—C8—C16—C15 48.1 (3) O1—C10—N3—C19 −0.1 (3) C10—C8—C16—C15 172.3 (2) C8—C10—N3—C19 178.67 (16) C9—C8—C16—C15 −70.7 (3) C12—C11—N3—C10 −174.6 (2) N2—C8—C16—C11 −131.37 (17) C16—C11—N3—C10 4.3 (2) C10—C8—C16—C11 −7.2 (2) C12—C11—N3—C19 −2.5 (3) C9—C8—C16—C11 109.90 (19) C16—C11—N3—C19 176.42 (18) N3—C19—C20—C25 52.9 (3) C20—C19—N3—C10 −122.3 (2) N3—C19—C20—C21 −130.3 (2) C20—C19—N3—C11 66.5 (3) C25—C20—C21—C22 0.7 (3) C27—C28—N5—C29 0.3 (3) C19—C20—C21—C22 −176.1 (2) C30—C29—N5—C28 179.6 (2) C20—C21—C22—C23 −0.6 (4) C34—C29—N5—C28 −0.4 (2) Hydrogen-bond geometry (Å, º) D—H···A D—H H···A D···A D—H···A N5—H5···N1i 0.86 1.96 2.812 (2) 170 C19—H19A···N4ii 0.97 2.46 3.370 (3) 157 Symmetry codes: (i) −x+1, y+1/2, −z+1/2; (ii) x−1/2, y, −z+1/2. data-7 data-7 IUCrData (2016). 1, x161686
https://openalex.org/W4320921048	https://www.microbiologyresearch.org/deliver/fulltext/acmi/10.1099/acmi.0.000493.v1/acmi.0.000493.v1.1.pdf?itemId=/content/reviewreports/10.1099/acmi.0.000493.v1.1&mimeType=pdf	English	null	Characterization of Shigella flexneri in Northern Vietnam in 2012 –2016	null	2,023	cc-by	2,161	How to cite the SciScore reports for this article: Nguyen DT, Morita M, Ngo TC, Le TH, Le DH, et al. SciScore reports for: Characterization of Shigella flexneri in Northern Vietnam in 2012 –2016. Access Microbiology. 2022. https://doi.org/10.1099/acmi.0.000493.v1.1 Nguyen DT, Morita M, Ngo TC, Le TH, Le DH, et al. SciScore reports for: Characterization of Shigella flexneri in Northern Vietnam in 2012 –2016. Access Microbiology. 2022. https://doi.org/10.1099/acmi.0.000493.v1.1 REPORTS: MDAR CHECKLIST FOR AUTHORS AND SCISCORE CORE REPORT SciScore® (https://sciscore.com) scans the methodology section of an article for important scientific rigour criteria and key biological resources and highlights if these are accessible or have problems associated. The Materials, Design, Analysis, and Reporting (MDAR) report and Core report generated from this are included here for transparency and can be cited independently using the DOI below. • Information on the MDAR report: https://sciscore.com/reports/MDAR-Report.php • Information on the Core report: https://sciscore.com/reports/Core-Report.php ITHENTICATE® REPORT iThenticate® (https://www.ithenticate.com) checks the submitted article against an extensive database of articles from the internet and scholarly publications and highlights where similar sentences or phrases have been used previously, including in the author’s own published work. Each individual match is given a percentage score based on how much it overlaps with the previously existing work, and an overall similarity score is given. The report generated from this are included here for transparency and can be cited independently using the DOI below. • FAQs: https://www.ithenticate.com/products/faqs • FAQs: https://www.ithenticate.com/products/faqs • Help pages: https://help.turnitin.com/ithenticate/ithenticate-user/ithenticate-user.htm#T UNDERSTANDING AND CONTEXTUALIZING THE REPORTS Readers of these automated manuscript Review Tool reports are encouraged to use them to support them in performing their own assessment and ‘health check’ on a preprint prior to it completing peer review. However, these should only be used as a guide, read within the overall context of the article itself, and should never replace full peer review. Please ensure you read the article fully alongside these and familiarize yourself with the tools and how they work, using the links provided below. These reports are published under the terms of the Creative Commons Attribution License How to cite the iThenticate report for this article: Nguyen DT, Morita M, Ngo TC, Le TH, Le DH, et al. iThenticate report for: Characterization of Shigella flexneri in Northern Vietnam in 2012 –2016. Access Microbiology. 2022. https://doi.org/10.1099/acmi.0.000493.v1.2 Nguyen DT, Morita M, Ngo TC, Le TH, Le DH, et al. iThenticate report for: Characterization of Shigella flexneri in Northern Vietnam in 2012 –2016. Access Microbiology. 2022. https://doi.org/10.1099/acmi.0.000493.v1.2 SciScore: 4 What's this? Document Identifier: 1891_63204eece1c482.38326653 Document Identifier: 1891_63204eece1c482.38326653 Document Identifier: 1891_63204eece1c482.38326653 SciScore Report SciScore Report Below you will find your SciScore report containing three tables. Your score is calculated based on adherence to scientific rigor criteria (Table 1) and identification of key biological resources (Table 2). Table 3 contains statistical tests and oligonucleotides but is not scored. If SciScore makes any mistakes, please contact us to help us learn and improve. Table 1: Rigor Adherence Table Table 1: Rigor Adherence Table Table 1: Rigor Adherence Table Ethics not required. Inclusion and Exclusion Criteria SNVs in recombinogenic , repeat , and prophage regions identified by Gubbins version 2.3.4 [ 13] , NUCmer [ 14] , and PHAST [ 15 ] were excluded. Attrition not required. Sex as a biological variable not required. Subject Demographics Age: not required. Weight: not required. Randomization not detected. Blinding not detected. Power Analysis not detected. Replication not required. Code Information Inclusion and Exclusion Criteria Identifiers: Single nucleotide variants ( SNVs ) were identified using Snippy version 4.3.6 ( https://github.com/tseemann/snippy). https://github.com/tseemann/snippy Data Information Availability: Nucleotide sequence data have been submitted to the DNA Data Bank of Japan ( DDBJ ) Sequence Read Archive under accession numbers DRR298131DRR298147 . Availability: The data have been deposited with links to DDBJ BioProject accession number PRJDB11754 in DDBJ . Identifiers: Single nucleotide variants ( SNVs ) were identified using Snippy version 4.3.6 ( https://github.com/tseemann/snippy). Table 2: Key Resources Table Your Sentences REAGENT or RESOURCE SOURCE IDENTIFIER Software and Algorithms 3.2 Molecular typing and data analyses The isolates were subjected to whole genome sequencing (WGS). WGS Genomic assembly was performed using SPAdes version 3.13.1 ( RRID:SCR_000131 ) with the careful option and a read coverage cutoff value of 10 [ 10] . SPAdes SPAdes ( RRID:SCR_000131)(link ) The assembled contigs were incorporated into the BioNumerics software ver . 8.0 BioNumerics Sequence comparisons were performed using BLASTN and visualized using Easyfig [ 17 ] . BLASTN Suggestion: (BLASTN, RRID:SCR_001598)( link) The data have been deposited with links to DDBJ BioProject accession number PRJDB11754 in DDBJ . BioProject Suggestion: (NCBI BioProject, RRID:SCR_004801)( link) SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore is not a substitute for expert review. 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W4385839579.txt	https://bmcpsychiatry.biomedcentral.com/counter/pdf/10.1186/s12888-023-05078-z	en		Study on the daily dose and serum concentration of clozapine in psychiatric patients and possible influencing factors of serum concentration	BMC psychiatry	2,023	cc-by	7,508	BMC Psychiatry (2023) 23:596 Liu et al. BMC Psychiatry https://doi.org/10.1186/s12888-023-05078-z Open Access RESEARCH ARTICLE Study on the daily dose and serum concentration of clozapine in psychiatric patients and possible influencing factors of serum concentration Taixiu Liu1, Peng Gao1* , Chuange Xie1, Heng Zhang1, Zheng Shi1 and Ruirui Chen1 Abstract Background Clozapine is the most effective drug for treatment-resistant schizophrenia, and the dosage and concentration of clozapine in the treatment of mental illness vary greatly in different populations and are affected by many factors. Methods The serum clozapine concentration of 3734 psychiatric patients was detected, and data on daily dose, sex, age and other medical records were collected for statistical analysis. Results The mean daily dose, mean serum concentration and mean C/D (concentration/dose) ratio of clozapine were 191.02 ± 113.47 mg/day, 326.15 ± 235.66 ng/mL and 1.94 ± 1.25 ng/mL per mg/day, respectively. There was difference in daily dose between sexes, and females had higher daily dose (p <0.01), higher serum clozapine concentrations (p < 0.01) and higher C/D ratios (p < 0.01). There were significant differences in daily dose (p < 0.001), serum drug concentration (p < 0.001) and C/D ratio (p < 0.001) among different age groups. The daily dose decreased with age (p for trend < 0.001), and the C/D ratio increased with age (p for trend < 0.001). Inpatients and outpatients had no difference in daily dose, but inpatients had higher serum concentration (p < 0.001) and C/D ratio (p < 0.001). There was no difference in daily dose among different occupations, but there were significant differences in serum concentration (p < 0.001) and C/D ratio (p < 0.001), and unemployed patients may have higher serum concentration and C/D ratio. Duration of disease, comorbidity, marital status, and psychotic type may influence the daily dose and serum concentration. Conclusions The effective daily dose and serum concentration of clozapine in the study area may be lower than recommended levels, and women have higher serum concentrations and slower metabolic rates. With increasing age, the daily dose decreases, and the metabolic rate slows. Inpatient status and occupation of patients may influence the serum concentration and metabolic rate of clozapine. Keywords Clozapine, Serum concentration, Daily dose, Therapeutic drug monitoring (TDM) Correspondence: Peng Gao gaopeng1965@163.com 1 Department of Clinical Laboratory, Shandong Daizhuang Hospital, Jining 272051, China Background Clozapine is a second-generation antipsychotic that mainly blocks serotonin (5-HT2A) and dopamine (DA1) receptors in the brain to treat psychosis. In addition, clozapine can directly inhibit the upwards activation system of the brain stem reticular structure and has a relatively © The Author(s) 2023. 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. Liu et al. BMC Psychiatry (2023) 23:596 powerful sedative and hypnotic effect. Clinically, clozapine is mainly used for treatment-resistant schizophrenia or treatment-resistant bipolar disorder; it can improve the positive symptoms of psychosis to a certain extent and have an effect on the negative symptoms [1]. Clozapine can be used for acute and chronic schizophrenia and has a certain therapeutic effect on fantasies, delusions and hallucinations. It is also useful for treating emotional symptoms such as depression, guilt, and anxiety, as well as mania and other psychiatric disorders [2]. Furthermore, clozapine is the only FDA-approved drug to prevent suicidal behaviour [3]. Major side effects of clozapine include strong sedative effects, more anticholinergic effects, common dizziness, fatigue, drowsiness, sweating, salivation, nausea, vomiting, dry mouth, tachycardia, constipation and postural hypotension. Second, the more common symptoms are increased appetite and weight gain. Third, it can also cause elevated blood sugar and lipids [4]. Fourth, the serious adverse effects are agranulocytosis and secondary infection. Clozapine is currently recognized as a fairly effective drug in the treatment of schizophrenia and represents the most effective pharmacotherapy for treatment-resistant psychosis [5]. In addition, clozapine was associated with a reduction in overall mortality compared with other second-generation antipsychotics [6]. Clozapine has these unique effects that are not matched by other antipsychotic drugs, such as risperidone and olanzapine [7–9]. Because clozapine reduces granulocytes, a serious side effect, it is not considered the first choice of antipsychotic drug at present. Therapeutic drug monitoring (TDM) quantifies and interprets the concentration of the drug in plasma or serum and examines the correlation between the dose of the drug and the concentration of the drug in the blood for each patient to yield the highest therapeutic effect and the lowest risk of adverse drug reactions/toxicity [10]. Based on empirical evidence, clozapine is a strongly recommended drug for TDM (Level 1) [11]. To ensure the efficacy of clozapine and reduce its side effects, blood clozapine concentrations should be monitored during clozapine therapy [12]. In this study, the relationship between clozapine dosage and serum drug concentration and the factors that may affect serum drug concentration were studied from the perspective of a population in eastern China to provide a basis and reference for the clinical application of clozapine. Methods Samples All samples in this study were collected from patients in Shandong Daizhuang Hospital. The included subjects were diagnosed with schizophrenia (paranoid Page 2 of 12 schizophrenia, undifferentiated schizophrenia and residual schizophrenia), affective (mood) disorder (bipolar disorder, recurrent depressive disorder, manic and major depressive episode) and others (organic, including symptomatic, mental disorders, dissociation [conversion] disorder, behavioural and emotional disorders that usually begin in childhood and adolescence, mental retardation, generalized anxiety disorder, obsessive compulsive disorder, specific personality disorder) by at least 2 psychiatrists according to the International Classification of Diseases, 10th Revision (ICD-10) or Diagnostic and Statistical Manual of Mental Disorders (DSM–5). The inclusion criteria were as follows: (1) patients with mental disorders diagnosed in our hospital, (2) patients with available medical records, and (3) patients taking clozapine. None of the subjects had obvious signs of clozapine poisoning or significant adverse drug reactions. The patients were instructed to take clozapine orally starting with a small dose; the initial dose was 25 mg/time, 2–3 times a day, and gradually increased to the conventional therapeutic dose according to the instructions. In a steady state, drug intake equals drug elimination over a period of time, and the steady state valley concentration (Cmin) is usually quantitative. The Cmin of most drugs in the steady state (fixed dose treatment with at least 4–6 half-lives) has been used as a standard procedure and is recommended in therapeutic drug monitoring (TDM). The elimination half-life of clozapine is approximately 12–16 h on average [11], and serum drug concentrations were measured after 4 to 6 metabolic halflives of fixed doses of clozapine. Patients took the drug at approximately 9–11 pm, and blood was drawn from the patient at approximately 6–8 am the next day. According to the clinical treatment requirements, the psychiatrist gave the patient a medical order for clozapine blood concentration monitoring, and the nurse drew 5 ml of fasting venous blood with a vacuum vessel without other components and immediately sent it for examination. Detection of serum clozapine concentration The clozapine serum concentration was measured using high-performance liquid chromatography (HPLC) at the Clinical Drug Concentration Monitoring Laboratory, Department of Clinical Laboratory, Shandong Daizhuang Hospital. The received blood samples were placed in a temperature box at 37 °C for several minutes to accelerate solidification and centrifuged at 4000 r/min for 10 min after solidification. One millilitre of the supernatant was added to a 10-ml glass tube, and 20 µl of the internal standard solution and 100 µL NaOH (concentration of 0.1 mol/L) were added and then shaken for several seconds. Then, 5 ml n-pentane was added and shaken for 1 min. The supernatant Liu et al. BMC Psychiatry (2023) 23:596 Page 3 of 12 was transferred to another 10-ml glass tube, dried in a water bath at 70 °C, removed, cooled to room temperature and re-dissolved in 100 µl of mobile phase. After shaking for a few seconds, the supernatant was centrifuged at 2000 r/min for 5 min, and 30 µl of the supernatant was taken in the intubation and sent to the machine for analysis. HPLC chromatographic conditions were as follows: mobile phase (methanol: water: tetramethylethylenediamine: acetic acid = 677:330:2.2:1.76), flow rate: 0.8 mL/ min, column: SB-C18, detection wavelength: 254 nm, column temperature: 40℃, injection volume: 20 µl, injection time: 12 min. Internal standard solution: 2-amino5-chloro-2-fluorophenone (5 µg/mL). 10 to 75 years old, with an average age of 38.18 ± 11.75 years old. A total of 2424 male patients and 1310 female patients were included. The duration of the diseases ranged from 1 to 600 months, with an average of 195.63 ± 118.56 months. In this study, the psychiatric disorders treated with clozapine were mainly schizophrenia (75.92%) and mood disorder (22.25%). Other psychiatric disorders treated with clozapine, including “organic, including symptomatic, mental disorders” and “behavioural and emotional disorders that usually begin in childhood and adolescence”, accounted for 1.83% of the included subjects. Paranoid schizophrenia, undifferentiated schizophrenia, and bipolar disorder were the most commonly treated disorders with clozapine (Table 1). C/D ratio and medical record information Results of daily dose, serum concentration and C/D ratio The results of clozapine serum concentration were looked up in the Laboratory Information System (LIS). The daily dose of clozapine for psychiatric patients was obtained by consulting the doctor’s orders in the medical records. The serum concentration/daily dose ratio (C/D ratio) was obtained by calculating the ratio of serum clozapine concentration (ng/mL) to daily dose (mg/day), which represented the metabolism and clearance rate of clozapine in vivo [13]. C/D ratios are inversely related to total clearance; the lower the C/D ratio is, the faster the clearance rate of clozapine in vivo; in contrast, the higher the C/D ratio is, the smaller the clearance rate of clozapine in vivo [14]. No names were collected in the case inquiry or medical advice in order to protect patient privacy, and the medical record number was used as the inquiry identifier. The medical records of each subject were collected and analysed, including gender, age, ethnicity, marital status, mental illness type, residence, medication amount, etc. Clozapine was prescribed once daily to three times daily by the psychiatrist, depending on the patient’s condition. The daily doses ranged from 12.5 mg/day to 825 mg/ day, and the average daily dose was 191.02 ± 113.47 mg/ day. The serum concentrations of clozapine ranged from 27.44 ng/mL to 1884.23 ng/mL, with an average of 326.15 ± 235.66 ng/mL. The C/D ratio ranged from 0.18 to 12.84, with an average of 1.94 ± 1.25. The Spearman correlation analysis showed a positive correlation between daily dose and serum concentration (r = 0.594, p < 0.01) (Table 2). Taking the clozapine daily dose as the independent variable and the serum drug concentration as the dependent variable, regression analysis showed that the regression coefficient was b = 1.067 (R = 0.513, p < 0.001) (Fig. 1). The subjects were divided into four groups according to the daily dose: <100 mg/day group, 100–200 mg/ day group, 201–400 mg/day group and > 400 mg/day group. The daily dose of clozapine in psychiatric patients between 100 and 400 mg/day accounted for 77.93% of the subjects, and the daily doses of 50–350 mg/d accounted for 87.50%. Through trend test analysis, the clozapine serum concentration showed an increasing trend with increasing drug dose (p < 0.001), and the C/D ratio showed a decreasing trend with increasing daily drug dose (p < 0.001) (Table 3). The subjects were divided into three groups according to the serum concentration: <350 ng/mL, 350–600 ng/ mL and > 600 ng/mL. The serum clozapine concentration in most patients was < 350 ng/mL, accounting for 62.77%. Only 28.07% of the patients had clozapine concentrations between 350 and 600 ng/mL. A total of 9.16% of patients had clozapine concentrations > 600 ng/mL, among which 62 patients (1.66%) had clozapine concentrations > 1000 ng/mL and 1 patient had clozapine concentrations > 1800 ng/mL. The concentration range of 50–600 ng/mL accounted for 86.20% of the population Statistical analysis Data with a normal distribution will be described by the mean ± SD, and data with a non-normal distribution will be described by the median (interquartile range). Spearman correlation was used for correlation analysis; the Mann‒Whitney test and Kruskal‒Wallis test were used to compare the data between groups. In addition, univariate and multivariate linear regression analyses and trend tests were also used to analyse the data. Values of p < 0.05 were considered statistically significant. Analysis was computed using SPSS Statistics version 22.0 (IBM Corporation, Armonk, NY, USA). Results Demographic characteristics A total of 3734 subjects were included in this study. All medical records were available and ranged in age from Liu et al. BMC Psychiatry (2023) 23:596 Page 4 of 12 Table 1 Types of psychiatric disorders treated with clozapine (n = 3734) Classification of psychiatric disorders Number Percentage (%) Schizophrenia (n = 2835) Paranoid schizophrenia 1425 38.16 Undifferentiated schizophrenia, 1403 37.57 Residual schizophrenia 7 0.19 Affective (mood) disorder(n = 831) Bipolar Disorder 779 20.86 Recurrent depressive disorder 24 0.64 Manic 19 0.51 Major depressive episode 9 0.24 Others (n = 68) Organic, including symptomatic, mental disorders 30 0.80 Dissociation [conversion] disorder 26 0.70 4 0.11 Behavioural and emotional disorders that usually begin in childhood and adolescence Mental retardation 4 0.11 Generalized Anxiety Disorder 2 0.05 Obsessive compulsive disorder 1 0.03 Specific personality disorder 1 0.03 Table 2 Clozapine dose, serum concentration and the C/D ratio Variables Mean ± SD Median (interquartile range) r Daily dose (mg/day) 191.02 ± 113.47 175.00(100.00–250.00) 0.594a Serum concentration (ng/mL) 326.15 ± 235.66 272.36(148.09–445.98) C/D ratio 1.94 ± 1.25 1.67(1.10–2.47) a Spearman correlation coefficient between daily dose and serum concentration * p < 0.01 in this study. Through trend test analysis, the C/D ratio showed an increasing trend with increasing serum drug concentration (p < 0.001) (Table 3). Comparison of clozapine daily dose, serum concentration and C/D ratios among different groups Subjects were grouped according to different attributes. According to age, the psychiatric patients were divided into four groups: the 10-17-year-old group, 18-44-yearold group, 45-59-year-old group and 60-75-year-old group. More than half of the subjects were in the 18–44 age range. According to the duration of the disease, the patients were divided into two groups: the < 180 months group and the ≥ 180 months group. According to marital status, the patients were divided into three groups: single, married and divorced. According to occupation, the patients were divided into four groups: unemployed, farmers, workers and others. The patients were divided into two groups according to whether the diseases were associated with somatic diseases: the psychiatric disorder group and the psychiatric disorder combined with somatic disease (comorbidities) group. Additional background information is presented in Table 4. Through statistical analysis, there was significant difference in the daily dose of clozapine between males and females (p < 0.01), there was a significant difference in the serum concentration of clozapine between males and females, and the serum concentration in females was higher (p < 0.01). There was also a statistically significant difference in the C/D ratio between males and females, and the C/D ratio in females was higher (p < 0.01). There were significant differences in the daily dose of clozapine in different age groups, and the daily dose in the 10–17 age group was the highest (p < 0.01). Correspondingly, there were significant differences in serum concentrations among different age groups. There were significant differences in the C/D ratio among different age groups, and the C/D ratio in the 10–17 age group was the lowest (p < 0.01). There were significant differences in the daily dose, serum concentration and C/D ratio between the two groups with different disease durations. Compared with the > 180-month Liu et al. BMC Psychiatry (2023) 23:596 Page 5 of 12 Fig. 1 Relationship between the daily dose of clozapine and serum concentration. The linear regression equation of clozapine daily dose and concentration was Y = 122.793 + 1.065*x, R = 0.513, p < 0.001 duration group, the daily dose in the ≤ 180-month duration group was higher (p < 0.01), and the serum concentration and C/D ratio in the < 180-month duration group were lower (p < 0.01). Psychiatric disorders treated with clozapine were mainly schizophrenia and bipolar disorder, with few other diseases. There were significant differences in daily dose, serum concentration and C/D ratio among schizophrenia, bipolar disorder and other types of mental diseases. The schizophrenia group had the highest daily dose (p < 0.01) and serum concentration (p < 0.01). There was no significant difference in daily dose between outpatients and inpatients, but there was a significant difference in serum concentration (p < 0.01) and C/D ratio (p < 0.01). The serum concentration and C/D ratio in inpatients were higher. There were significant differences in the daily dose serum concentration and C/D ratio between the psychiatric disorder group and the comorbidity group. The daily dose in the psychiatric disorder group was higher than that in the comorbidities group (p < 0.01), and the serum concentration and C/D ratio in the psychiatric disorder group were lower than those in the comorbidities group (p < 0.05 and p < 0.01, respectively). There were significant differences among different occupations in serum concentration (p < 0.01) and C/D ratio (p < 0.01), and the C/D ratio in the unemployed group was the highest (Table 5). Trend tests of dose, concentration, and C/D ratio between age groups The statistical results of this study showed that there were significant differences in the daily dose, serum concentration and C/D ratio among different age groups of psychiatric patients. According to trend analysis results, the daily dose of clozapine in psychiatric patients decreased with age (p < 0.001) (Fig. 2a). Serum concentration in the four age groups did not show an increasing trend with age (Fig. 2b1), but when the subjects were divided into 10–44 and 45–75 age groups according to their ages, there was a significant difference in serum drug concentration between the 10–44 and 45–75 age groups (p < 0.01), and the serum concentration in the 10–44 age group was lower (Fig. 2b2). The C/D ratio increased with age (p < 0.001) (Fig. 2c). Regression model and identifying factors We used a regression model to further examine the factors that influenced serum clozapine concentration. After univariate and multivariate regression analyses revealed that daily dose, sex, age, inpatients/outpatients, and occupation (worker) were significant factors (p = 0.000). The duration of the disease and the type of mental illness were significant factors in the univariate regression model (p < 0.05) but were not significant in the multivariate regression model (p > 0.05). Thus, daily dose, sex, age Groups n 1244 161 201–400 > 400 450.00(450.00–500.00) 486.65 ± 68.58 2344 1048 342 < 350 350–600 > 600 153.91 ± 101.16 296.53 ± 112.01 239.59 ± 99.17 300.00(225.00–375.00) 225.00(150.00–300.00) 150.00(75.00–200.00) 300.00(250.00–300.00) 150.00(100.00–175.00) 50.00(50.00–75.00) Median (interquartile range) 287.30 ± 52.27 145.37 ± 35.01 53.28 ± 18.12 Mean ± SD Serum concentration (ug/L) 663 1666 < 100 100–200 Daily dose (mg/day) Daily dose (mg/day) 0.000 0.000 p 858.70 ± 212.87 471.79 ± 82.37 183.33 ± 86.97 566.00 ± 326.99 448.54 ± 250.42 289.09 ± 175.81 131.38 ± 90.74 Mean ± SD Table 3 Trend test of different dose groups and different serum concentration groups 796.19(702.64–920.71) 462.06(400.09–535.34) 174.10(110.25–255.72) 501.03(283.86–805.97) 406.93(270.53–567.25) 252.99(155.09–379.81) 104.72(70.83–161.15) Median (interquartile range) Serum concentration (ng/mL) 0.000 0.000 p 3.28 ± 1.36 2.37 ± 1.25 1.56 ± 1.02 1.19 ± 0.70 1.60 ± 0.91 2.03 ± 1.21 2.57 ± 1.64 Mean ± SD 3.04(2.23–3.96) 2.05(1.52–2.89) 1.34(0.87–1.95) 1.07(0.59–1.66) 1.39(0.97–2.03) 1.75(1.13–2.69) 2.08(1.54–3.07) Median (interquartile range) C/D Ratio 0.000 0.000 p Liu et al. BMC Psychiatry (2023) 23:596 Page 6 of 12 Liu et al. BMC Psychiatry (2023) 23:596 Page 7 of 12 Table 4 Baseline characteristics of the included patients (N = 3734) Variables n Percentage (%) Sex Female 1310 35.08 Male 2424 64.92 Age group(year) 10–17 116 3.11 18–44 2418 64.76 45–59 1089 29.16 60–75 111 2.97 Duration (month) < 180 1924 51.53 ≥ 180 1810 48.47 Ethnicity Han 3692 98.88 Hui 42 1.12 Marital status Single 1435 38.43 Married 1992 53.35 Divorced 307 8.22 Occupation unemployed 1718 46.01 Farmer 1091 29.22 Worker 468 12.53 Others 457 12.24 Comorbidity or not psychiatric disorders 3424 91.70 Comorbidities 310 8.30 Psychotic type Schizophrenia 2835 75.92 Bipolar disorder 779 20.86 Other types 120 3.21 Inpatients/outpatients Inpatients 3209 85.94 Outpatients 525 14.06 Province Shandong 3674 98.39 Others 60 1.61 and occupation are factors that predict serum clozapine concentration (Table 6). Discussion Application prospects and therapeutic drug monitoring of clozapine With the increasing awareness of the need for personalized treatment, therapeutic drug monitoring (TDM), which combines quantitative blood drug concentrations, pharmacological interpretation, and therapeutic guidance, has been introduced into drug therapy as a precision medicine tool [15]. TDM allows for individualized pharmacokinetic therapy by taking into account individual differences in pharmacokinetics [11]. It has become routine practice to quantify the plasma concentration of a large number of neuro psychotropic drugs, especially first-generation and second-generation antipsychotics and mood stabilizers [16]. At the same daily dose, as patients differ in their ability to absorb, distribute, metabolize and excrete drugs due to concurrent disease, age, concomitant medication or genetic abnormalities, the homeostasis concentration of a drug in vivo can vary more than 20-fold between individuals [17–23]. This study found a correlation of 0.594 between the daily dose and serum concentration of clozapine, which was a moderate correlation level (0.5≤\|r\|<0.8), and this result is similar to the published study [24], in which r = 0.49 (p < 0.001). This reflects that the metabolism of clozapine varies greatly among individuals. Therefore, in the clinical application of clozapine, individualized medication and drug concentration monitoring become necessary and more meaningful. Discussion of clozapine dose, concentration, and C/D values Clozapine is mainly used for the treatment of treatmentresistant schizophrenia and schizoaffective disorder [25]; in addition, clozapine has also been used in the off-label treatment of bipolar disorder [3, 26], major depressive disorder (MDD) [27], and Parkinson’s disease (PD)[25, 28, 29]. The indications for clozapine in the drug label approved by China include that it may be effective for some patients who have failed or are not effective with traditional antipsychotics. It is also used for the treatment of agitation and hallucinatory delusions of mania or other psychotic disorders. The unique pharmacological properties and curative effects of clozapine make it gradually applied by psychiatrists beyond the drug label. In the study, clozapine was used by 75.92% of patients with schizophrenia, 22.25% of patients with affective (mood) disorders, and 1.83% of patients with other mental disorders. This indicates that clozapine is used in off-label treatment in the study area and suggests that clozapine has greater potential application value. Due to the side effects of clozapine, clozapine remains an underprescribed medication [28], and caution should be taken when using clozapine for off-label treatment. Following the instructions for clozapine (Shandong Renhetang Pharmaceutical Co. Ltd), the treatment dose of clozapine was 201–400 mg/day, and the maintenance dose was 100–200 mg/day. The average dose of clozapine in the study population was 191.02 ± 113.47 mg/day, and doses of 50–350 mg/d accounted for 87.50% of the 188.99 ± 116.63 2424 144.59 ± 71.78 111 1810 > 180 525 Outpatients 779 120 Bipolar disorder Other types 310 Comorbidities Kruskal‒Wallis test b 457 Others Mann‒Whitney test 204.05 ± 132.79 175.00 (100.00–300.00) 150.00 (100.00–250.00) 175.00 (100.00–250.00) 175.00 (100.00–250.00) 150.00 (100.00–225.00) 150.00 (100.00–250.00) 175.00 (100.00–300.00) 150.00 (100.00–200.00) 175.00 (100.00–250.00) 150.00 (50.00–225.00) 150.00 (100.00–225.00) 175.00 (100.00–275.00) 200.00 (100.00–300.00) 150.00 (100.00–250.00) 150.00 (100.00–225.00) 200.00 (100.00–300.00) 125.00 (100.00–200.00) 150.00 (100.00–250.00) 175.00 (100.00–275.00) 200.00 (100.00–300.00) 150.00 (100.00–250.00) 175.00 (100.00–250.00) Median (interquartile range) 0.426b 0.015b 0.000a 0.000b 0.097a 0.000a 0.000b 0.009a P 345.94 ± 277.56 278.84 ± 184.37 321.17 ± 244.27 336.94 ± 228.71 337.68 ± 265.36 335.07 ± 239.53 311.30 ± 222.63 329.60 ± 192.58 325.84 ± 239.20 264.62 ± 222.83 288.80 ± 209.00 339.02 ± 241.59 218.75 ± 177.34 343.72 ± 239.35 334.48 ± 231.82 318.31 ± 239.00 358.25 ± 207.07 361.99 ± 236.25 307.94 ± 233.20 338.65 ± 265.38 300.63 ± 224.24 373.37 ± 248.72 Mean ± SD 268.62 (128.74–483.43) 253.39 (129.83–390.40) 252.07 (140.51–432.51) 291.61 (161.67–462.02) 275.50 (150.02–465.99) 281.97 (152.88–450.50) 258.11 (138.40–429.40) 316.44 (179.28–434.09) 267.06 (145.14–447.17) 199.87 (109.82–331.52) 234.49 (129.68–388.50) 284.83 (157.56–459.65) 172.72 (95.02–279.92) 294.66 (161.98–465.91) 285.00 (152.64–458.75) 259.64 (142.60–427.60) 343.13 (185.77–472.19) 329.30 (174.70–489.60) 249.40 (137.74–410.38) 308.03 (113.66–490.33) 249.30 (131.06–403.78) 320.66 (179.30–508.36) Median (interquartile range) Serum concentration (ng/mL) 0.000b 0.027b 0.044a 0.000b 0.000a 0.003a 0.000b 0.000a P 1.87 ± 1.17 1.88 ± 1.43 1.88 ± 1.20 2.02 ± 1.25 2.17 ± 1.64 2.00 ± 1.19 1.82 ± 1.23 2.51 ± 1.69 1.89 ± 1.19 1.94 ± 1.31 1.82 ± 1.10 1.98 ± 1.29 1.25 ± 0.83 2.06 ± 1.27 2.15 ± 1.36 1.75 ± 1.11 2.67 ± 1.56 2.20 ± 1.42 1.81 ± 1.14 1.61 ± 0.92 1.83 ± 1.19 2.17 ± 1.34 Mean ± SD C/D Ratios 1.63 (1.04–2.31) 1.53 (1.00–2.41) 1.62 (1.05–2.39) 1.74 (1.17–2.58) 1.78 (1.12–2.70) 1.77 (1.15–2.57) 1.54 (1.01–2.29) 2.16 (1.44–3.28) 1.63 (1.07–2.40) 1.71 (0.99–2.37) 1.59 (1.08–2.29) 1.70 (1.11–2.51) 1.05 (0.65–1.62) 1.78 (1.19–2.59) 1.87 (1.24–2.73) 1.47 (1.01–2.18) 2.27 (1.55–3.38) 1.90 (1.27–2.73) 1.56 (1.02–2.29) 1.36 (0.86–1.98) 1.55 (1.02–2.32) 1.88 (1.25–2.72) Median (interquartile range) 0.000b 0.000b 0.000a 0.034b 0.000a 0.000a 0.000b 0.000a P (2023) 23:596 a 191.53 ± 131.32 188.10 ± 106.42 1091 468 Farmer 189.26 ± 106.65 Worker unemployed 1718 181.39 ± 103.90 307 Occupation divorced 186.31 ± 109.68 1435 1992 Single 199.61 ± 119.93 167.34 ± 120.58 193.16 ± 112.57 163.54 ± 111.34 176.29 ± 105.53 196.23 ± 116.14 204.29 ± 130.79 188.85 ± 110.24 176.91 ± 107.99 204.29 ± 116.87 married Marital status 3424 Psychiatric disorders Comorbidity or not 2835 Schizophrenia Psychiatric classification 3209 Inpatients Inpatients/outpatients 1924 ≤ 180 Duration (month) 60–75 2418 1089 18–44 45–59 193.00 ± 115.91 188.65 ± 108.78 116 216.27 ± 125.77 194.77 ± 107.31 Mean ± SD 1310 n 10–17 Age group (year) Male Female Gender Variables Daily dose (mg) Table 5 Comparison of clozapine dose, serum concentration and C/D ratio among different variable groups Liu et al. BMC Psychiatry Page 8 of 12 Liu et al. BMC Psychiatry (2023) 23:596 Page 9 of 12 Fig. 2 a Relationship between daily dose of clozapine and age groups. With increasing age, the daily dose of clozapine tended to decrease. b1 Relationship between clozapine serum concentrations and age groups. Although there were significant differences in the serum concentrations of clozapine among age groups, there was no significant trend in the daily dose of clozapine with age.b2 Relationship between clozapine serum concentration and age groups.When the subjects were divided into two age groups, 10-44 years old and 45-75 years old, the serum concentration of clozapine was significantly different between the two groups (P <0.01), and the serum concentration of clozapine in the 45-75 years old group was higher than that in the 10-44 years old group. c Relationship between the C/D ratio and age group. With increasing age, the C/D ratio tended to increase. With increasing age, the daily dose of clozapine tended to decrease population, which means that 50–350 mg/day may be the effective therapeutic dose for the population in the study region. This result is different from the recommended dose of 300–600 mg/day but is similar to the study conclusion of Jose et al. Jose’s study showed that the average clozapine dose in Asian countries was less than 300 mg/ day [30]. More research is needed to determine whether there are regional or ethnic differences in the therapeutic dose of clozapine. The average serum concentration of clozapine in this study was 326.15 ± 235.66 ng/mL, lower than the Arbeitsgemeinschaft für Neuropsychopharmakologie und Pharmakopsychiatrie (AGNP) recommended effective and safe concentration of 350–600 ng/mL [31, 32]. The population within the concentration range of 350–600 ng/mL only accounted for 28.07% of the population in this study, while the population with < 350 ng/mL concentration accounted for 62.77%. The serum concentration range of 50–600 ng/mL accounted for 86.20% of the subjects in this study; therefore, the treatment concentration of clozapine recommended in this study area may be more appropriate at 50–600 ng/mL. The therapeutic reference scope of clozapine in treatment-resistant schizophrenia is still controversial [33], and some existing studies suggest keeping clozapine levels above 350 ng/mL before considering dosing [34], which may not be appropriate for all populations. There were significant individual differences in the C/D ratio, which are affected by genetic, personal, and environmental factors [35, 36]. The C/D ratio indicates the clearance rate of drugs [37]; the smaller the C/D ratio is, the faster the drug clearance/metabolism is. In this study, the psychiatric patients were grouped according to the daily dose, the serum drug concentration increased and the C/D ratio decreased as the daily dose increased. The larger the daily dose was, the smaller the C/D ratio, indicating that the larger the dose was, Liu et al. BMC Psychiatry (2023) 23:596 Page 10 of 12 Table 6 Predictors associated with clozapine serum concentration in the linear regression model Variables Univariate model Daily dose (mg/day) Sex Male Age (year) Multivariate model B (95%CI) p B (95%CI) p 1.065 (1.007;1.122) 0.000 1.091 (1.036;1.146) 0.000 -72.738 (-88.411;-57.065) 0.00 -71.282 (-84.858;-57.706) 0.000 1.716 (1.075;2.357) 0.000 2.250 (1.488; 3.011) 0.000 Inpatients/outpatients Outpatients -124.971 (-146.352;-103.589) 0.000 -129.023 (-147.713;-110.333) 0.000 Duration (month) ≥ 180 16.170 (1.048;31.292) 0.036 -8.611 (-24.952; 7.730) 0.302 Comorbidity or not Comorbidity 3.760 (-23.647;31.167) 0.788 22.976 (-0.838; 46.790) 0.059 Occupation Unemployed -9.003 (-33.245; 15.245) 0.457 5.475(-14.465; 25.416) 0.590 Farmer -24.773 (-50.437; 0.892) 0.059 12.585 (-8.773;33.943) 0.248 Worker -67.096 (-97.387; -36.806) 0.000 -65.020 (-90.202; -39.838) 0.000 Others 1 Schizophrenia 74.396 (31.533; 117.259) 0.001 31.930 (-3.548; 67.408) 0.078 Bipolar disorder 24.177 (-20.925; 69.278) 0.293 19.101 (-17.901; 56.103) 0.312 Other types 1 Single -26.387 (-55.412; 2.638) 0.075 -4.381 (-29.126; 20.363) 0.729 Married -2.616 (-30.917; 25.685) 0.856 -8.678 (-31.990; 14.634) 0.466 Divorced 1 Psychiatric classification Marital status the faster the clearance. Therefore, it is more important to carry out drug concentration monitoring in people taking large doses than in people taking small doses to ensure effective doses. In this study, the average C/D ratio was 1.94 ± 1.25, which was higher than the 1.57 in the Asian population and 1.07 in Caucasians in the study results of Jose et al. [30]. The results indicate that the metabolic rate of clozapine in the population of the study area might be slower and that a lower daily dose may be required to achieve an effective serum concentration. In this study, both the serum concentration and C/D ratio of females were higher than those of males, which was similar to the results of scholars such as Castberg [38], Michaelaet [39] and Jönsson [40] and consistent with the drug instructions. Similarly, the metabolic rate of clozapine was higher in males than in females. Discussion of influencing factors In this study, the results showed that the older the age, the lower the required clozapine dose and the slower the metabolism, which suggests that serum drug concentration needs to be monitored among older individuals to prevent drug-related side effects caused by high serum concentration. The 10–17 year age group had the highest clozapine dose, the smallest C/D value and the smallest serum concentration. This may be because the metabolic rate of clozapine was faster in the 10–17 year age group, so a slightly higher dose was required to reach the serum effective therapeutic concentration. Our follow-up study 1 1 1 focused on the association between different ages and the concentration of effective psychiatric medications. Compared to outpatients, inpatients have similar daily doses, higher serum concentrations and slower metabolism rates. This may mean that there should be different standards for serum concentrations in inpatients and outpatients. The daily dose of clozapine did not differ among patients with different occupations, but unemployed patients had higher serum concentrations and slower metabolic rates. This may be because unemployed patients have less physical labor than employed patients, and physical activity increases blood circulation and drug metabolism, so unemployed people have the highest C/D ratio or lower metabolic rate. This suggests that occupation may be one of the factors that predict serum clozapine concentration, and psychiatrists should consider occupation factors when prescribing dosage of clozapine. Further studies are needed to explain the association between occupation and clozapine metabolism. Therefore, the monitoring of serum concentration should be strengthened in unemployed patients. Compared with psychiatric patients, patients with comorbidities have a smaller dose and slower metabolism; therefore, concentration monitoring in patients with comorbidities should be strengthened. Limitations of this study The major enzymes and efflux transporters involved in the metabolism and distribution of clozapine include Liu et al. BMC Psychiatry (2023) 23:596 CYP1A2, CYP2C19, CYP3A4, and P-gp (ABCB1) [21, 41–43]. Some drugs, such as carbamazepine, phenobarbital, phenytoin and rifampicin, can induce CYP1A2, CYP2C19, CYP3A4, P-gp (ABCB1) and other enzymes or ABC transporters [44–46]. The combination of these drugs with clozapine may accelerate the metabolism and distribution of clozapine. Such drugs were not included in this study and should be included in subsequent studies. Some drugs, such as fluvoxamine, enoxacin and phenylpropanolamine, fluoxetine and norfluoxetine, omeprazole, macrolobemide, and voriconazol, can inhibit the activity of the CYP1A2 enzyme and CYP2C19 enzyme [47–51]. These drugs can inhibit the metabolism of clozapine and increase the blood concentration of clozapine. These drugs were not included in this study, and the interaction between these drugs and clozapine could not be analyzed, and subsequent studies should be optimized. Future studies should include drug interactions and clozapine-related metabolic genes. The results of a previous study suggest that smoking is inversely associated with clozapine blood levels or decreases clozapine blood levels [39]. One mechanism that has been reported is that smoking can induce the CYP1A2 enzyme and accelerate the metabolism of clozapine [52]. Therefore, smoking patients should increase the dose of clozapine appropriately according to clinical needs and conduct blood concentration monitoring. Future clozapine-related studies should assess smoking. Conclusions In summary, the metabolism of clozapine in psychiatric patients is affected by a variety of factors and varies greatly among individuals. It is important to monitor the drug concentration when taking clozapine to determine the optimal therapeutic concentration range to guide treatment and achieve accurate treatment [17]. Abbreviations C/D concentration/ dose TDM Therapeutic drug monitoring Cmin steady state valley concentration HPLC high-performance liquid chromatography MDD major depressive disorder PD Parkinson’s disease Acknowledgements Thanks to all the researchers who participated in this study. Authors’ contributions PG and CX initiated the study and designed the protocol. HZ, ZS and RC conducted the serum concentration test. TL collected the information and wrote the paper. All authors read and approved the final manuscript. Funding Not applicable. Page 11 of 12 Availability of data and materials Data and material are available from the corresponding author upon reasonable request. Declarations Ethics approval and consent to participate The research protocol was approved by the ethics committee of Shandong Daizhuang Hospital. In this study, participants’ names were discarded, and existing data were analysed. The need for consent was waived by the Ethics Committee of Shandong Daizhuang Hospital. All experiments were performed in accordance with the Declaration of Helsinki and relevant guidelines and regulations. Consent for publication Not applicable. Competing interests Not applicable. Received: 25 April 2022 Accepted: 2 August 2023 References 1. Fitton A, Heel RC. Clozapine. A review of its pharmacological properties, and therapeutic use in schizophrenia. Drugs. 1990;40(5):722–47. 2. Khokhar JY, Henricks AM, Sullivan EDK, Green AI. Unique Effects of Clozapine: a pharmacological perspective. Adv Pharmacol (San Diego Calif ). 2018;82:137–62. 3. Kapczinski F, Pfaffenseller B, Dursun SM, de Azevedo Cardoso T. 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https://openalex.org/W2800599122	https://jmir.org/api/download?alt_name=resprot_v7i6e10525_app1.pdf	English	null	A Digital Decision Support Tool to Enhance Decisional Capacity for Clinical Trial Consent: Design and Development	JMIR research protocols	2,018	cc-by	1,307	Mobile Application Description AutisMate This app allows users to take their own videos, pictures, and voice recordings to share with others on the Autism spectrum. AutismXpress This app uses colorful, animated icons which can be used by those with Autism to help express their emotions. First Then Visual Schedule This app is used for people who require a structured environment and assists with activity transitions by providing a visual schedule. Grid Player This app allows users to build sentences by selecting a specific symbol or button. These sentences are then vocalized by the app. Look2Learn - AAC Individuals use photographs in this app to express their needs and wants. The app also comes with pre-loaded vocal outputs that users can pair with photographs. Pocket Cancer Care Guide This app was designed for cancer patients or caregivers of cancer patients to easily compile questions for their physician. Proloquo This app is a Multilingual Augmentative and Alternative Communication (AAC) solution designed for those who are unable to speak or have difficulty communicating. This software was designed for Mac OS X. Proloquo2Go This app allows users to build sentences by selecting a specific symbol or button. These sentences are then vocalized by the app. Sono Flex Lite This app allows users to build sentences by selecting a specific symbol or button. These sentences are then vocalized by the app. TalkingTILES This app allows users to build sentences by selecting a specific symbol or button. These sentences are then vocalized by the app. Autism Apps Behavior Scripts This app describes a variety of social situations children with Autism may encounter. The app also provides behavioral options for children to choose from. Conversation Social Stories and This app teaches communication skills Mobile Application Description AutisMate This app allows users to take their own videos, pictures, and voice recordings to share with others on the Autism spectrum. AutismXpress This app uses colorful, animated icons which can be used by those with Autism to help express their emotions. First Then Visual Schedule This app is used for people who require a structured environment and assists with activity transitions by providing a visual schedule. Grid Player This app allows users to build sentences by selecting a specific symbol or button. These sentences are then vocalized by the app. Look2Learn - AAC Individuals use photographs in this app to express their needs and wants. The app also comes with pre-loaded vocal outputs that users can pair with photographs. Pocket Cancer Care Guide This app was designed for cancer patients or caregivers of cancer patients to easily compile questions for their physician. Proloquo This app is a Multilingual Augmentative and Alternative Communication (AAC) solution designed for those who are unable to speak or have difficulty communicating. This software was designed for Mac OS X. Proloquo2Go This app allows users to build sentences by selecting a specific symbol or button. These sentences are then vocalized by the app. Sono Flex Lite This app allows users to build sentences by selecting a specific symbol or button. These sentences are then vocalized by the app. TalkingTILES This app allows users to build sentences by selecting a specific symbol or button. These sentences are then vocalized by the app. Autism Apps Behavior Scripts This app describes a variety of social situations children with Autism may encounter. The app also provides behavioral options for children to choose from. Conversation Social Stories and This app teaches communication skills Simple PECS Comm Tool through four social stories about different conversational skills. Hidden Curriculum for Kids This app includes entries about “unwritten social rules” that may result in anxiety or confusion for those with Autism. Living Safely This app provides self-directed learning sessions on home and personal safety skills topics. Luca Lashes Visits the Doctor This app helps children learn what to expect from a visit to the doctor through interactivity, touch, sight, and sound. My Health, My Choice, My Responsibility This is a cognitively accessible self- directed learning app that helps individuals with special needs learn about eight topics necessary for maintaining a healthy lifestyle. Mobile Application Description AutisMate This app allows users to take their own videos, pictures, and voice recordings to share with others on the Autism spectrum. AutismXpress This app uses colorful, animated icons which can be used by those with Autism to help express their emotions. First Then Visual Schedule This app is used for people who require a structured environment and assists with activity transitions by providing a visual schedule. Grid Player This app allows users to build sentences by selecting a specific symbol or button. These sentences are then vocalized by the app. Look2Learn - AAC Individuals use photographs in this app to express their needs and wants. The app also comes with pre-loaded vocal outputs that users can pair with photographs. Pocket Cancer Care Guide This app was designed for cancer patients or caregivers of cancer patients to easily compile questions for their physician. Proloquo This app is a Multilingual Augmentative and Alternative Communication (AAC) solution designed for those who are unable to speak or have difficulty communicating. This software was designed for Mac OS X. Proloquo2Go This app allows users to build sentences by selecting a specific symbol or button. These sentences are then vocalized by the app. Sono Flex Lite This app allows users to build sentences by selecting a specific symbol or button. These sentences are then vocalized by the app. TalkingTILES This app allows users to build sentences by selecting a specific symbol or button. These sentences are then vocalized by the app. Autism Apps Behavior Scripts This app describes a variety of social situations children with Autism may encounter. The app also provides behavioral options for children to choose from. Conversation Social Stories and This app teaches communication skills Stories2Learn This app uses stories to help promote social messages to individuals with autism and other developmental disabilities. The Social Express The app includes interactive lessons designed to teach children and young adults how to think about and manage social situations and help them to develop meaningful social relationships. Decision Support (n=7) AHRQ ePSS This app was developed to assist primary care clinicians to identify the screening, counseling, and preventive medication services that are appropriate for their patients. iDecisio This app is used to help adults optimize personal decision-making. inDecision This app is designed to simplify choices through the use of pros and cons. Informed Consent This app for researchers is a course that teaches about the key compliance requirements of the informed consent form and the administration process. MDS Risk This app assists physicians with risk stratification and selection of treatment options for patients with Myelodysplastic Syndrome (MDS). SecureConsent This app educates clinical trial candidates and records their informed consent. Tools 4 Students This app includes 25 graphic organizers for students to use to organize their thinking while reading or preparing to write. Clinical Trial (n=3) Clinical Trial Seek This app, sourced from clinicaltrials.gov, allows users to get information on cancer trials including how they work and searches by location, disease type, phase of trial and other filters. Clinical Trials Mobile This app, for prospective clinical trial participants or investigators, allows access to the National Library of Medicine database of available clinical trials. Guide to Clinical Trails This app provides clinical trial information and content that is easy for patients to use and understand. Behavior Modification (n=3) Behavior Tracker Pro This app allows teachers, therapists or parents to track behaviors and graph them. Skill Tracker Pro This app helps parents, teachers, therapists, and behavior analysts automate applied behavior analysis instruction for children with autism. Ther-Ad for Autism This evidence- based video self-modeling app includes motivating media, followed by a short clip of the individual with autism engaging in a behavior the parent or teacher wishes to encourage, followed by reinforced motivating content.
https://openalex.org/W2967270930	https://hess.copernicus.org/articles/24/489/2020/hess-24-489-2020.pdf	English	null	HESS Opinions: The myth of groundwater sustainability in Asia	Hydrology and earth system sciences	2,020	cc-by	9,460	Correspondence: Franklin W. Schwartz (schwartz.11@osu.edu) Correspondence: Franklin W. Schwartz (schwartz.11@osu.edu) Received: 1 August 2019 – Discussion started: 12 August 2019 Revised: 4 November 2019 – Accepted: 17 December 2019 – Published: 30 January 2020 Received: 1 August 2019 – Discussion started: 12 August 2019 Revised: 4 November 2019 – Accepted: 17 December 2019 – Published: 30 January 2020 Abstract. Across the arid regions of water-stressed coun- tries of Asia, groundwater production for irrigated agricul- ture has led to water-level declines that continue to worsen. For India, China, Pakistan, Iran, and others, it is unrealis- tic to expect groundwater sustainability in a veriﬁable sense to emerge. Fragmented governance and the general inability to bring traditional socio-economic tools to bear on reduc- ing groundwater demands have impeded progress to ground- water sustainability. For India and Pakistan, where opera- tional management is at the level of states and provinces, there is no capacity to regulate. Also in both China and In- dia, the tremendous numbers of groundwater users, large and small, confound regulation of groundwater. With business as usual, groundwater-related problems receive insufﬁcient at- tention, a situation referred to as an “accelerating and invisi- ble groundwater crisis” (Biswas et al., 2017). Another obsta- cle to sustainability comes from trying to manage something you do not understand. With sustainable management, there are signiﬁcant burdens in the needed technical know-how, in collecting necessary data, and in funding advanced technolo- gies. Thus, there are risks that Iran, India, and Pakistan will run short of groundwater from over-pumping in some places and will also be adversely affected by global climate change. ture and growing urbanization. However, progress to sustain- able development has been slow to non-existent. For many of these countries and even others outside of Asia, groundwa- ter sustainability is essentially just a myth. This paper makes a case that groundwater impacts in developing Asian coun- tries are already bad and getting worse. Further, it describes governmental and socio-economic impediments to sustain- able groundwater management, along with evident deﬁcien- cies in available data and, for most, an absent capacity to ei- ther regulate or undertake necessary projects. There is a huge gap between the full-blown water management approaches with demonstrated compliance, for example, in Singapore and California, USA, and those in Asia. Correspondence: Franklin W. Schwartz (schwartz.11@osu.edu) The concept of sustainability refers to the “development and use of groundwater in a manner that can be maintained for an indeﬁnite time without causing unacceptable, envi- ronmental, economic or social consequences” (Alley et al., 1999). It builds on the foundational concept of safe yield as “the limit to the quantity of groundwater which can be with- drawn regularly and permanently without dangerous deple- tion of the storage reserve” (Lee, 1915). A modern concept of groundwater sustainability recognizes the additional com- plexity provided by the inherent coupling of groundwater and surface water systems (Winter et al., 1998; Sophocleous, 1997) and a groundwater supply that is impaired because of contamination. 1 Introduction Progress toward aquifer sustainability also requires gov- ernments to provide a framework for action. The ﬁrst step is developing a vision for groundwater management, which leads to policies and laws (Smith et al., 2016). The laws pro- vide the authorities and tools needed to protect groundwa- ter resources. Examples include explicit powers to locate and register wells and tools to manage quantities pumped, using About 20 years ago, hydrogeologists began more fully to ap- preciate the extent of non-sustainable withdrawals of ground- water worldwide. However, recognizing a problem and doing something about it are two different things. Our focus here is Asia, where the need for sustainable groundwater man- agement is essential, given impacts from irrigated agricul- HESS Opinions: The myth of groundwater sustainability in Asia Franklin W. Schwartz1, Ganming Liu2, and Zhongbo Yu3 1School of Earth Sciences, Ohio State University, Columbus, OH 43210, USA 2School of Earth, Environment and Society, Bowling Green State University, Bowling Green, OH 43403, USA 3State Key Laboratory of Hydrology-Water Resources and Hydraulic Engineering, Nanjing, 210098, China 2 Trends in depletion and contamination of groundwater continue to worsen Successful implementation of management programs de- pends on the support and willing participation of local stake- holders. When stakeholder views are represented, especially during the drafting of legislation and implementation, there is broader compliance with regulations, a willingness to collect and share basic groundwater data, and active self-regulation (Smith et al., 2016; Garduno and Foster, 2010). In China, India, Pakistan, and other hotspots (Fig. 1), the impacts on groundwater due to depletion and contamination are continuing to worsen for reasons that we will discuss in Sect. 3. China’s most visible groundwater problem is associ- ated with the over-production of groundwater from aquifers underlying the core of the North China Plain (Fig. 2a). Groundwater withdrawals since 1960 have produced exces- sive drawdowns that began to receive attention about 15 years ago. Water-level declines of > 20 m were evident in the shal- low unconﬁned aquifer and > 40 m in the deep freshwater aquifer (Foster and Garduno, 2004). Estimated reductions in groundwater storage were about −8.8 km3 yr−1. By about 2010, drawdowns as high as ∼60 m were reported in the un- conﬁned aquifer (Cao et al., 2012) and > 80 m in the deep freshwater aquifer (Zheng et al., 2010) (Fig. 2a). Governments can use socio-economic tools to promote groundwater sustainability. For example, charging for the water will reduce its use, especially with large commercial users (Garduno and Foster, 2010). Of course, this step re- quires the difﬁcult task of metering of water used in irri- gation. A second example is strategic, governmental invest- ments to provide for efﬁcient irrigation. Another beneﬁcial direction is reversing common macro-policies that actually promote groundwater over-use (Garduno and Foster, 2010). Examples in India include price support for crops that need lots of water and reduced or no costs for agricultural neces- sities, such as rural electric power and agrochemicals. The aquifers of the North China Plain are essential to wheat production, to maintaining socio-economic contribu- tions associated with the agricultural economy, and to sup- plying water to cities (e.g., Beijing and Baoding) (Foster and Garduno, 2004). However, continuing water-level de- clines indicate limited progress to sustainability. A recent assessment using GRACE (Feng et al., 2018) indicates al- most constant declines in storage of −7.2 km3 yr−1 from 2002 to 2015. This result compares to an earlier estimate of −11.3 Gt yr−1 (−11.3 km3 yr−1) (Rodell et al., 2018). Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 490 F. W. Schwartz et al.: Groundwater sustainability in Asia Figure 1. Map showing the Asian countries and cities mentioned in the paper. licenses or “obligations” (Smith et al., 2016). They also could extend to (i) banning new-well construction in critical areas; (ii) “capping” withdrawal rates with existing wells (Garduno and Foster, 2010); and (iii) prosecutions for illegal pumping or waste discharges (Smith et al., 2016). Legislation might also provide strict controls on the use and disposal of haz- ardous chemicals or bans on the storage or use of hazardous chemicals in areas critical for groundwater. Laws and tools are ineffectual without an operational framework for managing groundwater in the ﬁeld. Oversight is required at national levels, together with other jurisdictions that make sense, for example, states, river basins, aquifers, or local communities (Smith et al., 2016). Governments also are responsible for funding various programs and coordinat- ing activities, with implications for groundwater in the areas of surface water, agriculture, energy, etc. It is also important to anticipate unintended consequences from actions taken in other parts of the government, a theme we will return to. Figure 1. Map showing the Asian countries and cities mentioned in the paper. Hydrol. Earth Syst. Sci., 24, 489–500, 2020 2 Trends in depletion and contamination of groundwater continue to worsen These impacts will continue even with water available from the South-to-North Water Transfer Project (Ye et al., 2014; Bloomberg, 2017). Had decision-makers acted on the basis of scientiﬁc knowledge, Asia would be far along to groundwater sus- tainability. Earlier studies pointed out problems and posited solutions. For example, in 2000, the World Bank formed the Groundwater Management Advisory Team (GW-MATE). Over the next decade, team members worked in Asia and elsewhere on groundwater-related issues and sustainable groundwater use. Their reports identiﬁed seriously impacted groundwater systems and provided practical approaches to sustainability. However, these ideas did not lead to substan- tive action because there was little in the way of operational frameworks and capacity for management. In China, there are other places with groundwater-related problems. Depletion is evident at some 164 locations, en- compassing three-fourths of China’s provinces (Wang et al., 2018). Declining water levels have increased the size of the area affected by land subsidence – 4.9×104 km2 in the 1990s to 7.9 × 104 km2 in the 2000s to 9 × 104 km2 in 2012 (Wang et al., 2018). Pumping of groundwater in coastal areas is also producing seawater intrusion. Hydrol. Earth Syst. Sci., 24, 489–500, 2020 www.hydrol-earth-syst-sci.net/24/489/2020/ F. W. Schwartz et al.: Groundwater sustainability in Asia 491 Figure 2. (a) is a shaded relief map of the core of the North China Plain. The red/orange colored areas have drawdowns > 20 m in the shallow aquifer (Cao et al., 2012). The yellow dots indicate areas with local drawdowns in the deep aquifer > 80 m (Zheng et al., 2012). (b) shows India, Pakistan, and Bangladesh. The red dots generally indicate the locations of hard-rock aquifers. The yellow dots point to the general location of the IGA system along the plains of the Indus and Ganges rivers. Figure 2. (a) is a shaded relief map of the core of the North China Plain. The red/orange colored areas have drawdowns > 20 m in the shallow aquifer (Cao et al., 2012). The yellow dots indicate areas with local drawdowns in the deep aquifer > 80 m (Zheng et al., 2012). (b) shows India, Pakistan, and Bangladesh. The red dots generally indicate the locations of hard-rock aquifers. The yellow dots point to the general location of the IGA system along the plains of the Indus and Ganges rivers. 2 Trends in depletion and contamination of groundwater continue to worsen pletion, estimated using GRACE data, range from 17.76 ± 4.5 km3 yr−1 (Rodell et al., 2009) to 14±0.4 km3 yr−1 (Long et al., 2016). The most realistic estimate (2000–2012) is somewhat lower, 8.0 ± 3.0 km3 yr−1 (5.2 ± 1.9 km3 yr−1 for northern India), based on actual groundwater measurements (MacDonald et al., 2016). The groundwater situation is also troubling in India, with an annual production of ∼250 km3, the largest in the world. For India, groundwater provides 85 % of drinking water and 60 % of water for irrigation (World Bank, 2010). There are two prototypical settings for groundwater in India. Shal- low hard-rock aquifers, like the Deccan Traps (basaltic lava ﬂows) or weathered granitic rocks, occur across the upland areas of the Indian peninsula (Fig. 2b). These low-yielding, weathered bedrock aquifers are important sources of water, which are being increasingly exploited with rates of with- drawal often greater than recharge (World Bank, 2010). Wa- ter typically occurs in fractures in the upper 25 m. During a typical year, increases in water levels due to recharge from monsoonal rains do not fully recover the withdrawals of pre- vious years. However, impacts from pumping are not the urgent prob- lem that some measurements (e.g., Rodell et al., 2009) im- ply. The relatively large quantities of groundwater stored in the upper 200 m of the IGA system coupled with 100 plus years of additional recharge from unintended canal leakage and irrigation return ﬂows means that depletion is restricted to certain local areas. However, what is concerning is that the greatest recent water-level declines are evident in north- ern Indian and Pakistan, areas essential for food production with irrigation. The Indo-Gangetic alluvial (IGA) aquifer system occurs across the top of India (Fig. 2b), extending into Pakistan, Nepal, and Bangladesh. It includes ﬂood plains along the Indus and Ganges rivers and their tributaries as a sequence of alluvial sediments > 200 m thick, derived from the Hi- malayan mountains (MacDonald et al., 2016). Often misunderstood, the threat to the sustainability of supplies from the IGA aquifer system is associated with water-quality issues – salinity, urban and industrial contam- inants, and arsenic in groundwater (MacDonald et al., 2016; Foster et al., 2018; Young et al., 2019). The origin of salinity in the shallow groundwater is complex but commonly associ- ated with effects of irrigation. www.hydrol-earth-syst-sci.net/24/489/2020/ F. W. Schwartz et al.: Groundwater sustainability in Asia 492 Elsewhere in Asia, the non-sustainable production of groundwater has resulted in even more serious problems. In Iran, the signiﬁcant loss of groundwater resources could ren- der major parts of the country uninhabitable with the pos- sibility of millions displaced as conditions worsen (Collins, 2017). In addition to widespread declines in water levels, there are signiﬁcant problems related to land subsidence and declining water quality (Madani et al., 2016). The largest cities of India exemplify the emerging prob- lems of water sustainability. A useful example is Delhi, whose population of ∼25 million is poised to double in the next 30 to 50 years. Most of Delhi’s drinking water comes from surface-water sources, but groundwater from the IGA aquifer system is both important and problematic. Almost ev- ery sustainability issue just discussed is a major problem for Delhi – rapidly declining water levels, salinity at depth, and nitrate concentrations commonly > 45 mg L−1 and as high as 1500 mg L−1. Various news outlets have been active in ex- pressing concerns about the local impacts of these problems (Box 1). Various factors have contributed to groundwater insecu- rity. Iran has a growing population of ∼80 million, which has doubled over the last 40 years (Bozorgmehr, 2014). The country is dry, making groundwater a growing source for drinking and irrigation water. A continuing trend towards urbanization has resulted in an urban population of 70 %, with 18 % in Tehran (Madani et al., 2016). Since 1999, there has been a succession of drought years. When coupled with an increase in annual temperature, the new normal is dryer and hotter weather with a likely decline in precipitation and recharge in the coming decades due to climate change (Go- hari et al., 2013; Nabavi, 2018). Political and policy failures associated with groundwater and surface water have created a crisis for India that bears di- rectly on food, water, and health (Biswas et al., 2017). With centuries of mismanagement of water resources and “institu- tional incompetence” (Biswas et al., 2017) in the context of a large growing population, there has been no willingness for action politically in India beyond “cosmetic changes” (Biswas et al., 2017). Although issues involved with sur- face waters (contamination, ﬁghts over allocation, and re- liability of public supplies) are worsening, “the groundwa- ter situation is even worse” (Biswas et al., 2017). However, data on groundwater are poor in quality or unavailable. F. W. Schwartz et al.: Groundwater sustainability in Asia Ram- pant growth in groundwater utilization is linked in part to the failure of government to provide surface water for irrigation (Biswas et al., 2017). This water crisis is also driven by socio-economic deci- sions of the late 1970s to become self-sufﬁcient in wheat, the country’s most important crop (Collins, 2017). The expan- sion in wheat production through irrigation has had signiﬁ- cant impacts on groundwater. However, there are few signs of movement to a more sustainable groundwater future (Collins, 2017). Pakistan is another country with groundwater issues threatening future sustainability. Its large population, ∼ 208 million and growing, contributes to its water-scarce sta- tus, with a per capita availability of water in the lowest 10 % of the world’s population (Young et al., 2019). The Indus River and its tributaries are signiﬁcant surface-water resources, used almost entirely to support irrigated agricul- ture. The use of water, however, is inefﬁcient, with signiﬁcant losses due to canal leakage, evaporation, and over-irrigation (Young et al., 2019). Another Asian hotspot for impacts associated with unsus- tainable groundwater production is Jakarta, Indonesia, on the island of Java. Approximately 25 %–30 % of the more afﬂu- ent residents of this large city receive piped-in surface wa- ter (Colbran, 2009). Others obtain drinking water from large numbers of groundwater wells, rainwater, vendors, bottled water, etc. The poor quality of piped water has pushed indus- tries and other large consumers to deep (∼150 m) ground- water (Colbran, 2009). However, this is not a drought story. Large, localized pro- duction from the shallow unconﬁned aquifer ∼50 m thick and a deeper conﬁned aquifer ∼100 m thick is not sustain- able even with signiﬁcant natural recharge (Kagabu et al., 2013). The over-use of groundwater has been evident for a long time. For example, in 1995, reported pumping rates were 3 times larger than recharge rates. By 2008, drawdowns in the deep aquifer were > 40 m, with hydraulic heads 25 m below sea level (Kagabu et al., 2013). Water quality in the shallow aquifer is impacted by urban contaminants like NO3 (Kagabu et al., 2013) because there are virtually no sanitary sewer systems. There is evident seawater intrusion landward within the deep aquifer caused by over-pumping. Declining water levels have also resulted in subsidence that in sev- eral places exceeds 2 m (IRIDeS, 2013). 2 Trends in depletion and contamination of groundwater continue to worsen Leaking canals, over more than a century in some instances, have led to waterlogging and salt accumulation in soil and the salinization of recharge (Foster et al., 2018). Large-capacity irrigation wells are also capable of mobilizing naturally salty water occurring at depth with up-coning. Estimates are that 18 000 km3 or 60 % of shallow groundwater in the IGA system suffers from water-quality impairment (MacDonald et al., 2016). Groundwater production from the IGA aquifer system in 2010 (including adjacent countries) was 205 km3 yr−1, in- creasing at 2–5 km3 yr−1 due to continued expansion of ir- rigated agriculture (MacDonald et al., 2016). These large withdrawals are offset by comparably large inﬂows as leak- age from irrigation canals, irrigation return ﬂows, and nat- ural recharge from monsoonal rains. Assessments are com- plicated by spatial variability in hydraulic parameters, var- ious water-quality impacts, and uncertainties in recharge estimates (MacDonald et al., 2016). Rates of storage de- www.hydrol-earth-syst-sci.net/24/489/2020/ Hydrol. Earth Syst. Sci., 24, 489–500, 2020 3 What are the hurdles to groundwater sustainability? Developing Asian countries have encountered signiﬁcant roadblocks hindering progress to groundwater sustainabil- ity. So far, it has been relatively painless for countries and large cities simply to ignore groundwater issues, which in the case of India has been called an “invisible” crisis (Biswas et al., 2017). Of greater concern in Asian countries is a col- lection of more critical national issues related, for example, to growing their economies, feeding their people, maintain- ing national security, and improving the social conditions for growing populations. Another drag on sustainability efforts in India is politi- cal sensitivities. For example, the free or nearly free elec- trical power for irrigation in rural areas is a signiﬁcant fac- tor in groundwater over-pumping. However, there is no po- litical will to change this policy (World Bank, 2010). It is also problematic that groundwater oversight exists in many government agencies, with no clear deﬁnition of responsi- bilities between the state and central governments. Thus, for India, there are no realistic possibilities for state intervention in groundwater management (World Bank, 2010). The best opportunities exist at the community level. In Asian countries, there much less deference to water se- curity than food security. India’s “Green Revolution” (GR) is a case in point. In the 1950s, government leaders in India were troubled by the deaths from the Bengal Famine of 1943 (Rahman, 2015). With their growing population, achieving food security became a top priority. In the 1960s, the GR began with an expansion in agricultural lands, new high- yielding seeds, expanded irrigation, double cropping, and vastly increased fertilizer and pesticide applications (Rah- man, 2015; Schmanski, 2008). India became food secure, with large increases in the production of food and cereal grains. The situation in Pakistan is quite similar to India, with little prospect for progress. Within their federal system of governance, provinces have responsibilities for managing groundwater. However, there are neither provincial regu- latory frameworks nor the capacity to regulate access to groundwater (Young et al., 2019). There are limited techni- cal capabilities in managing surface water and groundwater conjunctively (Young et al., 2019). Another concern is the unmitigated, long-term risk to groundwater sustainability in the lower Indus River basin and delta from inefﬁcient irriga- tion and seawater intrusion. Yet there is a dark side, which includes severe social, economic, and environmental problems, particularly in the amazingly productive Punjab region of India (Fig. 2b). 3 What are the hurdles to groundwater sustainability? Ex- amples include the high suicide rates of farmers, increasing cancer rates from pesticides, and especially the unsustain- able use of groundwater (Schumanski, 2008; Singh and Park, 2018). Groundwater impacts were slow to develop, but are now serious, with total water-level declines ranging from 4.5 to 35 m (Rahman, 2015). In China, there are legal/operational frameworks that have the potential to contribute to groundwater sustainability. A complex, multi-tiered system for water-resource administra- tion exists, with units represented all the way down to local water user associations (Doczi et al., 2014). Most progress in the management of agricultural water has been with tra- ditional surface-water irrigation systems, which has con- tributed to rises in production and irrigation efﬁciency (Doczi et al., 2014). Groundwater is more problematic, with water- level declines continuing (Doczi et al., 2014; Biswas and Hartley, 2017; Wang et al., 2018). The number of privately owned wells has increased signiﬁcantly, giving farmers the ability to “protect their crops” (Doczi et al., 2014) from drought and the inconsistent availability of surface water for irrigation. China, India, and Iran have been able to aggressively ramp up agricultural production to feed their people with- out adequately considering the impacts on groundwater. With recharge and the inherent capacity of large aquifers to store abundant groundwater, problems developed incrementally and have been difﬁcult to recognize in data-poor settings. Now, food production from irrigated agriculture is struc- turally part of the national economies of these countries, making it difﬁcult to reduce the production of food and groundwater. Doczi et al. (2014) point to problems in implementing na- tional policies, with regulation unable to catch up with the rapidly expanding use of groundwater. Speciﬁc measures like drilling permits, quotas on water, and fees have been imple- mented but neither broadly (Wang, et al., 2019) nor effec- tively (Doczi et al., 2014). Controlling water-level declines across the North China Plain is also complicated by the grow- ing need for groundwater to help support rapid urban and in- dustrial growth (Biswas and Hartley, 2017). However, China does have the ﬁscal and technical capacity to support projects focused on sustainability. The challenge in this respect is in ﬁnding water sources for managed aquifer recharge (MAR). The second major impediment to sustainable management is limitations in terms of a socio-economic framework for action and necessary data. F. W. Schwartz et al.: Groundwater sustainability in Asia enforce regulations, especially with many small water users. Thus, India’s estimated 20 million wells are unregulated and outside any regulatory framework (World Bank, 2010). F. W. Schwartz et al.: Groundwater sustainability in Asia Now approximately 40 % of the city’s land surface is below sea level, with only a seawall to protect land from inundation. However, there is no urgency around groundwater, with business as usual. The IGA aquifer system extends southward into Pakistan along the length of the Indus River. For now, levels of groundwater in the IGA aquifer system in Pakistan are sta- ble or even increasing (MacDonald et al., 2016). The main problems are associated with water-level declines of ∼10 m since the 1980s in the important food-growing area of Pun- jab province to the northeast (Young et al., 2019). Here, as in India, canal leakage and irrigation return ﬂow have con- tinued to provide an unmanaged aquifer recharge system that has banked water in the subsurface since the late 1800s to the point of waterlogging in some places (MacDonald et al., 2016). The greater threat to sustainability often comes from the kinds of water-quality problems mentioned previously. There is little progress in the development of a sustainabil- ity ethic for groundwater management in Pakistan. Assess- ments are frustrated by an absence of data and the lack of a quantitative understanding of groundwater–surface-water in- teractions along the major rivers (Young et al., 2019). www.hydrol-earth-syst-sci.net/24/489/2020/ Hydrol. Earth Syst. Sci., 24, 489–500, 2020 493 www.hydrol-earth-syst-sci.net/24/489/2020/ g y p y There are positive activities underway, which contribute to groundwater sustainability by promoting groundwater recharge and storage. However, just what that contribution actually comprises is unknown in a technical sense. For ex- ample, India is the world leader in the number of installed systems to store water in the subsurface (Dillon et al., 2019). There are several million traditional (often old) recharge structures, such as percolation tanks/ponds and streambed in- ﬁltration systems, with millions more planned (Dillon et al., 2019). However, there are few quantitative assessments of how well these systems work in promoting recharge (Dillon et al., 2019; Dashora et al., 2018) and whether they can be ef- fective without actions that manage demands and quantities pumped. Box 1. Headlines and articles reacting to Delhi’s worsening ground- water problems. Box 1. Headlines and articles reacting to Delhi’s worsening ground- water problems. systems. Figure 3 highlights the broad scope of data needs with an illustrative conceptual model of a complex coastal hydrologic system (CDWR, 2016). China, however, has a much smaller number of these kinds of traditional recharge projects in operation (Dillon et al., 2019). Instead, they are investing in their “sponge-city” con- cept, a collection of “low-impact” practices (Biswas and Hartley, 2017) designed primarily to reduce urban ﬂooding and water pollution. The idea is to store water in the subsur- face through pervious pavements or utilize/store storm water in rain gardens or rooftop gardens. These kinds of green in- frastructure projects are growing in China. While contribut- ing to groundwater sustainability through urban recharge, it is not yet clear what that contribution will be. Asian countries starting from scratch will need to antic- ipate costs associated with years of ﬁeld operations in, for example, groundwater mapping, aquifer testing, and water- quality measurements. Various monitoring networks will need to be designed and emplaced, as well as equipment to be purchased, installed, and operated. Provision must be made for data compilation and storage, interpretations, modeling, laboratory measurements, etc. What adds even more difﬁ- culty is an absolute need to monitor for 1 to several decades to provide an average set of baseline conditions (CDWR, 2016). The creation of conceptual models, water-balance cal- culations, and compliance assurance all require these kinds of data. A useful place to gain perspective is with a series of best practice reports of the California Department of Water Resources (e.g., CDWR, 2016). 3 What are the hurdles to groundwater sustainability? Following here is a discussion of these issues for three large countries: India, Pakistan, and China. As mentioned, centralized groundwater management re- quires appropriate policies, legislation, and a functional reg- ulatory framework. India has a useful collection of laws in place, but groundwater management is “weak to nonexistent” (World Bank, 2010). The most important limitation is that water is a state responsibility and many of the states in India have no capacity to monitor groundwater production or to The Asian countries we have examined also have prob- lems with data. Some water-level data are available in ar- eas most impacted by over-pumping. However, information www.hydrol-earth-syst-sci.net/24/489/2020/ Hydrol. Earth Syst. Sci., 24, 489–500, 2020 494 F. W. Schwartz et al.: Groundwater sustainability in Asia F. W. Schwartz et al.: Groundwater sustainability in Asia on which wells are pumping what quantities of water typ- ically does not exist. It is evident that the tens of millions of wells in both China and India provide a formidable op- erational challenge in monitoring. With the pervasive irriga- tion associated with rivers and systems of canals, unmanaged aquifer recharge is a signiﬁcant yet unknown factor inﬂuenc- ing water levels. There is even less information on threats to sustainability coming from groundwater contamination. Box 1. Headlines and articles reacting to Delhi’s worsening ground- water problems. Development of some “understanding” in relation to the sustainable management of groundwater depends upon pro- grams of hydrogeologic mapping, monitoring, and modeling. However, there is little discernable progress in data collection necessary to support sustainability initiatives in either India (Biswas et al., 2017) or Pakistan (Young et al., 2019). There may be somewhat more progress in China, but information there is siloed and lacking in the necessary transparency. F. W. Schwartz et al.: Groundwater sustainability in Asia F. W. Schwartz et al.: Groundwater sustainability in Asia Box 2. The yellow and green boxes list some of the strategies for increasing aquifer storage of groundwater by increasing inﬂows through managed recharge and/or decreasing the quantity of water pumped, respectively. The red arrows indicate associated issues. age does not change over the long term while maintaining appropriate natural discharges to rivers and springs. Reduc- tions in storage due to unsustainable production can only be reversed in two ways – increasing the quantity of inﬂows to the aquifer (e.g., recharge) or decreasing the outﬂows (e.g., pumping with wells). The yellow box in Box 2 lists four recharge schemes to increase inﬂows to aquifers (i.e., MAR) with links to the associated issues/problems, as indicated by the red arrows. or changing to crops that use less water, will lead to less groundwater utilization (iii to vi green, Box 2). On the one hand, with governments ﬁrmly committed to food security and poor farmers needing to maintain their livelihoods, such initiatives are unattractive. On the other hand, these strategies require minimal technical expertise. Governments can pass a law, check the sustainability box, and plan to spend money to import food. Finally, more efﬁcient irrigation technologies might lead to reduced pumping while also leading to reduced recharge (Garduno and Foster, 2010). Clogging is a problem reducing the quantities of water in- ﬁltrated or injected into the subsurface, but can be managed with regular maintenance to maintain performance. In an Asian context, the other issues affecting MAR (Box 2) also provide formidable challenges. Finding water to recharge an aquifer can be difﬁcult. Surface water can be scarce be- cause excess water is often only available with summer mon- soons. Treated municipal sewage, another important source of water, is often not available or of appropriate quality. For example, ∼50 % of Delhi’s population has no sewers (Sengupta, 2015), with signiﬁcant quantities of wastewater dumped into the nearby Yamuna River or left to seep into the ground. In addition, there tends to be declining interest in projects involving long transfers of water. Farmers in India (and China) prefer groundwater for irrigation as compared to government-supplied surface water (World Bank, 2010). In- frastructure like reservoirs, pipelines, or canals is needed to transfer water to where it is needed. F. W. Schwartz et al.: Groundwater sustainability in Asia g ( ) The feasibility of sustainable groundwater management is on display with projects of Orange County Water District (OCWD) in southern California. This case study illustrates that in arid areas with modest recharge and signiﬁcant with- drawals, groundwater sustainability will require MAR. It also shows how wastewater recycling can provide a source of wa- ter when there are limited prospects for new surface water. However, this source is expensive in terms of physical infras- tructure and advanced technologies for puriﬁcation. OCWD distributes water to ∼2.4 million people. Sustainable oper- ation of the aquifer systems produces ∼345 Mm3 yr−1 of groundwater, which is ∼4.7 times the natural recharge of 74 Mm3 yr−1 (Hendron and Markus, 2014). MAR, using in- ﬁltration basins, makes up the deﬁcit, with 185 Mm3 yr−1 coming from the Santa Ana River and 86 Mm3 yr−1 from pu- riﬁed urban wastewater (Hendron and Markus, 2014). Mu- nicipal wastewater is collected and treated conventionally and then puriﬁed with additional advanced treatment with re- verse osmosis and more. Some of this puriﬁed water is used to maintain a hydraulic barrier in the subsurface to prevent seawater intrusion. This kind of system, providing evidence- based sustainability and high-quality water, is expensive to A variety of strategies exists to reduce groundwater with- drawals. Replacing groundwater (i and ii green, Box 2) in irrigation with imported surface water or treated wastewater is often challenging and would require re-imagining of water support systems. Decreasing agricultural production through acreage reductions, growing one crop per year instead of two, They are intended to provide technical assistance for California’s new state-wide initiative in sustainable groundwater management. In the megacities, like Jakarta, Delhi, and Karachi, our re- views found the status of groundwater data to be meagre to non-existent and inadequate to support technical or socio- economic efforts to sustainability. The kinds of technical knowledge and data needed for sustainable groundwater management are well known. They include a robust qualitative and quantitative understand- ing of how the land-based portion of the hydrologic sys- tem functions, physically, chemically, and biologically. Basic data collection involves metering or other approaches to es- tablish water utilization, groundwater–surface-water interac- tions, aquifer characterizations, testing, sampling, and mea- surements in the ﬁeld, supported by various monitoring net- works, data acquisition systems, laboratories, and database The third major obstacle is that technically oriented initia- tives on sustainability require expensive infrastructure with continuing operating costs, especially with the development of new water sources for MAR, for example, treated sewage, rainwater, or imported surface water. Consider a problem where the key issue with sustainable management is water- level declines from excessive pumping. The operational ob- jective is to end up with an aquifer system where water stor- www.hydrol-earth-syst-sci.net/24/489/2020/ Hydrol. Earth Syst. Sci., 24, 489–500, 2020 495 F. W. Schwartz et al.: Groundwater sustainability in Asia 495 Box 2. The yellow and green boxes list some of the strategies for increasing aquifer storage of groundwater by increasing inﬂows through managed recharge and/or decreasing the quantity of water pumped, respectively. The red arrows indicate associated issues. F. W. Schwartz et al.: Groundwater sustainability in Asia 496 build and operate. It is also critically dependent on monitor- ing (OCWD, 2015). et al., 2019). The continuing trend towards urbanization at all scales up to megacities is localizing water demands and exacerbating groundwater problems. The existence of nec- essary data as a prerequisite for problem understanding and management decisions remains a problem for all countries except perhaps China. Such sophisticated water management systems are un- common in Asia. Yet there are several extraordinary exam- ples. The island state of Singapore is home to an innova- tive collection of management activities creating near self- sufﬁciency from water imports from Malaysia (Irvine et al., 2014). Drinking and industrial waters come from capturing and treating rainwater captured with urban catchments, the advanced puriﬁcation of urban wastewater, creating a prod- uct called NEWater, and the addition of desalination plants (Irvine et al., 2014). MAR projects in Israel also provide other useful examples. The Dan Region Reclamation Project (also known as Shafdan) uses treated wastewater from Tel Aviv and environs for MAR (Cikurel et al., 2012). The system yields 140 Mm3 yr−1 of high-quality water that is pumped 100 km south for irrigation. As of 2012, this was the largest project of its kind in Europe and the Middle East (Cickurel et al., 2012). Israel also depends on the reverse os- mosis of seawater with periodic storage of excess water in the Israeli Coastal Aquifer (Ganot et al., 2018). There is, however, some hope that new technologies may create sufﬁcient visibility of the severity of the groundwa- ter problems to ﬁnally spur action (Fogg, 2019). For exam- ple, there are relatively inexpensive, wireless technologies available for monitoring water levels in real time. Expected improvements in satellite remote sensing, particularly future GRACE missions, are also expected to enhance our under- standing of aquifers worldwide (Fogg, 2019). However, ﬁeld- based hydrogeological campaigns (e.g., MacDonald et al., 2016) and comprehensive water-quality monitoring will be necessary. y With China as an obvious exception, groundwater-related research is not close to where it needs to be. Our review is evidently cursory, focused mainly on available informa- tion in journal papers and government reports. Much of what is known about the groundwater in Asian countries (China again excepted) comes from international researchers but with in-country collaborators and cooperators. F. W. Schwartz et al.: Groundwater sustainability in Asia Inﬂuen- tial in this respect are long-term studies by the World Bank on speciﬁc strategies for management, large-scale interpreta- tions of water-storage changes with GRACE, and a few re- gional groundwater investigations. One area of critical need is research to address water-quality issues in both India and Pakistan. Work by MacDonald et al. (2016) and Foster et al. (2018) has identiﬁed the threat to sustainability related to problems of salinity and groundwater contamination. For example, with the IGA aquifer system, this threat, around issues of water quality, is more serious than over-pumping (MacDonald et al., 2016). Problems of arsenic pollution are widespread, including Pakistan, while human activities have led to groundwater salinization and urban/agricultural con- tamination (MacDonald et al., 2016). There are also hints of broad groundwater contamination in China (Biswas and Hartley, 2017), although information is scarce. The common characteristics of all three of these success- ful implementations include (i) extreme shortages of water to the point of exhausting local surface water and groundwater supplies, (ii) technologically advanced and prosperous soci- eties, with modern and reliable water/power infrastructures, and (iii) a manageable problem scope stemming from rela- tively small populations. www.hydrol-earth-syst-sci.net/24/489/2020/ www.hydrol-earth-syst-sci.net/24/489/2020/ Hydrol. Earth Syst. Sci., 24, 489–500, 2020 www.hydrol-earth-syst-sci.net/24/489/2020/ 4 Groundwater management: a call to action Complexities arise from (i) the number of different processes operat- ing within the basin and their associated parameters, (ii) a need to quantify the diverse array of water exchanges within the hydrologic cycle, (iii) water uses that need to be metered, (iv) potential groundwater contamination from irrigation return ﬂows, and (v) constraints dictated by sustainable groundwater management (with permission, California Department of Water Resources, 2016). Figure 3. Conceptual model of a hypothetical coastal hydrologic system featuring a major river, cities, agricultural irrigation, an alluvial aquifer system being recharged using various MAR systems, and more. Complexities arise from (i) the number of different processes operat- ing within the basin and their associated parameters, (ii) a need to quantify the diverse array of water exchanges within the hydrologic cycle, (iii) water uses that need to be metered, (iv) potential groundwater contamination from irrigation return ﬂows, and (v) constraints dictated by sustainable groundwater management (with permission, California Department of Water Resources, 2016). of the countries involved. While these approaches represent an important ﬁrst step to groundwater sustainably, they are no panacea. For example, traditional approaches to water harvesting in India are not well suited for hard-rock areas, impact downstream users, and often lead to more pumping (World Bank, 2010). In addition, there is signiﬁcant uncer- tainty as to whether these approaches will contribute mean- ingfully to sustainability, especially with uncontrolled with- drawals. All of these technologies would beneﬁt from analy- ses to identify strengths and weaknesses and to optimize the beneﬁts for groundwater sustainability. Studies sponsored by the World Bank (2010) suggest that there is hope for community-based management in the hard-rock areas of In- dia and perhaps elsewhere. support complex projects. There is some thinking that after many decades of aggressively exploiting aquifers, societies have begun to wake up to the need to manage and recharge aquifers (Fogg, 2019). Nevertheless, there is a signiﬁcant risk that progress will be slow in many Asian countries given the problems of capacity and socio-economic constraints. A major transition will be required to move from water poli- cies, viewed widely as muddling along from one crisis to the next without substantive action to full-blown projects with demonstrated compliance (i.e., Singapore, California). Only time will tell as to whether the successful water management schemes in places with relatively small and economically ad- vantaged populations are practically scalable to many tens of millions of people in developing countries. 4 Groundwater management: a call to action There are compelling arguments to explain the slow devel- opment of a sustainability ethic in some Asian countries. In Pakistan, India, and China, fragmented governance and the general inability to bring traditional socio-economic tools to bear on reducing groundwater demands impede progress to groundwater sustainability. The indictment for India, “cen- turies of mismanagement, political and institutional incom- petence; indifference at central, state, and municipal levels, and steadily increasing population” (Biswas et al., 2017), applies as well to other countries. For India and Pakistan, where operational management is at the level of states and provinces, there is no capacity to regulate. Also in both China and India, the tremendous numbers of groundwater users, large and small, confound regulation of groundwater. Thus, groundwater that has always been freely available for irriga- tors remains so. Adding water-quality issues to the mix of sustainability issues reveals even greater deﬁciencies and a need for water- quality monitoring as an essential step for sustainable man- agement. Salinity problems are complicated because impacts can occur in so many ways. In Pakistan, saline water exists at depth in addition to salinized recharge caused by waterlog- ging. Moreover, this deep groundwater water can be remo- bilized by pumping (Foster et al., 2018). In China, shallow groundwater across the eastern half of the North China Plain is salinized (Foster and Garduno, 2004). This creates the pos- sibility of eventual water-quality impairment in the underly- ing deep freshwater aquifer as over-pumping there continues. Groundwater-related problems are largely invisible (Biswas et al., 2017) and seemingly irrelevant to a greater agenda. It may also be that groundwater is so plentiful that it has never been a concern (Fogg, 2019). For China, India, Pakistan, and Iran, there is an undeniable focus on food production to support growing populations and changing food preferences of increasingly afﬂuent societies (Young We hope that countries in Asia will begin to address sus- tainability problems more aggressively in critical areas, in- cluding the practical hydrogeologic investigations needed to www.hydrol-earth-syst-sci.net/24/489/2020/ Hydrol. Earth Syst. Sci., 24, 489–500, 2020 F. W. Schwartz et al.: Groundwater sustainability in Asia F. W. Schwartz et al.: Groundwater sustainability in Asia 497 Figure 3. Conceptual model of a hypothetical coastal hydrologic system featuring a major river, cities, agricultural irrigation, an alluvial aquifer system being recharged using various MAR systems, and more. www.hydrol-earth-syst-sci.net/24/489/2020/ Hydrol. Earth Syst. Sci., 24, 489–500, 2020 F. W. Schwartz et al.: Groundwater sustainability in Asia Competing interests. The authors declare that they have no conﬂict of interest. Collins, G.: Iran’s Looming Water Bankruptcy, Rice University’s Baker Institute for Public Policy, 19 pp., 2017. Dashora, Y., Dillon, P., Maheshwari, B., Soni, P., Dashora, R., Da- vande, S., Purohit, R. C., and Mittal, H. K.: A simple method using farmers’ measurements applied to estimate check dam recharge in Rajasthan, India, Sustainable Water Resources Man- agement, 4, 301–316, 2018. Acknowledgements. We thank the editor and reviewers for their helpful comments and suggestions. Dillon, P., Stuyfzand, P., Grischek, T., Lluria, M., Pyne, R. D. G., Jain, R. C., Bear, J., Schwarz, J., Wang, W., Fernandez, E., Ste- fan, C., Pettenati, M., van der Gun, J., Sprenger, C., Massmann, G., Scanlon, B. R., Xanke, J., Jokela, P., Zheng, Y., Rossetto, R., Shamrukh, M., Pavelic, P., Murray, E., Ross, A., Bonilla Valverde, J. P., Palma Nava, A., Ansems, N., Posavec, K., Ha, K., Martin, R., and Sapiano, M.: Sixty years of global progress in managed aquifer recharge, Hydrogeol. J., 27, 1–30, 2018. Review statement. This paper was edited by Alberto Guadagnini and reviewed by Olaf Arie Cirpka, Graham Fogg, and one anony- mous referee. F. W. Schwartz et al.: Groundwater sustainability in Asia 498 of groundwater. No doubt, such analyses would be difﬁcult and uncertain, given the absence of data. However, with po- tentially hundreds of millions of people at risk, it would be prudent to better understand the scope and scale of future problems. world-s-largest-water-diversion-plan-won-t-slake-china-s-thirst (last access: 15 May 2018), 2017. Bozorgmehr, N.: Iran: Dried out, Financial Times, available at: http://www.ft.com/content/ 5a5579c6-0205-11e4-ab5b-00144feab7de (last access: 3 May 2018), 2014. y Cao, G., Zheng, C., Scanlon, B. R., Liu, J., and Li, W.: Use of ﬂow modeling to assess sustainability of groundwater resources in the North China Plain, Water Resour. Res., 49, 159–175, 2013. Data availability. The production of the digital elevation maps for Fig. 2 used the following data sources: SRTM 90 m DEM Version 4, accessed and downloaded from http://srtm.csi.cgiar.org/srtmdata/ (last access: 15 May 2019) (Jarvis et al., 2008), GIS data for admin- istrative area boundaries from https://www.diva-gis.org/gdata (last access: 15 May 2019) (Hijmans et al., 2012), and country bound- aries made with Natural Earth: free vector and raster map data at https://www.naturalearthdata.com (last access: 15 May 2019). North China Plain, Water Resour. Res., 49, 159–175 CDWR (California Department of Water Resources): Best Manage- ment Practices for the Sustainable Management of Groundwater Water Budget, California Department of Water Resources, available at: http://water.ca.gov/-/media/DWR-Website/ Web-Pages/Programs/Groundwater-Management/ Sustainable-Groundwater-Management/ Best-Management-Practices-and-Guidance-Documents/Files/ BMP-4-Water-Budget.pdf (last access: 6 April 2019), 51 pp., 2016. Sustainable-Groundwater-Management/ Author contributions. FS conceived the idea and wrote much of the manuscript. GL created the colored, three-dimensional elevation maps for China and India and prepared Figs. 1 and 2. Both GL and ZY contributed to the paper, especially insights and material with respect to China. All the authors reviewed early manuscript drafts and the ﬁnal draft. Author contributions. FS conceived the idea and wrote much of the manuscript. GL created the colored, three-dimensional elevation maps for China and India and prepared Figs. 1 and 2. Both GL and ZY contributed to the paper, especially insights and material with respect to China. All the authors reviewed early manuscript drafts and the ﬁnal draft. Cikurel, H., Guttman, J., and Aharoni, A.: Managed aquifer recharge for agricultural reuse in Shafdan, Israel, edited by: Kazner, C., Wintgens, T., Dillon, P., Water Reclamation Tech- nologies for Safe Managed Aquifer Recharge, IWA Publishing, 83–102, 2012. Colbran, N.: Will Jakarta be the next Atlantis? Excessive ground- water use resulting from a failing piped water network, Law En- vironment and Development Journal, 5, 20–37, 2009. 4 Groundwater management: a call to action In any case, lo- gistical constraints mean that it will be decades before sus- tainable systems are up and running. Such a delay increases the possibilities of predictable surprises – problems (e.g., cli- mate change) that are anticipated but ignored (Bazerman and Watkins, 2004). There are risks that Iran, India, and Pakistan will run short of groundwater from over-pumping in some places and be adversely affected by global climate change, espe- cially ﬂoods and droughts. The dimensions of these risks are not well deﬁned. However, in the case of climate change, a forward-looking study has examined the extent of in-country migration in order that countries “can plan and prepare” (Rigaud et al., 2018). The study involved modeling future migration in arid regions of the world due to climate change. The results for South Asia suggested that by 2050, there could be 35.7 million in-country climate migrants under a pessimistic future climate scenario. In the context of ground- water sustainability, we envision a need for similar scoping studies to examine the threats associated with running out There are basic technical approaches that have the poten- tial to contribute to sustainability. For example, several coun- tries are already invested in recharge projects, India with their traditional MAR (Dillon et al., 2018) and China with their “sponge-city” concepts. Signiﬁcant opportunities ex- ist in identifying strengths and weaknesses in these methods and in optimizing the beneﬁts for groundwater sustainabil- ity. To be most useful, studies should focus on best prac- tices appropriate to the economic and technical capacities www.hydrol-earth-syst-sci.net/24/489/2020/ Hydrol. Earth Syst. Sci., 24, 489–500, 2020 F. W. 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https://openalex.org/W3009735491	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0229929&type=printable	English	null	Cue-dependent effects of VR experience on motion-in-depth sensitivity	PloS one	2,020	cc-by	8,233	PLOS ONE RESEARCH ARTICLE Cue-dependent effects of VR experience on motion-in-depth sensitivity Jacqueline M. FulvioID1☯, Mohan Ji1☯, Lowell Thompson1,2, Ari RosenbergID2‡, Bas Rokers1‡ 1 Department of Psychology, University of Wisconsin–Madison, Madison, Wisconsin, United States of America, 2 Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin– Madison, Madison, Wisconsin, United States of America ☯These authors contributed equally to this work. ‡ These authors also contributed equally to this work. jacqueline.fulvio@wisc.edu a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Data Availability Statement: The data are available in an Open-Science Framework repository, with DOI: 10.17605/OSF.IO/DY9QX Data Availability Statement: The data are available in an Open-Science Framework repository, with DOI: 10.17605/OSF.IO/DY9QX Funding: This work was supported by Facebook Reality (BR, JMF), Google Daydream (BR, JMF), Whitehall Foundation (2016-08-18) (AR), and National Institutes of Health (EY029438) (AR). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Abstract The visual system exploits multiple signals, including monocular and binocular cues, to determine the motion of objects through depth. In the laboratory, sensitivity to different three-dimensional (3D) motion cues varies across observers and is often weak for binocular cues. However, laboratory assessments may reflect factors beyond inherent perceptual sensitivity. For example, the appearance of weak binocular sensitivity may relate to exten- sive prior experience with two-dimensional (2D) displays in which binocular cues are not informative. Here we evaluated the impact of experience on motion-in-depth (MID) sensitiv- ity in a virtual reality (VR) environment. We tested a large cohort of observers who reported having no prior VR experience and found that binocular cue sensitivity was substantially weaker than monocular cue sensitivity. As expected, sensitivity was greater when monocu- lar and binocular cues were presented together than in isolation. Surprisingly, the addition of motion parallax signals appeared to cause observers to rely almost exclusively on monocu- lar cues. As observers gained experience in the VR task, sensitivity to monocular and binoc- ular cues increased. Notably, most observers were unable to distinguish the direction of MID based on binocular cues above chance level when tested early in the experiment, whereas most showed statistically significant sensitivity to binocular cues when tested late in the experiment. This result suggests that observers may discount binocular cues when they are first encountered in a VR environment. Laboratory assessments may thus underes- timate the sensitivity of inexperienced observers to MID, especially for binocular cues. Editor: Christopher R. Fetsch, Johns Hopkins University, UNITED STATES Editor: Christopher R. Fetsch, Johns Hopkins University, UNITED STATES Received: August 23, 2019 Accepted: February 18, 2020 Published: March 9, 2020 Copyright: © 2020 Fulvio 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. OPEN ACCESS Citation: Fulvio JM, Ji M, Thompson L, Rosenberg A, Rokers B (2020) Cue-dependent effects of VR experience on motion-in-depth sensitivity. PLoS ONE 15(3): e0229929. https://doi.org/10.1371/ journal.pone.0229929 PLOS ONE PLOS ONE Introduction Three-dimensional (3D) visual perception is made more precise by integrating multiple cues (e.g., [1–8]). In the case of discriminating 3D motion, observers rely on monocular cues such as optic flow, as well as object size and density changes [9–11]. They can also rely on binocular cues including changing disparity and interocular velocity differences [12–17]. However, sen- sitivity to different motion-in-depth (MID) cues varies across observers [16, 18–23]. Even Competing interests: The authors have declared that no competing interests exist. Support from 1 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0229929 March 9, 2020 PLOS ONE Cue-dependent effects of VR experience on motion-in-depth sensitivity these funders does not alter our adherence to PLOS ONE policies on sharing data and materials. within observers, sensitivity to MID cues can vary considerably across the visual field [23–26]. In addition, sensitivity to binocular MID cues is often relatively poor in the laboratory, espe- cially for observers with little prior psychophysical experience [23, 27,28]. Applied research further suggests that displays which present binocular cues, such as 3D TVs, have little effect on the perceptual experience [29]. These findings call the relative importance of binocular cues to 3D motion perception into question and have substantial implications for the 3D media industry. The appearance of poor sensitivity to binocular motion cues may be related to prior experi- ence. A typical observer has extensive experience with visual displays (e.g., televisions, tablets, and cell phones) which convey 3D motion through monocular but not binocular cues. The presentation of MID in 3D displays also requires the apparent movement of visual elements through a physical screen, which conflicts with the real world experience that solid objects do not seamlessly pass through each other. As such, when observers are first presented with bin- ocular MID cues in a display, the cues may simply be discounted. Thus, poor sensitivity to bin- ocular cues may not be an inherent feature of visual processing, but instead reflect cue discounting. We recently found that the performance of inexperienced observers on a virtual reality (VR)-based MID task only improved with feedback [30]. There, the stimuli always contained monocular and binocular cues, and in a separate condition, also contained motion parallax cues. Thus, the effect of feedback on sensitivity to individual MID cues presented in a VR dis- play could not be determined. Here we evaluated how the sensitivity of observers to individual MID cues depended on feedback-based VR experience. Introduction We presented MID stimuli in a head-mounted VR display. Observers judged the motion direction of stimuli in four conditions in which monocular cues, binocular cues, combined (monocular and binocular) cues, or the combined cues plus motion parallax were presented in a randomized blocked order. On each trial, they were provided feedback about their perfor- mance. Observers varied in their sensitivity to monocular and binocular cues, and showed greater sensitivity when the cues were combined. Sensitivity to both monocular and binocular cues significantly improved with feedback-based experience in the VR experiment. However, for binocular cues, the improvement had a more fundamental impact. Consistent with the hypothesis that observers without prior VR experience may discount binocular cues, the majority of observers tested early in the experiment appeared insensitive to binocular cues pre- sented in isolation. In contrast, the majority of observers exhibited sensitivity to binocular cues when tested later in the experiment. This result is consistent with feedback-based VR experi- ence leading the observers to stop discounting the cues. Furthermore, the addition of motion parallax appeared to cause the observers to rely almost exclusively on monocular cues. This result may be a consequence of viewing geometry, since the observers’ head movements tended to make the monocular cues available to one eye more reliable than the other cues [23]. These results help explain poor sensitivity to binocular MID cues reported in previous studies and underwhelming reactions to 3D media. In particular, providing naturally occurring visual cues does not guarantee that observers will immediately exploit those cues, even in immersive VR environments. Observers Ninety-five members of the University of Wisconsin-Madison community gave informed written consent. Stereoacuity was assessed using the Randot Stereotest (Stereo Optical Co., Inc.). All observers passed the Randot Forms test and achieved a stereoacuity of at least 100 2 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0229929 March 9, 2020 PLOS ONE Cue-dependent effects of VR experience on motion-in-depth sensitivity arcsec in the Randot circles test. Five observers did not complete the experiment due to techni- cal issues (n = 3), experimenter error (n = 1), or difficulty seeing the stimuli (n = 1). Data from ten observers who completed the study were excluded from analysis because they either reported prior VR experience (n = 4) or did not achieve above chance performance in any of the MID conditions (n = 6). Therefore, data from 80 observers were included for analysis. We determined that at least 52 observers would be needed to detect an effect size of .8 typical of prior results in our laboratory with 80% power at a significance level of α = .05 (two-tailed). All observers were naive to the purpose of the study. Experimental procedures were approved by the University of Wisconsin-Madison Institutional Review Board and carried out in accor- dance with the Declaration of Helsinki. PLOS ONE \| https://doi.org/10.1371/journal.pone.0229929 March 9, 2020 Apparatus and display Observers viewed the stimuli in an Oculus Rift Development Kit 2 (DK2), a stereoscopic head- mounted VR system (Fig 1A) with a 14.5 cm low-persistence AMOLED screen (Samsung) embedded in the headset providing a resolution of 1920x1080 pixels (960x1080 pixels per eye) with a refresh rate of 75 Hz. The horizontal field of view of the device was ~85˚ (100˚ diago- nal). Stimuli were presented at a simulated viewing distance of 120 cm, the focal distance of Fig 1. Task schematics. (a) Experimental setup. Observers viewed MID stimuli in a virtual reality headset. The stimuli simulated dots moving toward/away from the observer through a cylindrical volume. Observers reported the perceived motion direction. (b) Motion-in-depth stimuli and temporal sequence. Binocular cues stimuli contained binocularly opposite horizontal motion cues. Monocular cues stimuli contained optic flow patterns shown to one eye only. Combined cues stimuli contained both cues at the same time. Full VR stimuli contained binocular and monocular cues, as well as motion parallax. Stimuli were presented for 250 ms. Auditory and visual feedback were provided to observers after they responded. The next trial started 750 ms after feedback. The number of dots, their sizes, and optic flow patterns shown here are intended to convey the MID cues, rather than to portray the exact stimuli. Movies illustrating the actual stimuli are provided in the Supporting Information. https://doi org/10 1371/journal pone 0229929 g001 Fig 1. Task schematics. (a) Experimental setup. Observers viewed MID stimuli in a virtual reality headset. The stimuli simulated dots moving toward/away from the observer through a cylindrical volume. Observers reported the perceived motion direction. (b) Motion-in-depth stimuli and temporal sequence. Binocular cues stimuli contained binocularly opposite horizontal motion cues. Monocular cues stimuli contained optic flow patterns shown to one eye only. Combined cues stimuli contained both cues at the same time. Full VR stimuli contained binocular and monocular cues, as well as motion parallax. Stimuli were presented for 250 ms. Auditory and visual feedback were provided to observers after they responded. The next trial started 750 ms after feedback. The number of dots, their sizes, and optic flow patterns shown here are intended to convey the MID cues, rather than to portray the exact stimuli. Movies illustrating the actual stimuli are provided in the Supporting Information. Stimuli We presented random dot stimuli that moved either toward or away from the observer. Sti- muli were presented in a 3˚ diameter aperture at the center of the visual field (Fig 1A). A stim- ulus consisted of a volume containing 12 bright (64.96 cd/m2) dots on a dark (0.01 cd/m2) background. Each dot was 0.1˚ in diameter when located at the fixation distance. Dots were initialized with random x, y, and z positions and moved either toward or away from the observer through a cylindrical volume perpendicular to the fixation plane. The volume spanned ±0.15˚ of horizontal disparity (a depth range of ~24 cm at the 120 cm viewing dis- tance). Stimuli moved through the entire length of the volume during the 250 ms presentation time, producing an average retinal speed of 1.2˚/s. If a dot reached a disparity of ±0.15˚, it wrapped to the opposite end of the volume. Dot wrapping can cause an apparent MID signal in the direction opposite of the intended stimulus direction. To reduce this effect, each dot was assigned new x and y positions when it wrapped. To ensure binocular correspondence was not interrupted due to occlusion of a dot by the edge of the aperture, the x and y dot coordinates were restricted to lie within the central 2.4˚ of the aperture (Fig 1B). To assess MID sensitivity, we manipulated motion coherence by varying the proportion of signal to noise dots. On each stimulus frame, we randomly selected a subset of dots as signal dots, which moved coherently. The remaining dots (noise dots) were assigned random x, y, and z coordinates within the cylindrical volume. Signal and noise dots were selected on a frame-by- frame basis to discourage observers from tracking the direction of motion of individual dots. Directionally-signed motion coherences of ±1, ±0.5, and ±0.17 were presented (corresponding to 12/12, 6/12, and 2/12 signal/total dots, respectively). The stimuli appeared as a cloud of dots with signal dots moving perpendicular to the screen and noise dots moving randomly [23]. To help with observer immersion, the stimuli were displayed in the center of a virtual room (3 m in height, 3.52 m in width, and 3.6 m in depth). The virtual walls, ceiling, and floor were mapped with tiles (except for the back wall, which was dark). Apparatus and display https://doi.org/10.1371/journal.pone.0229929.g001 https://doi.org/10.1371/journal.pone.0229929.g001 3 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0229929 March 9, 2020 PLOS ONE Cue-dependent effects of VR experience on motion-in-depth sensitivity the display. Prior to the experiment, the display was calibrated for each observer’s interpupil- lary distance. Observers were free to move their head but were instructed to sit still. The sti- muli were rendered in MATLAB (MathWorks, Inc., R2015a) using the Psychophysics Toolbox 3 [31]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0229929 March 9, 2020 Procedure Each of the cue conditions was presented in a separate experimental block. The order was ran- domized and counterbalanced across observers. Each block contained 15 repetitions of two MID directions and three motion coherences (15×2×3 = 90 trials). On each trial, a stimulus was displayed for 250 ms. There was no time restriction on observer responses. Auditory and visual feedback were provided following each response (Fig 1B). When observers responded correctly, a “cowbell” sound was played. If they made a mistake, a “swoosh” sound was played. A running tally of the observer’s performance (percent correct) was displayed at the center of the stimulus aperture. If the observer responded correctly, the percent correct was displayed in green, otherwise in red. A new trial began 750 ms after the onset of feedback. All blocks were completed in a single session and were preceded by 5–10 trials of the full VR condition with experimenter guidance to familiarize the observer with the task and response keys. Observers reported the direction of MID (toward or away) on each trial using the up (away) and down (toward) arrow keys on a computer keyboard. The 1/f noise pattern of the textured panel changed between blocks to reduce perceptual fading. Stimuli The wall, ceiling, and floor elements were visible if the observer moved their head but were typically out of view during MID trials. To facilitate version and vergence, we presented the MID stimuli within a 3˚ diameter cir- cular aperture cutout of a 1/f noise textured panel (1.76 m x 1.76 m). The textured panel was oriented perpendicular to the stimulus motion direction and was located at the simulated viewing distance. A fixation point was shown in the center of the aperture between, but not during, trials. We instructed observers to maintain fixation on this point when it was present but did not enforce fixation. We presented stimuli in three cue conditions in which the direction of MID was defined by monocular cues, binocular cues, or both cues combined (Fig 1B). A fourth condition included monocular and binocular cues, as well as motion parallax (made available by enabling the VR system’s capacity to update the visual display according to head position). Movies of all condi- tions are found in the Supporting Information. Combined MID cues were created using projective geometry and stereoscopic presentation. Full VR MID cues were created the same way, however, head movements resulted in a corre- sponding change in the viewpoint of the scene, as occurs in the real world. Monocular MID cues were provided by changes in retinal dot size and the pattern of optic flow due to projective geometry. For these stimuli, binocular cues were eliminated by presenting single eye views of 4 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0229929 March 9, 2020 PLOS ONE Cue-dependent effects of VR experience on motion-in-depth sensitivity the combined cues stimuli [1,23]. The virtual room and 1/f noise texture were visible to both eyes. Binocular MID cues included interocular velocity differences (IOVD) and changing dis- parity (CD) signals. For these stimuli, monocular cues that signal MID were eliminated by: (1) horizontally translating the left and right eye dot pairs with equal and opposite speeds (1.2˚/s), (2) using orthographic projection, and (3) drawing the dots with a fixed size (0.1˚ of visual angle) regardless of the simulated distance. Thus, there was no perspective information signal- ing MID in the binocular cues condition. Data analysis For each cue condition, we calculated the proportion of ‘toward’ responses as a function of the directionally signed motion coherence. We fit the data with a psychometric function (a cumu- lative Gaussian, g(x)), allowing for a non-zero lapse rate [23, 32] using maximum likelihood estimation in MATLAB: g x ð Þ ¼ l þ 1 2l ð Þ 1 2 1 þ erf x m s ffiffiffi 2 p ; Eq 1 Eq 1 fififi where x is the directionally signed motion coherence, μ is the observer bias, σ reflects the preci- sion of the responses, and λ is the lapse rate. To stabilize fits when precision was low, we enforced a bound of ±0.5 on μ. We assumed a maximum lapse rate of 2%. Sensitivity (1/σ) was our measure of interest. We enforced bounds on σ such that sensitivity was constrained between 0.01 and 100. To ensure stable estimates, we computed median fit parameters using a bootstrap procedure. For each observer, we resampled the “toward”/“away” response data with replacement 1,000 times and fit a psychometric function to each resampled data set. We then obtained median parameters from the fits. To identify sensitivities associated with above chance performance, we bootstrapped a 95% confidence interval on the sensitivity of a simulated observer who responded randomly [23]. We simulated 10,000 data sets in which the responses at each coherence level had a 50% chance of being toward or away. We then fit psychometric functions to the 10,000 simulated data sets and obtained the sensitivity from each. We classified sensitivities above the upper 95% confi- dence bound of the simulated data (0.43) as above chance performance. Observers were included in the analyses if they performed above chance in at least one of the four conditions (n = 6 excluded). 5 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0229929 March 9, 2020 PLOS ONE Cue-dependent effects of VR experience on motion-in-depth sensitivity In the monocular cues condition, we pseudo-randomly presented stimuli to one eye on each trial. Left and right eye sensitivities were not significantly different (Wilcoxon signed rank test: Z = 1.46, p = .14), so we merged the responses to left and right eye presentations and estimated a single measure of monocular cue sensitivity [1]. It is unknown how left and right eye monocular MID cues contribute to observer sensitiv- ity. Data analysis At one extreme is the possibility that left and right eye monocular representations are completely redundant. In that case, if monocular and binocular cue representations are inde- pendent and optimally integrated using maximum likelihood, combined cue sensitivity would be: 1 s^c ¼ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 1 s2 M þ 1 s2 B s ; Eq 2:1 Eq 2:1 where 1 s^c is the optimal combined cue sensitivity, and 1 s2 M and 1 s2 B are the monocular and binocu- lar cue reliabilities (squared sensitivities), respectively. We refer to this as the ‘two-cue model’. At the other extreme, left and right eye monocular cues might make independent contribu- tions to MID sensitivity. In that case, if monocular and binocular cue representations are inde- pendent and optimally integrated using maximum likelihood, combined cue sensitivity would be: 1 s^c ¼ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 1 s2 ML þ 1 s2 MR þ 1 s2 B s ; Eq 2:2 Eq 2:2 where 1 s2 ML and 1 s2 MR are the left and right eye monocular cue reliabilities (which we assumed to be equivalent since the sensitivities did not significantly differ), respectively. We refer to this as the ‘three-cue model’. The two- and three-cue models define a range of sensitivities between which left and right eye monocular signals make partially independent contributions to MID perception [5]. Upper- (defined by the three-cue model) and lower- (defined by the two-cue model) bounds of optimal combined cue sensitivity were estimated using the observer averaged sensitivities to monocular and binocular cues. PLOS ONE \| https://doi.org/10.1371/journal.pone.0229929 March 9, 2020 MID cue sensitivity in observers inexperienced with VR Sensitivity to each of the cue conditions varied across observers. Behavioral performance for three representative observers is shown in Fig 2A. Some observers, like Observer 75 in the left panel of Fig 2A showed relatively high sensitivity across all cue conditions, while other observers were less sensitive (e.g., Observer 7 in the center panel of Fig 2A). Although overall sensitivity differed between these two observers, both showed greater sensitivity in the combined cues condition than in either of the cue-isolated conditions, consistent with cue integration. Surprisingly, sensi- tivity in the full VR condition was lower than in the combined cues condition for both of these observers. As shown below, this finding was characteristic of our sample. Lastly, some observers that were sensitive to the monocular cues, combined cues, and full VR conditions seemed insensi- tive to MID based on binocular cues (e.g., Observer 80 in the right panel of Fig 2A). This apparent insensitivity to binocular cues was characteristic of a large portion of our sample. Indeed, across all binocular cues blocks, 50% (40/80) of observers did not perform above chance. The pattern of sensitivities across observers is summarized in Fig 2B. Sensitivity was great- est in the combined cues condition (Mcombined = 1.45; SDcombined = 0.78). An intermediate level PLOS ONE \| https://doi.org/10.1371/journal.pone.0229929 March 9, 2020 6 / 14 PLOS ONE Cue-dependent effects of VR experience on motion-in-depth sensitivity Fig 2. Sensitivity to MID cues. (a) Representative observers. Sensitivity to the visual cues that signal MID varied across individuals. Positive (negative) coherences indicate that signal dots moved toward (away from) the observer. Curves are cumulative Gaussian fits to the psychometric data. To visualize overlapping figure elements, we dashed some lines and enlarged some data points. (Left) Observer with relatively high sensitivity in all four conditions. (Center) Observer with poorer sensitivity in all four conditions. While sensitivity for these two observers differed, sensitivity in the combined cues condition was greater than when either cue was presented in isolation. This pattern indicates cue integration and was shared by the majority of observers. However, sensitivity in the full VR condition, which added motion parallax, was often less than combined cues sensitivity. (Right) Example observer sensitive to monocular cues, combined cues, and full VR conditions, but not binocular cues alone. (b) Comparison of monocular, binocular, combined, and full VR sensitivities across our sample (n = 80). MID cue sensitivity in observers inexperienced with VR The width of the shaded areas in the violin plots represents the proportion of the data at that level of sensitivity. The solid line in each shaded area marks the mean sensitivity and the dashed line marks the median sensitivity. (c) Relationship between stereoacuity and sensitivity to MID cues. Static stereoacuity did not predict sensitivity to MID based on monocular (blue), binocular (red), combined (purple), or full VR (green) cues. The horizontal black dashed line corresponds to the upper sensitivity bound associated with chance performance. Stereoacuity is plotted on a logarithmic scale, and datapoints are jittered at each stereoacuity level for visualization. Fig 2. Sensitivity to MID cues. (a) Representative observers. Sensitivity to the visual cues that signal MID varied across individuals. Positive (negative) coherences indicate that signal dots moved toward (away from) the observer. Curves are cumulative Gaussian fits to the psychometric data. To visualize overlapping figure elements, we dashed some lines and enlarged some data points. (Left) Observer with relatively high sensitivity in all four conditions. (Center) Observer with poorer sensitivity in all four conditions. While sensitivity for these two observers differed, sensitivity in the combined cues condition was greater than when either cue was presented in isolation. This pattern indicates cue integration and was shared by the majority of observers. However, sensitivity in the full VR condition, which added motion parallax, was often less than combined cues sensitivity. (Right) Example observer sensitive to monocular cues, combined cues, and full VR conditions, but not binocular cues alone. (b) Comparison of monocular, binocular, combined, and full VR sensitivities across our sample (n = 80). The width of the shaded areas in the violin plots represents the proportion of the data at that level of sensitivity. The solid line in each shaded area marks the mean sensitivity and the dashed line marks the median sensitivity. (c) Relationship between stereoacuity and sensitivity to MID cues. Static stereoacuity did not predict sensitivity to MID based on monocular (blue), binocular (red), combined (purple), or full VR (green) cues. The horizontal black dashed line corresponds to the upper sensitivity bound associated with chance performance. Stereoacuity is plotted on a logarithmic scale, and datapoints are jittered at each stereoacuity level for visualization. https://doi.org/10.1371/journal.pone.0229929.g002 of sensitivity was observed in the monocular cues (Mmonocular = 1.13; SDmonocular = 0.47) and full VR conditions (Mfullvr = 1.19; SDfullvr = 0.53). https://doi.org/10.1371/journal.pone.0229929.g002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0229929 March 9, 2020 MID cue sensitivity in observers inexperienced with VR Sensitivity was poorest in the binocular cues condition (Mbinocular = 0.54; SDbinocular = 0.50). To test for differences between cue sensitivities, we fit a two-factor linear mixed effects model to the full dataset of measured sensitivities (n = 80 observers x 4 sensitivities = 320 data points) with cue condition and block number as fixed factors. The main effects of cue condi- tion and block were both significant (F(3,313) = 62.374, p < .0001, and F(3,313) = 10.043, p < .0001 respectively). We first examined the main effect of cue condition. All post-hoc paired- sample t-tests revealed significant differences in observer sensitivity (p < .0083, the Bonfer- roni-corrected alpha-level), except for monocular cues vs. full VR (p = .24). Combined cues sensitivity was significantly greater than monocular cues and binocular cues sensitivities, con- sistent with cue integration. 7 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0229929 March 9, 2020 PLOS ONE Cue-dependent effects of VR experience on motion-in-depth sensitivity Only 50% (40/80) of the observers performed above chance in the binocular cues condition, compared to 88.75% of observers (71/80) in the monocular cues condition. One possible expla- nation for poor binocular cues sensitivity is that sensitivity to binocular MID cues is limited by stereoacuity. However, given that the binocular disparity in our MID stimuli (±0.15˚ of dispar- ity) greatly exceeded the stereoacuity of even our worst observers (100 arcsec, or equivalently 0.03˚), it is unlikely that stereoacuity was a limiting factor [24]. Indeed, stereoacuity did not predict sensitivity to any of the conditions (all p > .09; Fig 2C). Thus, limitations in binocular disparity processing did not account for poor performance with binocular MID cues. Somewhat surprising was that adding motion parallax in the full VR condition had a nega- tive impact on sensitivity compared to the combined cues condition. In particular, sensitivity was reduced to roughly the level of the monocular cues condition. Depending on the visual field location and direction of 3D motion, the monocular cues available to one eye can be sub- stantially more reliable than the other cues. Under such conditions, performance with com- bined cues can be dominated by the more reliable monocular cue [23]. We hypothesized that the decreased sensitivity was not due to motion parallax per se. Instead, the observers’ self- motion changed the viewing geometry, resulting in large differences in the reliabilities of the left and right eye monocular cues. MID cue sensitivity in observers inexperienced with VR We therefore quantified the effect of the head’s 3D pose on the left and right eye signal strengths, noting that the observers’ movements did not result in the occlusion of dots by the 1/f noise textured panel in either eye. For each trial, we computed the mean 3D head position (x-y-z translations) and orientation (yaw, pitch, and roll) during the 250 ms trial period. Given the head pose and interpupillary distance, we calculated the reti- nal speed of a dot traversing the center of the stimulus volume for each eye. On average, the retinal speeds in the two eyes differed by a factor of 1.79 (larger retinal speed = 0.84˚/s; smaller retinal speed = 0.47˚/s). This difference may have created a large enough difference in the reli- abilities of the left and right eye monocular cues that the observers relied predominantly on the faster signal. In sum, we found variability in sensitivity to different MID cues across a sample of inexperi- enced VR observers, relatively poor sensitivity to binocular cues, and evidence for monocular and binocular cue integration. Moreover, we found that an observer’s 3D pose can differen- tially affect left and right eye signal strengths, which can impact the relative contributions of different visual cues to MID perception. We next explored the role of feedback-based VR expe- rience on sensitivity to MID cues. Experience-dependent changes in MID cue sensitivity Lines are regression fits. The shaded area marks the range of sensitivities between the two-cue (lower-bound) and three-cue (upper-bound) model predictions (see Results for details). (b) Proportion of observers who performed above chance in each condition as a function of block. https://doi.org/10.1371/journal.pone.0229929.g003 https://doi.org/10.1371/journal.pone.0229929.g003 https://doi.org/10.1371/journal.pone.0229929.g003 between the average sensitivity in block 4 and block 1 for the binocular cues (Δ sensitiv- ity = 0.334) and monocular cues (Δ sensitivity = 0.331) conditions were nearly equivalent. Sim- ilarly, the difference between the average sensitivity in block 4 and block 1 for the combined cues was 0.329. Lastly, the difference between the average sensitivity in block 4 and block 1 for the full VR condition was 0.27. Thus, sensitivity improved with feedback-based VR experience in all conditions, but the improvements were only significant in the monocular cues and bin- ocular cues conditions. Together, these results indicate that sensitivity to both binocular cues and monocular cues improved with feedback-based VR experience. However, when we considered the proportion of observers sensitive to each cue as a function of the block number, the impact on binocular cues sensitivity stood out (Fig 3B). For the monocular cues, combined cues, and full VR condi- tions, the vast majority of observers performed significantly above chance regardless of which block the condition was presented. For the binocular cues condition, however, the majority of observers performed at chance level in the first two blocks. Only in blocks 3 and 4 did a major- ity of observers perform above chance. These results suggest that the feedback-based VR expe- rience may have reduced the observers’ tendency to discount binocular cues in the display. We ruled out an alternative explanation for sensitivity differences to binocular cues across blocks. Observers who by chance happened to complete the binocular cues condition in blocks 3 and 4 might have been more sensitive to MID in general. We compared the monocular cues, combined cues, and full VR sensitivities based on the block in which the binocular cues condi- tion was completed and found no significant differences in sensitivity between observers who completed the binocular cues condition in the first two blocks versus the last two blocks: mon- ocular: Z = -1.16, p = .10; combined: Z = -.30, p = .76; full VR: Z = -1.99, p = .05, at the Bonfer- roni-corrected alpha-value = .0167. Experience-dependent changes in MID cue sensitivity To assess the effect of feedback-based VR experience on sensitivity to MID cues, we examined the main effect of block number from the linear mixed effects model. We first considered the effect of exposure on sensitivity using post-hoc paired-sample t-tests to compare sensitivity across blocks independent of cue condition. Sensitivity differences between block 1 and block 3 as well as between block 1 and block 4 were significant (p < .001 for both, Bonferroni-cor- rected alpha-value = .0083). There were no other significant differences (p > .03 for all others). Thus, under the conditions used here, two blocks of 90 trials with feedback were sufficient to see significant improvements in MID cue sensitivity in VR. We next assessed how feedback-based experience in the VR experiment affected sensitivity in each cue condition. To address this, we carried out a linear regression for each condition to describe the relationship between sensitivity and block (Fig 3A). We found significant improvements in the binocular cues (regression line slope: β = .133, t(78) = 2.85, p = .006, Bon- ferroni-corrected alpha-value = .0125) and monocular cues (β = .118, t(78) = 2.56, p = .0123) conditions, but not the combined cues or full VR conditions (both p > .09). The difference 8 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0229929 March 9, 2020 PLOS ONE Cue-dependent effects of VR experience on motion-in-depth sensitivity Fig 3. Relationship between sensitivity and experimental block number. (a) Sensitivity to MID based on monocular cues (blue) and binocular cues (red) significantly improved with experimental block. Numerically similar trends were observed in the combined cues (purple) and full VR (green) conditions, but the improvements were not significant. Data points correspond to between-subject mean sensitivities. Error bars correspond to ±1 standard error of the mean (SEM). Lines are regression fits. The shaded area marks the range of sensitivities between the two-cue (lower-bound) and three-cue (upper-bound) model predictions (see Results for details). (b) Proportion of observers who performed above chance in each condition as a function of block. https://doi.org/10.1371/journal.pone.0229929.g003 Fig 3. Relationship between sensitivity and experimental block number. (a) Sensitivity to MID based on monocular cues (blue) and binocular cues (red) significantly improved with experimental block. Numerically similar trends were observed in the combined cues (purple) and full VR (green) conditions, but the improvements were not significant. Data points correspond to between-subject mean sensitivities. Error bars correspond to ±1 standard error of the mean (SEM). PLOS ONE \| https://doi.org/10.1371/journal.pone.0229929 March 9, 2020 Experience-dependent changes in MID cue sensitivity In sum, we found that sensitivity to binocular cues and monocular cues to MID improved with feedback-based VR experience. However, that experience had fundamentally different effects on the sensitivities measured for the two cues. Only in the binocular cues condition were the majority of inexperienced observers unable to distinguish the direction of MID above PLOS ONE \| https://doi.org/10.1371/journal.pone.0229929 March 9, 2020 9 / 14 PLOS ONE Cue-dependent effects of VR experience on motion-in-depth sensitivity chance level. This finding is consistent with the hypothesis that observers discount binocular cues when they are first encountered in a VR environment, and that discounting is reduced by feedback-based VR experience in which MID is signaled stereoscopically. Observers integrate monocular and binocular MID cues We lastly tested if we could predict observers’ combined cues sensitivity based on their sensi- tivities to monocular cues and binocular cues. We considered two models. In the ‘two-cue model’, left and right eye monocular cues were assumed to have completely redundant repre- sentations. This model predicts that sensitivity based on both eyes’ monocular cues will be equal to the sensitivity based on a single eye’s monocular cues [1,5]. The monocular cues and binocular cues were further assumed to produce independent estimates that are optimally inte- grated (Eq 2.1). This model accounts for the perception of 3D surface orientation based on binocular cues and monocular cues [1–3]. Another possibility is that two independent monoc- ular MID estimates (one from each eye) are both integrated with an independent binocular MID estimate in a ‘three-cue model’ (Eq 2.2). Partial dependence of the two eyes’ monocular cues would result in sensitivities intermediate to the two-cue and three-cue models. The two models thus define sensitivity bounds, assuming independent monocular and binocular esti- mates. If the monocular and binocular estimates are not independent, combined cues sensitiv- ity would be lower. Average observer sensitivity to the combined cues across all blocks (M = 1.45; SD = 0.78) fell within the bounds defined by the two models: [1.31, 2.01]. The observed sensitivity was not significantly different from the two-cue prediction in any individual block (all p > .04, with Bonferroni-corrected alpha-level = .0063; see Fig 3A). The observed sensitivity was also not significantly different from the three-cue prediction in blocks 1, 2, or 4 (all p > .01), but was lower in block 3 (p = .0048, with Bonferroni-corrected alpha-level = .0063). Together, the cur- rent findings indicate that monocular cues and binocular cues are integrated to improve the precision of MID perception, and suggest that left and right eye monocular cues may make partially independent contributions to MID estimates. Discussion In three out of the four experimental blocks, the average observer sensitivities were in-between the predictions of a two-cue model (in which a single monocular cues repre- sentation was integrated with an independent binocular cues representation) and a three-cue model (in which left eye monocular, right eye monocular, and binocular cues make indepen- dent contributions to perception). This result is consistent with the possibility that left and right eye monocular signals make partially independent contributions to MID perception. This contrasts with static 3D orientation perception, which is consistent with the two-cue model [1–3]. If subsequent data support this difference, it would suggest that left and right eye monocular signals may be integrated differently within the neural circuits supporting 3D ori- entation and motion processing. Since the current study was performed with inexperienced observers and few motion coherence values, sensitivity estimates were sometimes noisy. Furthermore, as is a common limitation with cue-combination studies, discrepancies between observed and predicted sensi- tivities may arise in part due to underestimating sensitivity to cue-isolating conditions or ceil- ing effects in estimates of sensitivity to combined cues. Future work that obtains more precise estimates from experienced observers can help further elucidate how different MID cues are perceptually integrated. We previously found that the performance of inexperienced observers in a MID task only improved with feedback [30]. However, we could not identify whether the improvements were due to changes in sensitivity to monocular and/or binocular cues because the cues were not presented in isolation. The current results suggest that feedback likely improves sensitivity to both cues. Enabling motion parallax cues had a surprisingly negative effect on sensitivity. Our analysis revealed that changes in observer head position resulted in retinal speeds in the two eyes that differed by a factor of 1.79. We previously found that differences in left and right retinal speeds can result in a “winner-take-all” scenario in which combined cues perception is dominated by the single most reliable monocular cue [23]. We speculate that this effect underlies the detri- mental impact of enabling motion parallax in the current study. In contrast, previous work found that head movements can enhance MID perception due to motion parallax above and beyond perception due to the combination of monocular and binocular cues [30]. However, there are a number of differences between these studies. Discussion We investigated sensitivity to MID cues in a large cohort of observers who were inexperienced with VR. Consistent with previous results, we found that sensitivity to the visual cues that sig- nal MID varied across individuals [23]. Sensitivity was greater when monocular cues and bin- ocular cues were presented together than in isolation, indicating cue integration. While the majority of observers performed well above chance in the monocular cues, com- bined cues, and full VR conditions, half of the observers appeared insensitive to binocular cues. Previous work similarly reported relatively poor sensitivity to binocular MID cues, espe- cially in inexperienced observers [23,27–28]. Poor sensitivity to binocular cues may be due in part by observers’ prior experience with 2D displays which convey MID using monocular cues only. Indeed, stimuli that only depict MID through binocular cues provide conflicting infor- mation since they will also contain monocular cues that signal that the stimuli are constrained to the image plane at all times (e.g., no size changes). Such conflicts do not appear to affect sen- sitivity to binocular cues for static 3D orientation judgments [1], but may be more prevalent with moving stimuli. Thus, poor sensitivity to binocular cues may partially reflect the dis- counting of binocular MID cues in 3D displays. We hypothesized that feedback-based VR experience would lead observers to stop dis- counting binocular cues. Experience in the VR task was associated with significant improve- ments in sensitivity to both monocular cues and binocular cues. However, there was a PLOS ONE \| https://doi.org/10.1371/journal.pone.0229929 March 9, 2020 10 / 14 PLOS ONE Cue-dependent effects of VR experience on motion-in-depth sensitivity fundamental difference in how sensitivities to these cues were affected. With monocular cues, the majority of observers showed significant sensitivity in all four blocks. In contrast, the majority of observers performed at chance level in the binocular cues condition in blocks 1 and 2. Only in blocks 3 and 4, did a majority show significant binocular cues sensitivity. Sensi- tivity to binocular cues remained substantially weaker than sensitivity to monocular cues throughout the experiment. We note, however, that sensitivity to binocular MID cues is speed dependent [33,34], so the relative sensitivity to monocular and binocular cues may also be speed dependent. We found that monocular and binocular cues are integrated to improve the precision of MID perception. PLOS ONE \| https://doi.org/10.1371/journal.pone.0229929 March 9, 2020 Discussion In the previous study, a single target moved for 1000 ms as opposed to multiple dots for 250 ms, reducing the availability of monoc- ular optic flow (expansion/contraction) cues, and increasing the availability of motion parallax cues. The stimuli in the previous study also moved along a variety of trajectories in the x-z plane and contained much larger disparities (up to ~1.2˚), compared to the current study in which only toward and away motions were shown with a disparity range of 0.3˚. Future work can evaluate how factors such as the stimulus configuration and presentation time affect the relative contributions of MID cues, and in particular the effect of motion parallax on MID perception. 11 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0229929 March 9, 2020 PLOS ONE Cue-dependent effects of VR experience on motion-in-depth sensitivity In conclusion, these findings suggest a reinterpretation of the performance of inexperienced observers. Rather than interpreting poor sensitivity to binocular MID cues as a deficiency in stereoscopic processing, poor sensitivity may reflect a reasonable discounting of binocular cues given their experience with visual displays. We speculate that feedback-based experience with stereoscopic displays may update observers’ expectations about the 3D information that can be contained in visual displays. Similar effects may occur with other cues that are typically absent in visual displays but can be provided in virtual and augmented reality environments, such as motion parallax and accommodative blur. Until prior expectations are appropriately updated, observers may be limited in their ability to take advantage of those cues, even if they rely on them in the natural environment. Supporting information S1 Movie. Movie illustrating the monocular cues stimulus condition. (MP4) S1 Movie. Movie illustrating the monocular cues stimulus condition. (MP4) S1 Movie. Movie illustrating the monocular cues stimulus condition. (MP4) S2 Movie. Movie illustrating the binocular cues stimulus condition. (MP4) S2 Movie. Movie illustrating the binocular cues stimulus condition. (MP4) S3 Movie. Movie illustrating the combined cues stimulus condition. (MP4) S4 Movie. Movie illustrating the full VR stimulus condition. (MP4) Author Contributions Conceptualization: Jacqueline M. Fulvio, Mohan Ji, Lowell Thompson, Ari Rosenberg, Bas Rokers. Data curation: Jacqueline M. Fulvio, Mohan Ji. Formal analysis: Jacqueline M. Fulvio, Mohan Ji, Lowell Thompson, Ari Rosenberg, Bas Rokers. Funding acquisition: Ari Rosenberg, Bas Rokers. Software: Jacqueline M. Fulvio, Lowell Thompson, Bas Rokers. Supervision: Jacqueline M. Fulvio, Ari Rosenberg, Bas Rokers. Visualization: Jacqueline M. Fulvio, Mohan Ji, Lowell Thompson, Ari Rosenberg, Bas Rokers. Writing – original draft: Jacqueline M. Fulvio, Mohan Ji, Ari Rosenberg, Bas Rokers. Writing – review & editing: Jacqueline M. Fulvio, Mohan Ji, Lowell Thompson, Ari Rosen- berg, Bas Rokers. References 1. Chang T-Y, Thompson L, Doudlah R, Byounghoon K, Sunkara A, Rosenberg R. 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https://openalex.org/W4245624517	https://www.qeios.com/read/QK3OG9/pdf	English	null	Hypopharyngeal Cancer TNM Finding v8	Definitions	2,020	cc-by	90	Qeios · Definition, February 2, 2020 Open Peer Review on Qeios Hypopharyngeal Cancer TNM Finding v8 National Cancer Institute National Cancer Institute Qeios ID: QK3OG9 · https://doi.org/10.32388/QK3OG9 Source National Cancer Institute. Hypopharyngeal Cancer TNM Finding v8. NCI Thesaurus. Code C132910. A finding about one or more characteristics of hypopharyngeal cancer, following the rules of the TNM AJCC v8 classification system. This staging system applies to all cancers of the hypopharynx. Minor salivary gland carcinomas and neuroendocrine carcinomas of the hypopharynx are included in this classification. (from AJCC 8th Ed.) 1/1
https://openalex.org/W2521227179	https://link.springer.com/content/pdf/10.1007%2Fs10336-016-1394-7.pdf	English	null	Nestling diet optimization and condition in relation to prey attributes and breeding patch size in a patch-resident insectivorous passerine: an optimal continuum and habitat constraints	Journal of ornithology	2,016	cc-by	12,280	J Ornithol (2017) 158:169–184 DOI 10.1007/s10336-016-1394-7 ORIGINAL ARTICLE Nestling diet optimization and condition in relation to prey attributes and breeding patch size in a patch-resident insectivorous passerine: an optimal continuum and habitat constraints Grzegorz Orłowski1 • Joanna Frankiewicz2 • Jerzy Karg3 Received: 20 February 2016 / Revised: 10 June 2016 / Accepted: 12 September 2016 / Published online: 20 September 2016 The Author(s) 2016. This article is published with open access at Springerlink.com Received: 20 February 2016 / Revised: 10 June 2016 / Accepted: 12 September 2016 / Published online: 20 September 2016 The Author(s) 2016. This article is published with open access at Springerlink.com Abstract Direct observational studies are needed to address dietary adjustment in species breeding in isolated non-forest habitat islands with respect to the energy demands of growing nestlings and breeding patch size. Using new dietary records determined for nestlings of Whinchat Saxicola rubetra, a dramatically declining insectivorous passerine and an indicator species of the cessation of agricultural activity, we investigated the rela- tionships between changes in the main dietary character- istics, numerical and biomass contributions of major taxonomic and functional prey groups (expressing chitin content, vertical distribution, habitat preference and vagi- lity within the landscape) and brood age, nestling condition and size of abandoned ﬁelds (i.e. breeding patches). Broods from larger abandoned ﬁelds received more sedentary and heavier prey like Orthoptera and soil-dwelling inverte- brates, whereas the proportion of caterpillars, aerial insects and prey from vegetation decreased with increasing patch size. Nestling condition was positively correlated with the proportion of caterpillars and Orthoptera or sedentary prey taxa, but negatively with the proportion of Coleoptera or vagile prey taxa in the diet, though not with patch area. This suggests that parent Whinchats can overcome the habitat constraints resulting from the small area of an abandoned ﬁeld by interchangeably incorporating the two major prey groups (Orthoptera or Lepidoptera) into the diet they feed to their nestlings. This implies a continuum in dietary optimization that is a trade-off between a brood’s nutritional demands and the parents’ ability to deliver top- ranked invertebrates present mostly within the breeding patch. Keywords Nestling development Dietary adjustment Invertebrate prey Optimal foraging theory Single-prey loaders 3 Department of Nature Conservation, Faculty of Biological Sciences, University of Zielona Go´ra, Prof. Z. Szafrana 1, 65-516 Zielona Go´ra, Poland & Grzegorz Orłowski orlog@poczta.onet.pl 1 Institute for Agricultural and Forest Environment, Polish Academy of Sciences, Bukowska 19, 60-809 Poznan´, Poland 2 Ornithological Society of Silesia, Zoological Institute of the University of Wrocław, Sienkiewicza 21, 50-335 Wrocław, Poland Communicated by F. Bairlein. Zusammenfassung Optimierung der Nestlingsnahrung und -kondition im Verha¨ltnis zu Beuteeigenschaften und Brutreviergro¨ße bei einem ortstreuen insektivoren Singvogel: ein optimales Kontinuum und habitatbedingte Zwa¨nge Communicated by F. Bairlein. Communicated by F. Bairlein. Electronic supplementary material The online version of this article (doi:10.1007/s10336-016-1394-7) contains supplementary material, which is available to authorized users. Um Erna¨hrungsanpassungen bei Arten, die in isolierten unbewaldeten Habitatinseln leben, im Hinblick auf den Energiebedarf wachsender Nestlinge und die Brutreviergro¨ße zu untersuchen, sind direkte Beobachtungen erforderlich. Mithilfe neuer Erna¨hrungsdaten, die an Nestlingen von Braunkehlchen Saxicola rubetra, einem dramatisch zuru¨ckgehenden insektenfressenden Singvogel und einer Indikatorart fu¨r die Einstellung landwirtschaftlicher Aktivita¨ten, erhoben wurden, untersuchten wir die Beziehungen zwischen A¨ nderungen der prima¨ren Erna¨hrungsparameter, zum 1 Institute for Agricultural and Forest Environment, Polish Academy of Sciences, Bukowska 19, 60-809 Poznan´, Poland 12 170 J Ornithol (2017) 158:169–184 Beispiel der Anteile bezu¨glich Anzahl und Biomasse der taxonomischen beziehungsweise funktionalen Hauptbeutegruppen (als Maß fu¨r den Chitingehalt, die vertikale Verteilung, Habitatpra¨ferenzen und die Beweglichkeit innerhalb der Landschaft), und dem Brutalter, der Nestlingskondition sowie der Gro¨ße aufgelassener Felder (d. h. Brutreviere). Bruten von gro¨ßeren, brachliegenden Feldern bekamen mehr ortsgebundene und schwerere Beuteobjekte wie Heuschrecken und bodenlebende Wirbellose, wohingegen der Anteil an Raupen, Fluginsekten und auf Vegetation gefangener Beute mit zunehmender Gebietsgro¨ße abnahm. Die Nestlingskondition korrelierte positiv mit dem Anteil an Raupen und Heuschrecken beziehungsweise ortsgebundener Beutetaxa, jedoch negativ mit dem Anteil an Ka¨fern oder beweglichen Beutetaxa an der Nahrung, allerdings nicht mit der Revierﬂa¨che. Dies legt nahe, dass Braunkehlcheneltern die Habitatnachteile, die aus der geringen Fla¨chengro¨ße eines aufgegebenen Feldes entstehen, u¨berwinden ko¨nnen, indem sie wechselweise die zwei Hauptbeutegruppen (Heuschrecken oder Schmetterlinge) in die Nahrung aufnehmen, mit der sie ihre Nestlinge fu¨ttern. Dies bedeutet ein Kontinuum der Erna¨hrungsoptimierung, die einen Kompromiss zwischen dem Na¨hrstoffbedarf einer Brut und der Fa¨higkeit der Eltern, die hochwertige, u¨berwiegend im Brutrevier vorkommende Wirbellose herbeizuschaffen, darstellt. There are many studies looking at the effects of the quality of diet (prey species) on reproductive success (e.g. Guillod et al. 2016; reviewed in Lourenc¸o et al. 2015). Early nutrition is often a strong predictor of offspring size, morphology and survival (cf. Wilkin et al. 2009). The quality of early diets is difﬁcult to assess in wild popula- tions and as such, quality is often inferred from alternative factors such as the length of the rearing period, observable parental care, natal habitat quality, offspring growth rates or condition at independence (reviewed by Wilkin et al. 2009). Zusammenfassung The food supply is a critical factor for growing nestlings (Martin 1987; Wilkin et al. 2009), the diets of which can differ from those consumed by adult/parent birds (Radford 2008; Wilson et al. 2004; Orłowski et al. 2014a). Therefore, not only the amount of food, but its quality, e.g. in terms of the availability of soft-bodied invertebrate prey such as spiders or caterpillars, may be more important for developing nestlings (Radford 2008; Ramsay and Houston 2003; Orłowski et al. 2014a, 2015). Moreover, according to a theory developed to examine how animals might be expected to behave when foraging to maximise their biological ﬁtness (Krebs and Davies 1991), the optimal diet of nestlings and foraging strategy of parent birds are determined by the simultaneous solution of var- ious cost-beneﬁt functions that ultimately affect the ﬁtness of the foragers. This primarily involves foraging costs, the handling and ingestion of food, the risk of predation, increased thermoregulatory costs, reduced time for terri- torial activities, and the potential consumption of toxic or inhibitory compounds (reviewed in Brodmann and Reyer 1999). Introduction Most of the few studies analysing the relationship between breeding patch size or edge effect and diet or condition (including studies of immune function and stress- induced hormones) in adults and/or dependent young birds were conducted in woodland areas (Burke and Nol 1998; Zanette et al. 2000; Suorsa et al. 2003; Weldon and Haddad 2005; Wilkin et al. 2009). Analogous studies in non-forest, open habitat islands, like remnant or non-cropped habitats (i.e. various grassland or steppe-like sites with or without limited human activity) within an agricultural matrix are exceptions [involving adult males (Keyel et al. 2012); for review relating to North American grassland birds, see Ribic et al. (2009)]. It is still rare, however, to come across studies providing evidence that the limited availability of food is a major detrimental effect of habitat fragmentation or area sensitivity of species living in habitat fragments. The food shortage hypothesis does not appear to have been taken into account as a potential major explanation for the population decrease of ground-nesting birds breeding in isolated grassland patches [analysis of the stomach con- tents of adult birds (Wiens and Rotenberry 1979; reviewed by Ribic et al. 2009)]. Habitat fragmentation can adversely affect the reproduc- tive success of birds primarily through reducing pairing success, lowering the survival of adults through nest predation and parasitism, and reducing the availability of critical resources such as food (Le Tortorec et al. 2013). In particular, small habitat patches may supply little food owing to their being completely isolated or of inferior quality. This may adversely affect reproductive success, since habitat patch size limits territory size and hence food availability. Even if the territory size in poor habitats could be enlarged, this would come at a cost because of the longer foraging distances required (reviewed in Le Tortorec et al. 2013). Therefore, the size of the breeding patch can inﬂuence the nutritional status of growing nestlings because adults feeding altricial young will be constrained in their foraging by the location of the nest (Hinsley et al. 1999; Zanette et al. 2000). To date, how- ever, studies of area sensitivity in birds have basically not been oriented towards food resources or their potential changes mediated by patch size (Ribic et al. 2009; Bayard and Elphick 2010). Introduction 123 123 J Ornithol (2017) 158:169–184 171 In the current global context, many species of grassland birds associated both with natural open non-forest habitats such as prairie grasslands (North America) and semi-nat- ural ones (like meadows extensively managed in Europe during recent centuries) are of conservation concern owing to their dramatic population decline caused by land use change, agricultural intensiﬁcation and climate change (Murphy and Moore 2003; Sanderson et al. 2006; Møller et al. 2008; Ribic et al. 2009). Therefore, direct observa- tional studies focusing on the question of dietary adjust- ment in species breeding in isolated non-forested habitat islands with respect to the energy demands of growing nestlings and the physical and landscape features of a breeding patch, such as its size, are urgently needed. Such knowledge is central to understanding the processes that drive area sensitivity in birds through potential changes of their body condition and/or survival of predator popula- tions within highly fragmented landscapes, and their extinction when resources become insufﬁcient (Zanette et al. 2000; Vickery and Herkert 2001; Ribic et al. 2009). It is also indispensable in the application of diet/foraging optimality theory to conservation science. 20–30 years, Whinchats have successfully recolonized abandoned crop ﬁelds and its populations have increased considerably in certain areas (Orłowski 2004, 2005; Try- janowski et al. 2009; Sanderson et al. 2013; Shitikov et al. 2015). 20–30 years, Whinchats have successfully recolonized abandoned crop ﬁelds and its populations have increased considerably in certain areas (Orłowski 2004, 2005; Try- janowski et al. 2009; Sanderson et al. 2013; Shitikov et al. 2015). 20–30 years, Whinchats have successfully recolonized abandoned crop ﬁelds and its populations have increased considerably in certain areas (Orłowski 2004, 2005; Try- janowski et al. 2009; Sanderson et al. 2013; Shitikov et al. 2015). Our earlier studies of the ecology and biology of Whinchats in this new habitat type had shown that during the breeding period the species was area-sensitive and the probability of occupancy of an abandoned ﬁeld was posi- tively correlated with ﬁeld area (Orłowski 2004). Adult Whinchats search for invertebrate prey from a perch, ﬂying to and taking prey mainly from the ground or in vegetation, sometimes in ﬂight, then return to the perch (Andersson 1981; Bastian and Bastian 1996; Suter 1988; Pudil and Exnerova´ 2015). Introduction Moreover, an earlier investigation showed that in Whinchats breeding in grasslands, hunting in the air was less effective than ground foraging; prey taxa taken in the air (ca. 20 % of all foraging ﬂights), mostly during calm weather (when ﬂying insects are more active), are less proﬁtable compared to prey items obtained from the soil surface or vegetation on cooler days (Bastian and Bastian 1996; Suter 1988). Overall, in grassland habitats Whinchats feed their young larger and more proﬁtable (i.e. more highly chitinized) prey as they grow older; during the brood-rearing period, adults can ﬂy as far as 400 m in order to acquire the optimal food type (i.e. caterpillars) (Bastian and Bastian 1996). Furthermore, the mowing of grasslands (compared to abandoned farmland) is detrimental to the invertebrate community at such sites, particularly to the larger and less vagile species like Orthoptera or Lepi- doptera (Siemann et al. 1999; Humbert et al. 2009). Therefore, the diet of Whinchats may differ substantially between grasslands and abandoned farmland. Hitherto, all dietary studies of nestling Whinchats were restricted to different grassland types (reviews in Bastian and Bastian 1996; Suter 1988; Britschgi et al. 2006), with hardly any detailed dietary data for Whinchats (and other bird species) breeding in abandoned farmland. Importantly, however, owing to the progressive increase in the acreage of aban- doned farmland in some temperate areas of the northern hemisphere (Kamp et al. 2015), such knowledge is essen- tial in order to diagnose the biodiversity and viability of bird populations in this habitat (Kamp et al. 2015; Try- janowski et al. 2009; Sanderson et al. 2013; Plieninger et al. 2014; Zakkak et al. 2015). In this paper we present the results of a dietary inves- tigation into a drastically declining insectivorous passerine bird, the Whinchat Saxicola rubetra. Since 1980 it has undergone an estimated 71 % long-term decline in abun- dance in Europe, which has resulted in an exceptionally high level of interest in ecological/conservation investiga- tions of this species (Henderson et al. 2014; Strebel et al. 2015; cf. Bastian and Feulner 2015). The primary breeding habitat of Whinchat used to be invertebrate-rich grasslands, especially those lying within traditionally managed agri- cultural landscapes, where the abundance and diversity of arthropod prey were higher than in intensively managed grasslands (Oppermann 1999; Bastian and Bastian 1996; Britschgi et al. 2006; Broyer et al. 2012; Strebel et al. 2015). Introduction The recent population decline of grassland birds, including the Whinchat, has been ascribed mainly to nest losses/female mortality resulting from more intensive agriculture practices, primarily earlier and more frequent mowing (Gru¨ebler et al. 2008, 2012), deteriorating food/foraging conditions in semi-natural grasslands/mead- ows and the loss of marginal habitats (Mu¨ller et al. 2005; Britschgi et al. 2006; Perlut et al. 2008; Broyer 2009; Broyer et al. 2012, 2014; Henderson et al. 2014; Strebel et al. 2015). A very recent investigation has found evidence that mortality in Whinchats occurs primarily outside the wintering period, i.e. mostly during the migratory and breeding stages; overwintering conditions thus exert a minimal inﬂuence on the survival of this declining species (Blackburn and Cresswell 2016). As a result of the socioeconomic transformation of agriculture and resulting land abandonment in Central and Eastern Europe in the last Based on the above framework and considering the conditions of the natural experiment of habitat fragmen- tation of Whinchats nesting in isolated patches of non- cropped vegetation in abandoned ﬁelds, we make a number of predictions linking certain features of the diet of nest- lings with their physiological state, food demands and the environment. In this study we explore the following major objectives: 12 3 3 172 J Ornithol (2017) 158:169–184 1. The differences in dietary composition between nest- lings in various age classes. Whinchats bred: Artemisia-Tanacetum vulgaris (54 % of all abandoned ﬁelds), Convolvulus arvensis-Agropyron repens (23 %) and Koeleria glauca-Corynephorus canes- cens (1 %). The meadows belonged to the Arrhenathere- talia order (Malawian-Arrhenatheretea class) (Frankiewicz 2010). Whinchats bred: Artemisia-Tanacetum vulgaris (54 % of all abandoned ﬁelds), Convolvulus arvensis-Agropyron repens (23 %) and Koeleria glauca-Corynephorus canes- cens (1 %). The meadows belonged to the Arrhenathere- talia order (Malawian-Arrhenatheretea class) (Frankiewicz 2010). 2. The effect on nestling diet of the area of abandoned crop ﬁelds where Whinchats bred. 3. The inﬂuence of diet on the body condition of Whinchat nestlings. 3. The inﬂuence of diet on the body condition of Whinchat nestlings. During the brood-rearing period the parent Whinchats collected food primarily from abandoned ﬁelds, to which 68–89 % of all the parent birds’ foraging trips were made (determined for 260 ﬂights of both adult birds from ten different nests). In our study population the average dis- tance of a ﬂight from the nest by parent Whinchats was 33 m [±SD 21.2 m, range 4–80 m (Frankiewicz 2008)]. Determination of nestling diet and body condition Our description of the Whinchat nestling diet is based on faecal analysis, the material for which was sampled from 47 nests examined in 2004–2007. In these 4 years, 177 faecal sacs were sampled: 63 (in 2004), 66 (2005), 20 (2006) and 28 (2007). The faecal sac sampling dates were as follows: 24 May–16 June 2004, 30 May–30 June 2005, 5–14 June 2006, 26 May–16 June 2007. We sampled between one and 13 faecal sacs (on average four) from each nest, which were then used in our analysis. Introduction This value was smaller compared with previous data for Whinchats foraging in extensive grasslands in Sweden [43.8 m (Andersson 1981)] or Alpine meadows in Switzerland [42.2–54.7 m (Britschgi et al. 2006)]. More- over, pairs of Whinchats breeding in narrow, long-aban- doned ﬁelds often foraged in adjacent crop ﬁelds (mostly oil-seed rape, but avoiding winter cereals) compared to pairs breeding in large patches of non-cropped vegetation (Frankiewicz 2010). In particular, we investigate the relationships between changes in the main dietary characteristics, i.e. the numerical and biomass contribution of major taxonomic and functional prey groups (expressed by their chitin content, vertical distribution, habitat preference and vagi- lity within the landscape) and brood age, nestling condition and breeding patch size. The functional prey group approach (i.e. the classiﬁcation of invertebrates based on their individual features) was used by earlier researchers (Wiens and Rotenberry 1979; Crossley et al. 1989), as well as by ourselves in our investigations of bird diets (Orłowski et al. 2014b). We hypothesized that the contribution of some prey groups (mostly more highly chitinized prey) would increase as the nestlings grew older, but that to some degree these changes would also co-vary with patch area. Thus, some invertebrate taxa, such as large-bodied, sedentary species (or less vagile ones potentially severely negatively affected by agricultural practices/mowing like non-ﬂying/soil-dwelling Orthoptera or Arachnidae), might be over-represented in the diet of nestlings from large patches of non-cropped vegetation, whereas mobile spe- cies, highly vagile within a landscape (Siemann et al. 1999; Tscharntke et al. 2005; Humbert et al. 2009) or living in a crop habitat, might be over-represented in smaller patches. Materials and methods Their ages ranged between 3 and 11 days old; the sample size of particular nestling age classes is shown in Fig. 1. Since some faecal sacs were collected from the same broods on consecutive days, the dietary data for these broods were treated sepa- rately in various age categories in the subsequent analysis. The age of the nestlings from which we sampled faecal sacs varied among the four study years (Kruskal–Wallis test, H3,177 = 24.3, P \ 0.001). old broods. For the graphical presentation of dietary data for nestlings with different body condition, and to prevent pseudoreplication, we arbitrarily divided these nestlings into three groups according to the residuals obtained from the regression analysis (see above). Hence, we classiﬁed the condition of nestlings with the lowest residuals as ‘poor’, i.e. \0 (samples from 19 broods; 74 faecal sacs); those with residuals between 0 and 1 as ‘medium’ (nine broods; 38 faecal sacs); and those with residuals [1 as ‘good’ (ﬁve broods; 23 faecal sacs). Before analysis the faecal sacs were crushed manually and separated on Petri dishes. The food components in the faecal sacs were identiﬁed under a binocular microscope at 409 magniﬁcation. The number of prey items representing particular invertebrate taxa present in each individual fae- cal sac was established from the numbers of fragments of chitin parts, chieﬂy the elytra (for different families and genera of Coleoptera, Homoptera or Heteroptera), wings (in the case of Diptera, Hymenoptera), mouthparts (most of the orders) and other preserved organs (e.g. limbs, perilous, clypeus, mandible). During the determination of the num- ber of prey items belonging to a particular taxon, a rule summing the different chitin parts to the level of one individual was applied: two or more different fragments of chitin parts (e.g. head, mandibles, six legs and other parts in the case of ants) from one faecal sac were treated as belonging to the same individual of a given species (Orłowski and Karg 2011, 2013). The mass of prey was calculated as dry mass (milligrams dry weight); these We estimated the index of nestling body condition (hereafter for simplicity referred to as ‘nestling body con- dition’) based on the residuals from a regression of body mass on the length of the second primary (Bortolotti et al. 2000; Frankiewicz 2010). Materials and methods The nests from which we sampled faecal sacs were distributed in 22 abandoned ﬁelds, ranging in area from 0.1 to 6.8 ha (average = 1.52 ha). Six of these ﬁelds were \0.5 ha, four were 0.5–1 ha, seven were 1–2 ha, and four 2–4 ha; one ﬁeld was[5 ha. In order to graphically present and prevent pseudoreplication of dietary data derived from the same broods, we allocated the dietary data to ﬁelds of four size classes: \0.75 ha (39 faecal sacs), 0.75–1.5 ha (36), 1.5–3 ha (57) and [3 ha (45). The results are part of an extensive study of the breeding biology and ecology of Whinchats conducted in a ca. 500-ha area of agricultural landscape in south-west Poland (Frankiewicz 2008, 2010, 2015). The dominant form of land use in the region was arable, with crop ﬁelds covering about 87–93 % of the total area studied. The study area was managed extensively, with small-scale farming pre- dominant. Within the study area Whinchats breed in nar- row, elongated abandoned ﬁelds, dissected by numerous ﬁeld margins, fallow ﬁelds and plantations of young trees, usually not exceeding 30 m in width. In 2003–2007, a total of 246 occupied territories (including those of territorial single males which occupied their territories for more than 2 weeks) and 117 nests of Whinchats were found in the study area on 44 different abandoned ﬁelds, the optimal habitat for the species (Frankiewicz 2008, 2010). Three types of plant associations (based on Matuszkiewicz 2005) could be discerned on the abandoned ﬁelds where the The nests were inspected several times during the season in order to establish the onset of egg-laying and clutch size (Frankiewicz 2010). Throughout the expected hatching period nests were monitored daily to determine the exact hatching date. In the case of nests discovered only after egg-laying or hatching, the hatching date was back-calcu- lated based on the stage of the nestlings’ development. Over the years of the entire study (2003–2007), nestlings from 96 broods were weighed and the length of the second primaries measured (Frankiewicz 2010). The nestlings 123 123 173 J Ornithol (2017) 158:169–184 from which the faecal sacs were sampled were aged according to parallel measurements of body weight and feather development of other individually marked nestlings in nests located in the same area. Data analysis Initially, we identiﬁed invertebrate prey items to the lowest possible taxonomic level. However, we assumed that the taxonomic composition of the prey alone would be insuf- ﬁcient to fully explain the relationship between breeding patch habitat/size in non-cropped vegetation and/or chan- ges in the composition of prey. Thus, in order to provide an adequate description of these changes and a meaningful biological interpretation, including the food requirements of growing nestlings, we arbitrarily grouped the identiﬁed prey species/taxa into the following different classes based on their individual features and functionality within the landscape/habitat (hereafter referred to as ‘functional prey groups’): Second, we assessed the effect of the area of the aban- doned crop ﬁelds (expressed in hectares) where Whinchats bred on the four main dietary characteristics and compo- sition of nestlings. Third, we tested the inﬂuence of the main dietary characteristics and composition, i.e. the con- tribution of the seven major food types and different functional prey groups, on the body condition (the residuals from a regression of body mass on the length of the second primary) of Whinchat broods. Both of these analyses were performed separately for each of the dietary characteristics and individual taxonomic and functional prey groups (in terms of their %biomass) using the generalized linear model module (GLZ) with mixed design in Statistica 7.0 (Statsoft 2007) with normal distribution and logarithmic link function. To control for the non-independence in the generalized linear mixed models (GLMMs), the individual brood identity was nested within the age of nestlings. The model statistics were the estimate (±SE) and Wald v2- value. We believe that our approach treating each indi- vidual prey group separately in these two analyses is jus- tiﬁed because in 90 % of cases parent Whinchats brought individual invertebrate prey items to the nestlings (Fran- kiewicz 2010). 1. Chitin content (less, intermediately and highly chi- tinized prey). 2. Vertical distribution (soil-dwelling invertebrates, soil/ vegetation up to ca. 1 m and aerial invertebrates). 3. Habitat preference (eurytopic taxa, associated with crops or non-cropped habitats). 4. Vagility of species/taxa within the landscape (seden- tary or vagile taxa). These classiﬁcations were based on our previous eco- logical studies of various groups of invertebrate taxa in an agricultural landscape, including the classiﬁcation of invertebrates into functional prey groups and the use of such an approach in dietary studies of insectivorous birds (Orłowski et al. 2014b). Materials and methods The index of body condition was calculated on the basis of average values for the entire brood, which most often comprised nestlings in one age class, i.e. nestlings hatched within 24 h in 94 % of the 37 nests investigated (Frankiewicz 2010). In order to analyse in detail the relationship between nestling condition and diet, we used data for a smaller set of broods of known body condition: 32 different broods aged between 4 and 10 days investigated in 2004–2007 (n = 135 faecal sacs). The number of faecal sacs sampled were as follows: 13 from 4-day-old broods, 30 from 5-day-old broods, 30 from 6-day-old broods, 41 from 7-day-old broods, 4 from 8-day- old broods, 5 from 9-day-old broods and 12 from 10-day- 123 Fig. 1 The four main dietary characteristics (average ± SE) determined for individual faecal sacs (n = 177 in total) of nestling Whinchats Saxicola rubetra vs. nestling age sampled in abandoned crop ﬁelds in south-west Poland, 2004–2007. Number of prey taxa = diet diversity. The sample sizes for consecutive age classes of nestlings are: 3 days (d) old (n = 6 faecal sacs), 4 days old (n = 10), 5 days old (n = 35), 6 days old (n = 30), 7 days old (n = 43), 8 days old (n = 13), 9 days old (n = 18), 10 days old (n = 16) and 11 days old (n = 6) 12 3 J Ornithol (2017) 158:169–184 174 values were obtained from detailed measurements of insect weights based on the analysis of 479,087 individuals of different insect taxa (Karg 1989). of the seven major food types and the different functional prey groups [multivariate ANOVA (MANOVA)] between nestlings in nine age classes ([3–11 days old) using Sta- tistica 7.0 (Statsoft 2007). To resolve the problem of pseudoreplication, these analyses were performed using ANOVA/MANOVA with a nested design and a mixed- model approach with brood identity as a random term; the independent variables were nestling age and individual brood identity (nested within nestling age). Data analysis A detailed classiﬁcation of all the identiﬁed prey taxa is presented in Table S1. To meet the assumption of normality some data were log transformed; in addition, all percentage data were square root-arcsine transformed prior to analysis. The sta- tistical analyses were done using Statistica 7.0 (Statsoft 2007) and Excel software. The probability of P \ 0.05 was considered statistically signiﬁcant. For each faecal sac we determined the four main dietary characteristics (diet diversity expressed as the number of prey taxa, total number of prey, total biomass of prey and individual prey weight) and the composition of the diet expressed as the number, percent number (%number), biomass and percent biomass (%biomass) representing the seven major taxonomic prey groups [classes/orders of invertebrates: Heteroptera, Coleoptera, Hymenoptera/Dip- tera, Lepidoptera (larvae), Orthoptera, Aranaea; other invertebrates comprising Mollusca, Diplopoda and unidentiﬁed insects] and prey groups with four different classiﬁcations of the functionality of invertebrate taxa (i.e. chitin content, vertical distribution, habitat preference and vagility within the landscape). Effect of nestling age on diet composition Nestlings received more highly chitinized prey as they grew older. This was especially evident in the %biomass of highly chitinized prey, which increased signiﬁcantly with nestling age (Spearman rank correlation coefﬁcient, rs = 0.717, P = 0.030; Fig. 3). Interestingly, the contri- bution pattern of %number and %biomass of the three other functional prey groups, expressing their vertical distribution, habitat preference and vagility within the landscape, showed no clear age-related trend (Fig. 3). So nestlings were fed predominantly prey items from vegeta- tion ([70 % of prey biomass), and eurytopic prey items constituted [50 % of the prey biomass in each age class, whereas typical soil-dwelling invertebrates made up no more than 10 % of the biomass consumed (Fig. 3; Fig. S2). Lastly, over the entire brood-rearing period the number and %number of prey representing sedentary taxa were con- sistently higher compared to vagile taxa; however, the biomass and %biomass of both these prey groups showed large day-to-day variations (Fig. 3; Fig. S2). Three of the four main dietary characteristics analysed (Fig. 1), i.e. dietary diversity, the number of prey items and the biomass of prey exhibited signiﬁcant variations in relation to both partial effects tested, i.e. Brood identity (Age) and Age, and showed a highly signiﬁcant and close ﬁt in the overall model and apparent increase with age (Table 1). In contrast, the average mass of prey (Fig. 1) was signiﬁcantly related only to the Brood identity (Age) (Table 1). Furthermore, although the ﬁt in the overall model for the average mass of prey was also statistically signiﬁcant (Table 1), the trend of the changes, especially in nestlings older than 4 days suggests a decrease in the average mass of prey (Fig. 1). The results of the MANOVA showed that the dietary composition, expressed as the number, %number, biomass and %biomass of the seven major taxonomic prey groups/food types (class/orders of invertebrates) (Fig. 2; Fig. S1) and four functional prey groups in terms of chitin content, vertical distribution, habitat preference and vagility of a species/taxa within the landscape (Fig. 3; Fig. S2), varied markedly both with the age of nestlings and between various broods, as indicated by the output of the partial effect of brood identity nested within age (Table 2). In consecutive age classes of nestlings we observed a large variation in the contribution of each of the seven major taxonomic prey/food types. Effect of nestling age on diet composition In particular, the %biomass of prey between the 3rd and 4th day dis- played the most pronounced differences in variation: Orthoptera (up to ca. eightfold increase), Aranaea (ca. sevenfold decrease), Coleoptera (ca. twofold decrease) (Fig. 2). Results The analysis yielded 1778 individual invertebrate prey items representing 58 various taxa in all the faecal sacs examined. The most numerous prey items were Coleoptera (47.6 %), followed by Arachnidae (for simplicity, here- after, Araneae; 13.4 %), Heteroptera (10.2 %), Hyme- noptera (10.2 %), Orthoptera (6.5 %), Diptera (5.9 %), Lepidoptera larvae (2.8 %), Diplopoda (2.3 %) and other prey (Mollusca, Nematoda and unidentiﬁed insects; 1.1 %) (Table S1). Three major objectives were explored in the statistical analyses. First, we assessed the age-related differences in the main dietary characteristics (ANOVA), the contribution 123 123 175 J Ornithol (2017) 158:169–184 Relationship between diet and nestling condition, and breeding patch size The results of the GLMMs showed the statistically sig- niﬁcant effect of the area of the abandoned ﬁeld where the Whinchats bred on 17 of the 23 dietary characteris- tics/contributions of %biomass of major prey groups analysed, including nine positive and eight negative inﬂu- ences (Table S2). In particular, we found evidence that broods from larger abandoned ﬁelds received heavier prey items and consumed more Araneae, Diplopoda/Mollusca, Orthoptera, Hymenoptera, sedentary taxa, soil-dwelling invertebrates, eurytopic taxa or highly chitinized prey. On the other hand, the contribution of Coleoptera, Heteroptera, Lepidoptera larvae, intermediately chitinized, crop-speci- ﬁc, non-crop speciﬁc, vagile prey or prey from vegetation, as well as aerial insects, decreased in their diet (Fig. 4; Table S2). Lastly, the post hoc contrast (Tukey’s test) from the MANOVA showed that the numerical contribution of three of the seven major taxonomic prey groups/food types (Aranaea, Lepidoptera larvae and Orthoptera) to the diet varied signiﬁcantly among the four study years. ype III SS) testing the effect (F-values) of age (days) and brood identity (nested within age) on the main dietary cal sacs of Whinchat Saxicola rubetra nestlings (see Fig. 1) Table 1 Results of ANOVA (type III SS) testing the effect (F-values) of age (days) and brood identity (nested within age) on the main dietary characteristics identiﬁed for faecal sacs of Whinchat Saxicola rubetra nestlings (see Fig. 1) Source of variation Effect Model Brood identity(Age)a Ageb R2 P Diet diversity (n taxa) 2.37*** 2.65* 0.473 \0.0001 Number of prey items 3.59* 5.02* 0.543 \0.0001 Biomass of prey 4.65* 6.73* 0.612 \0.0001 Average mass of prey 1.50* 1.65 (n.s.) 0.345 0.045 * P \ 0.05, *** P \ 0.001 (statistical signiﬁcance for partial effects) a df = 38 b df = 8 Table 1 Results of ANOVA (type III SS) testing the effect (F-values) of age (days) and brood identity (nested w characteristics identiﬁed for faecal sacs of Whinchat Saxicola rubetra nestlings (see Fig. 1) J Ornithol (2017) 158:169–184 176 Fig. 2 The average (±SE) percent number (%number) and percent biomass (%biomass) of seven major prey/food types (class/orders of invertebrates) identiﬁed in faecal sacs of nestling Whinchats S. rubetra vs. nestling age. Other prey are Diplopoda, Mollusca, Nematoda and unidentiﬁed insects; for the number and biomass of prey, see Fig. S1 Fig. Relationship between diet and nestling condition, and breeding patch size 2 The average (±SE) percent number (%number) and percent biomass (%biomass) of seven major prey/food types (class/orders of invertebrates) identiﬁed in faecal sacs of nestling Whinchats S. rubetra vs. nes Nematoda and u prey, see Fig. S1 er (%number) and percent food types (class/orders of of nestling Whinchats S. rubetra vs. nestling age. Other prey are Diplopoda, Mollusca, Nematoda and unidentiﬁed insects; for the number and biomass of prey, see Fig. S1 Fig. 2 The average (±SE) percent number (%number) and percent biomass (%biomass) of seven major prey/food types (class/orders of invertebrates) identiﬁed in faecal sacs of nestling Whinchats S. rubetra vs. nestling age. Other prey are Diplopoda, Mollusca, Nematoda and unidentiﬁed insects; for the number and biomass of prey, see Fig. S1 Nestling condition was signiﬁcantly correlated with six of the 23 dietary characteristics/contributions of %biomass of major prey groups analysed, including four positive and two negative relationships (Table S3; Fig. 5). Evidence was forthcoming that the %biomass of Lepidoptera larvae, Orthoptera, less chitinized or sedentary prey were posi- tively correlated with nestling condition, while the %bio- mass of Coleoptera and vagile prey were negatively correlated with nestling condition (Fig. 5; Table S3). Finally, nestling condition was not correlated with the area of the abandoned ﬁeld where Whinchats bred (GLMM, with nest identity as a random term, estimate = -0.022, SE = 0.026, v2 = 0.74, P = 0.390). the speciﬁc nutritional needs of offspring in different age classes (Flinks and Pfeifer 1988; Magrath et al. 2004; Radford 2008; Mitrus et al. 2010; Garcı´a-Navas et al. 2012), and agrees with earlier dietary studies on Whinchats breeding in grasslands (reviewed by Bastian and Bastian 1996; Suter 1988). The three main dietary characteristics, i.e. diversity of diet, number of prey items and biomass of prey—the last mentioned being primarily a consequence of provisioning with heavy orthopterans—apparently increased with nestling age, which generally seems to be a function of the progressive gain in body mass, and the concurrent growing volume/quality of food ingested/di- gested and increasing volume of faecal sacs produced by growing nestlings (Flinks and Pfeifer 1988). In this respect, the most relevant variable describing a priori dietary changes in nestlings of increasing age and size is the average mass of individual prey items, which is only related to brood identity and is least biased (if at all) as a result of the progressive gain in body mass by nestlings. Relationship between diet and nestling condition, and breeding patch size Moreover, there may exist some differences in the fre- quency of defecation between younger and older nestlings, which is a consequence of their receiving different amounts or quality of food (Quan et al. 2015), as well as of ther- moregulation and perhaps also water regulation. Ther- moregulation only comes into play in nestling Whinchats when they are 4–5 days old (Bastian and Bastian Discussion Our study demonstrated several major results documenting the effect of open non-forest habitat fragmentation or patch size effect on individual ﬁtness and dietary optimization in passerine nestlings, as well as complementing basic dietary information on a declining insectivorous grassland passerine in a novel breeding habitat. First, we found that nestling Whinchats received more chitinized prey as they grew older, which overall is in line of the idea of the selective foraging of parent birds to meet 123 123 177 J Ornithol (2017) 158:169–184 1993, 1996). In addition, the defecation efﬁciency in nestlings of different ages is presumably different, which may inﬂuence the passage of some prey remains to the f l average mass of invertebrate prey should, as in three previous cases, increase progressively with nestling age, but we observed such an increase only between 3- and 4 d ld li I h hi di l Fig. 3 The average (±SE) %number and %biomass of four functional prey groups expressing (from the top) the chitin content, vertical distribution, habitat preference and vagility of species/taxa within the landscape identiﬁed in faecal sacs of nestling Whinchats S. rubetra vs. nestling age. The identiﬁed prey items are listed in Table S1. For the number and biomass of functional prey groups, see Fig. S2 the defecation efﬁciency in presumably different, which of some prey remains to the parent inconsistency was the f invertebrate prey, observed he nestlings. As expected, the average mass of invertebrate prey should, as in three previous cases, increase progressively with nestling age, but we observed such an increase only between 3- and 4-day-old nestlings. It seems that this contradictory result may illustrate the overall ability of some insectivorous passerine nestlings to ingest large prey at an early stage of their post-natal development, which in practice can mean 1993, 1996). In addition, the defecation efﬁciency in nestlings of different ages is presumably different, which may inﬂuence the passage of some prey remains to the faecal sacs. average mass of invertebrate prey should, as in three previous cases, increase progressively with nestling age, but we observed such an increase only between 3- and 4-day-old nestlings. Discussion It seems that this contradictory result may illustrate the overall ability of some insectivorous passerine nestlings to ingest large prey at an early stage of their post-natal development, which in practice can mean On the other hand, an apparent inconsistency was the decrease in average mass of invertebrate prey, observed from the 4th day of life of the nestlings. As expected, the 12 178 J Ornithol (2017) 158:169–184 Table 2 Results of multivariate ANOVA testing the effect of age (days) and brood identity (nested within age) on dietary composition expressed as the number, percent number, biomass and percent biomass of seven major prey/food types (class/orders of inverte- brates = Araneae, Heteroptera, Diptera/Hymenoptera, Coleoptera, Lepidoptera larvae, Orthoptera and other invertebrates; see Fig. 2) Table 2 Results of multivariate ANOVA testing the effect of age (days) and brood identity (nested within age) on dietary composition expressed as the number, percent number, biomass and percent biomass of seven major prey/food types (class/orders of inverte- brates = Araneae, Heteroptera, Diptera/Hymenoptera, Coleoptera, Lepidoptera larvae, Orthoptera and other invertebrates; see Fig. 2) and four functional prey groups expressing chitin content (less, intermediately and highly chitinized), vertical distribution (soil surface, soil/vegetation, aerial insects), habitat preference (eurytopic, crops and non-cropped) and vagility of a species/taxa within the landscape (sedentary and vagile species) (see Fig. 3) based on analysis of faecal sacs of nestling Whinchats S. Discussion rubetra Source of variation Brood identity(Age) Age Wilk’s k df F P Wilk’s k df F P Ho Error Ho Error Major prey/food types Number of prey items 0.013 266 867.9 2.86 \0.0001 0.141 56 673.1 5.28 \0.0001 Percentage of prey 0.027 266 867.9 2.53 \0.0001 0.389 56 673.1 2.30 \0.0001 Biomass of prey 0.010 266 867.9 3.31 \0.0001 0.389 56 673.1 6.20 \0.0001 Percentage of prey biomass 0.017 266 867.9 2.64 \0.0001 0.355 56 673.1 2.55 \0.0001 Chitin content Number of prey items 0.139 114 384.3 3.14 \0.0001 0.466 24 371.8 4.66 \0.0001 Percentage of prey 0.223 114 384.3 2.19 \0.0001 0.674 24 371.8 2.56 0.001 Biomass of prey 0.187 114 384.3 2.53 \0.0001 0.473 24 371.8 4.55 \0.0001 Percentage of prey biomass 0.215 114 384.3 2.26 \0.0001 0.753 24 371.8 1.59 0.039 Vertical distribution Number of prey items 0.145 114 384.3 3.04 \0.0001 0.633 24 371.8 2.64 \0.0001 Percentage of prey 0.304 114 384.3 1.64 0.0003 0.840 24 371.8 0.96 0.513 Biomass of prey 0.136 114 384.3 3.19 \0.0001 0.598 24 371.8 3.01 \0.0001 Percentage of prey biomass 0.171 114 384.3 2.71 \0.0001 0.746 24 371.8 1.64 0.030 Habitat preference Number of prey items 0.187 114 384.3 2.52 \0.0001 0.633 24 371.8 1.64 \0.0001 Percentage of prey 0.266 114 384.3 1.88 \0.0001 0.714 24 371.8 1.91 0.007 Biomass of prey 0.260 114 384.3 1.91 \0.0001 0.623 24 371.8 2.71 \0.0001 Percentage of prey biomass 0.261 114 384.3 1.91 \0.0001 0.754 24 371.8 1.58 0.043 Vagility of a species/taxon Number of prey items 0.247 76 258 3.44 \0.0001 0.675 16 258 3.50 \0.0001 Percentage of prey 0.341 76 258 2.42 \0.0001 0.844 16 258 1.43 0.129 Biomass of prey 0.334 76 258 2.47 \0.0001 0.737 16 258 2.65 0.0007 Percentage of prey biomass 0.376 76 258 2.14 \0.0001 0.835 16 258 1.52 0.092 The identiﬁed prey items are listed in Table S1 sudden increase in the average mass of invertebrate prey, prey abundance and biomass between 3- and 4-day-old nestlings is that this may have been due in part to the small sample size of the youngest (3 days old) nestlings compared with older (5–7 days old) nestlings. Indeed, the contribution of orthopteran prey did increase between days 3 and 4, but then decreased between days 4 and 5; the same applied to the decrease in Coleoptera. Discussion Moreover, the contribution of Orthoptera and two other prey groups (Aranaea and Lepidoptera larvae) varied signiﬁcantly between the 4 study years, which most likely resulted in part from the unequal age distribution among the nestlings in the 4 study years, although some differences in the availability of these invertebrates at the parent Whinchats’ that 4-day-old nestling Whinchats can already exceed the threshold of the gape-size constraint hypothesis. Basi- cally, the gape-size constraint hypothesis states that only nestlings of small passerines older than 7 days are not limited by gape size (cf. Garcı´a-Navas et al. 2012). In the case of the Whinchat, a species relying primarily on single invertebrate prey items caught during discrete hunting events (Andersson 1981; Bastian and Bastian 1996; Frankiewicz 2010; Pudil and Exnerova´ 2015), the early ability of nestlings to ingest prey of large size might therefore be behaviourally justiﬁed. On the other hand, the lowest prey weight measured in 3-day-old nestlings suggests that this is the intentional work of parent Whinchats. An alternative explanation for the observed 123 J Ornithol (2017) 158:169–184 179 Fig. 4 Statistically signiﬁcant dietary changes (average ± SE) in nestling Whinchats S. rubetra averaged across broods of different ages in relation to the area of abandoned crop ﬁelds. Summary of statistical analysis (generalized linear mixed models; GLMMs) testing the effect of abandoned crop ﬁeld area on these and the other dietary variables listed in Table S2 foraging sites are also possible. On the other hand, the analysis of the contribution of the functional prey groups seems more convincing, mostly because of the relatively smaller variation between the consecutive nestling age classes, compared to the more variable taxonomical division of prey. We found evidence that nestling condition was posi- tively correlated with the contribution of Lepidoptera lar- vae and Orthoptera or sedentary (mostly large-bodied) prey taxa, and negatively with the contribution of Coleoptera or vagile prey taxa in the diet, but not with the area of the patch where Whinchats bred. In this respect, in so far as Fig. 5 Statistically signiﬁcant dietary changes (average ± SE) in nestling Whinchats S. rubetra in relation to their body condition based on the residuals from a regression of body mass on the length of the second primary. The condition of nestlings with the lowest residuals, i.e. Discussion the largest ingested prey items in our study. Owing to their high caloric value, Orthoptera are presumably as beneﬁcial as lepidopteran larvae for nestling condition. A similar ﬁnding, namely, a positive relationship between nestling body mass and the availability of certain orthopterans occurring in larger rectangular patches of early successional stages of woodland, was reported by Weldon and Haddad (2005). In contrast, we observed a negative relationship between nestling condition and the contribution of highly chitinized Coleoptera. Overall, this could be due to the poorer digestibility and lower water content of these insects (Studier and Sevick 1992), which translate into a smaller energy gain compared to the ingestion of more energy-rich caterpillars and Orthoptera (Brodmann and Reyer 1999). Moreover, apart from the fact that Orthoptera and Lepidoptera larvae supply a lot of water and are easier to digest, they presumably contain a certain amount of indigestible plant remains for nestling Whinchats, which may appear to increase their body mass. On the other hand, our study did not conﬁrm the relation- ship between nestling condition and breeding patch area. It may be that parent birds compensate for the negative impacts of a deteriorating environment (i.e. a smaller breeding patch area) by working harder to get the same or reduced amount of food for their young, thereby main- taining a standard reproductive output (Brickle et al. 2000; Morris et al. 2001; Britschgi et al. 2006); our previous investigation in this study area did not provide any evi- dence that the area of abandoned ﬁelds where the Whin- chats bred affected their breeding success or productivity (Frankiewicz 2010). We found evidence that Whinchat broods from larger patches of non-cropped vegetation received more sedentary and heavier prey such as Orthoptera, Araneae and soil- dwelling invertebrates (such as Diplopoda/Mollusca). These results seem to conﬁrm our prediction that large patches of vegetation that are not cropped (i.e. larger abandoned ﬁelds) but are managed agriculturally are refuges for less vagile or more sedentary species of invertebrates, which (mostly due to the severe mortality caused by farm machinery) did not normally occur in cropped areas. In particular, this applies to Araneae and orthopterans, which are very vulnerable to machinery-in- duced mortality, and which thus occur in considerably higher abundance in transitional habitats with successional vegetation, like abandoned grasslands or crop ﬁelds, after the cessation of agricultural management (Siemann et al. 1999; Humbert et al. 2009; Marini et al. Discussion However, it should be remembered that parent Whinchats [like other bird species preying on large orthopterans (Kaspari 1991; Ban´bura et al. 1999)] most probably pre- pared these large insects before feeding them to their nestlings by removing highly chinitized body parts, such as legs (this seems particularly necessary in the largest taxa consumed, e.g. Metrioptera), since these insect parts were not recovered in the analysed faeces of nestling Whinchats. To date, in our sample the overall contribution (by number) of orthopterans in the diet of nestling Whinchats was 6.5 % (Table S1), although it varied between different age classes and its maximum percentage contribution was nearly 20 % (by number) or as much as 42 % (by biomass) in 4-day-old nestlings (see Fig. 2). This corresponds to previous detailed dietary data reported, namely, a 3–12.8 % numerical share of orthopterans in the diet of Whinchat nestlings (Suter 1988; Kleinschmidt 2001). Another potential explanation for the lesser use of orthopteran prey by Whinchats in grasslands (Britschgi et al. 2006) may be the relatively longer handling and preparation time with this prey type compared to the easily swallowed caterpillars and spiders (Banbura et al. 1999). In addition, Aranaea are primarily fed to the youngest nestlings (Magrath et al. 2004; Ramsay and Houston 2003): such a pattern of provisioning nestlings with spiders (35 % of all prey by number in 3-day-old nestlings) was conﬁrmed in our study. This implies that different developmental stages of nestlings should be considered in dietary/foraging studies in order to acquire the full picture of the dietary/food requirements of Whin- chats and perhaps also other insectivorous passerine nest- lings. Finally, it should borne in mind that Whinchats adapt rapidly to increased food density and that they are under strong selection for efﬁciency (Andersson 1981). Thus, the presence of some abundant invertebrate prey in our sam- ples, such as orthopterans or large beetles like the Garden Chafer Phyllopertha horticola (nearly 24 % of all prey identiﬁed; Table S1), presumably indicates that parent Whinchats make use of the largest invertebrate prey available near the perching site (Kleinschmidt 2001). In turn, we found that nestling Whinchats from smaller several earlier studies had already discovered a positive relationship between the contribution of lepidopteran lar- vae (or insect larvae/soft-bodied prey) and nestling condi- tion (Donald et al. 2001; Ban´bura et al. 1999; Garcı´a-Navas and Sanz 2011), we conﬁrmed an analogous relationship for Orthoptera, i.e. Discussion \0, was classiﬁed as ‘poor’ (19 broods/74 faecal sacs), for those with residuals between 0 and 1 as ‘medium’ (9/38), and for those with residuals [1 as ‘good’ (5/23). The statistical analysis (GLMMs) testing the effect of these and the other dietary variables on the nestling body condition is summarized in Table S3 Fig. 5 Statistically signiﬁcant dietary changes (average ± SE) in nestling Whinchats S. rubetra in relation to their body condition based on the residuals from a regression of body mass on the length of the second primary. The condition of nestlings with the lowest residuals, i.e. \0, was classiﬁed as ‘poor’ (19 broods/74 faecal sacs), for those with residuals between 0 and 1 as ‘medium’ (9/38), and for those with residuals [1 as ‘good’ (5/23). The statistical analysis (GLMMs) testing the effect of these and the other dietary variables on the nestling body condition is summarized in Table S3 We found evidence that nestling condition was posi- tively correlated with the contribution of Lepidoptera lar- vae and Orthoptera or sedentary (mostly large-bodied) prey taxa, and negatively with the contribution of Coleoptera or vagile prey taxa in the diet, but not with the area of the patch where Whinchats bred. In this respect, in so far as foraging sites are also possible. On the other hand, the analysis of the contribution of the functional prey groups seems more convincing, mostly because of the relatively smaller variation between the consecutive nestling age classes, compared to the more variable taxonomical division of prey. foraging sites are also possible. On the other hand, the analysis of the contribution of the functional prey groups seems more convincing, mostly because of the relatively smaller variation between the consecutive nestling age classes, compared to the more variable taxonomical division of prey. 12 J Ornithol (2017) 158:169–184 180 traditionally managed grasslands (Britschgi et al. 2006). Interestingly, earlier dietary studies of nestling Whinchats from different grassland habitats showed that orthopterans (Saltatoria) and Aranaea were rather less-preferred prey types and that the selectivity of these prey types was apparently lower or similar (Bastian and Bastian 1996; Britschgi et al. 2006) compared to their occurrence in the habitat. Britschgi et al. (2006) claimed that orthopterans were under-represented in the diet of nestling Whinchats primarily because of the high level of chitin they contain. Discussion One should bear in mind the fact that the structure and composition of vegetation, and thus indirectly also the availability of soil surface and the potential soil-dwelling invertebrate prey present there, vary between grasslands and abandoned ﬁelds: in the former the vegetation is more condensed and access to the soil surface is limited. Con- sequently, owing to the lack of the compact layer of veg- etation normally occurring in grasslands, the soil on abandoned farmland was drier. Presumably this is beneﬁ- cial for some soil-dwelling invertebrates, such as ants, some coleopterans and orthopterans (which are abundant in the diet of the nestling Whinchats we examined), which are also easier to catch on bare ground or in lower vegetation (Andersson et al. 2009; Schaub et al. 2010). In addition, it should be stressed that Whinchats hunt primarily from a perch, so both the denser vegetation (i.e. more concealed prey) and the perch height (or even the lack of perches in modern grasslands) are of critical importance for access to prey and hunting efﬁciency (Andersson 1981; Andersson et al. 2009). This concurs with the assumption that the mobility of ground-foraging insectivorous passerines and access to food are of greater importance during the selec- tion of a foraging site than the abundance of food per se, which translates into preferential foraging in short vege- tation, even though more invertebrate prey were available in tall vegetation (discussed in van Oosten et al. 2014; Schaub et al. 2010). Therefore, it is possible that the recent successful colonization of abandoned farmland by the Whinchat and its population increase in this new habitat are in part simply the consequence of favourable foraging conditions and the excellent accessibility of prey in the loose vegetation or bare ground present there. Four major conclusions with some critical implications for future ecological/dietary studies of Whinchats and other grassland passerines can be drawn from our investigation. On the other hand, the overall low percentage of crop- speciﬁc invertebrate prey in the nestling diet (\10 % of each analysed dietary characteristic across all analysed age classes; see Fig. 3) might conﬁrm the strong preference of Whinchats for foraging within the occupied patches of abandoned cropland (Frankiewicz 2010). Further, our dietary analysis showed that for Whinchats soil-dwelling invertebrates tended to be of secondary importance as food because the majority of prey items ([70 % in each age class) were those associated with vegetation. Discussion 2009), as well as in In turn, we found that nestling Whinchats from smaller patches received lighter prey, primarily the consequence of a lower proportion of large-bodied orthopterans, and that the contribution of Coleoptera, Heteroptera, 123 123 J Ornithol (2017) 158:169–184 181 Lepidoptera larvae, intermediately chitinized, crop-speci- ﬁc, non-crop-speciﬁc, vagile or prey from vegetation, as well as aerial insects, was greater in these patches. This might be indicative of an edge effect, due mostly to an increasing abundance of some edge invertebrate species and/or differences in the composition of the invertebrate community representing both the taxa living in non- cropped vegetation and ﬂoaters from adjacent crops (such as some Coleoptera) (Siemann et al. 1999; Holland and Luff 2000; Tscharntke et al. 2005). Alternatively, there may be such a large amount of all these prey items near the nest that it is useful for the parents to exploit them. Loading time is thus shorter, and it makes sense to deliver smaller prey and raise the feeding frequency without having to expend more energy (Andersson 1981). Con- sequently, such changes in the invertebrate community might lead to a behavioural/dietary adjustment in the parent Whinchats, primarily in their hunting technique, i.e. more aerial hunts/insects, when other prey groups are scarce owing to the small patch area, as an alternative to foraging in adjacent crops. For example, it may be a very economical method to catch bigger ﬂies when on a warm, sunny day with a light breeze these insects are driven in the direction of the perch (Bastian and Bastian 1996). Therefore, weather conditions and the exact time of day when the faecal sacs were collected would also have an important inﬂuence on the dietary composition in nestling Whinchats, especially in the context of the functional aspect of prey. Furthermore, differences in the collection timing of faecal sacs between different years may also have had an inﬂuence on the dietary composition: this generally seems to explain the observed signiﬁcant dif- ferences in the contributions of the three prey groups (Aranaea, Lepidoptera larvae and Orthoptera) during the 4 years of the study. hampered a more detailed analysis and the drawing of further inferences. Discussion This seems to imply indirectly the importance of a mosaic-like structure of the microhabitat with low vegetation or vegetation-free patches and bare ground, where prey are more accessible and the hunting of foraging Whinchats is more efﬁcient (Bastian et al. 1994; Bastian and Bastian 1996; Andersson et al. 2009). Lastly, we were able to identify some inver- tebrate prey items only to the level of class/order (such as Araneae or Lepidoptera larvae), which to some degree We found that the diet of nestling Whinchats from larger patches contained basically more proﬁtable (mostly heav- ier) prey. Evidence demonstrating that food limitation is a likely mechanism of area sensitivity in Whinchats breeding in patches of abandoned farmland therefore seems to be in part justiﬁed. However, this needs further detailed inves- tigations analysing food supply/quality in patches of abandoned crop ﬁelds of different sizes and vegetation types. The dietary composition and presumably the speciﬁc nutritional needs of nestlings in different age classes vary primarily with respect to the contribution of soft-bodied prey. Consequently, dietary records (and perhaps concur- rent potential food resources) need to be sampled throughout the brood-rearing period. Dietary composition and the prey attributes of nestlings (between various 12 3 3 J Ornithol (2017) 158:169–184 182 world, such studies are imperative. They should analyse the dietary, behavioural and physiological responses and demography of birds towards size, habitat composition and landscape conﬁguration of patches of non-cropped vege- tation growing on fallow land. populations/areas) should be compared only for similar age classes of offspring. Otherwise, sampling without reference to nestling age will not lead to correct inferences about relationships between food supply and environment. Besides the previously reported additional factors affecting optimal diet and foraging strategy, which are a simultaneous resolution of various cost-beneﬁt functions (sensu Brodmann and Reyer 1999), our results add to this dietary optimization (compensation strategy), as it is a trade-off between a brood’s nutritional demands and patch size. This implies a continuum in dietary optimization in that the parent birds deliver the top-ranked invertebrates present mostly within a patch. In other words, this suggests that parent Whinchats can overcome habitat constraints resulting from the small area of a breeding patch by incorporating interchangeably the two major prey groups (Orthoptera and Lepidoptera) in the diet they feed to their nestlings. This is the ultimate justiﬁcation for stating that their condition is independent of patch size. References Andersson M (1981) Central place foraging in whinchats, Saxicola rubetra. 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Indirectly, given the colonization success and overall increase in populations of the Whinchat in abandoned farmland areas, this suggests that progressive climate change (mostly shortages of rainfall and reductions in temperature) are not disadvantageous for this species [e.g. in the Scottish Highlands Whinchats preferred warmer south-facing hillsides (Calladine and Bray 2012)]. One can assume that the major threats to Whinchats and other birds breeding in grasslands nowadays are the direct mortality/ nest losses of birds due to mowing and the intensiﬁcation of agricultural management as a result of the encroachment of tall grass species resulting from nitrogen deposition and acidiﬁcation, which diminish habitat suitability and reduce prey accessibility (Vickery et al. 2001; Broyer et al. 2014; Fischer et al. 2013; van Oosten et al. 2014). Bastian H, Bastian A (1993) Entwicklung der Ko¨rpermasse nestjunger Braunkehlchen (Saxicola rubetra). 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Discussion On the other hand, breeding on small patches may only be possible if there is a sufﬁciency of available prey items. Consequently, it is also possible that some prey groups will be more abundant in adjacent crops (such as lepidopteran larvae). Therefore the quality of small patches chosen for breeding could be similar or even higher (e.g. when agriculturally subsidized prey are numerous) than that of larger patches. Acknowledgments Peter Senn kindly corrected the English of the manuscript. All procedures regarding this study were conducted in compliance with Polish legislation. The permits for conducting the study were accepted by the II Ethics Commission in Wrocław. Licences for bird ringing were issued by the Ornithological Station of the Polish Academy of Sciences based on decisions of the Polish Ministry of Environment. 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https://openalex.org/W2593217535	https://escholarship.org/content/qt2pm58210/qt2pm58210.pdf?t=omsvpf	English	null	The impact of human development on individual health: a causal mediation analysis examining pathways through education and body mass index	PeerJ	2,017	cc-by	6,264	Permalink https://escholarship.org/uc/item/2pm58210 Journal PeerJ, 5(3) ISSN 2167-8359 Authors Wang, Aolin Arah, Onyebuchi A Publication Date 2017 DOI 10.7717/peerj.3053 Peer reviewed UCLA UCLA Previously Published Works UCLA UCLA Previously Published Works UCLA Previously Published Works Title The impact of human development on individual health: a causal mediation analysis examining pathways through education and body mass index Powered by the California Digital Library University of California eScholarship.org eScholarship.org ABSTRACT Background. The macro environment we live in projects what we can achieve and how we behave, and in turn, shapes our health in complex ways. Policymaking will benefit from insights into the mechanisms underlying how national socioeconomic context affects health. This study examined the impact of human development on individual health and the possible mediating roles of education and body mass index (BMI). Methods. We analyzed World Health Survey data on 109,448 participants aged 25 or older from 42 low- and middle-income countries with augmented human development index (HDI) in 1990. We used principal components method to create a health score based on measures from eight health state domains, used years of schooling as education indicator and calculated BMI from self-reported height and weight. We used causal mediation analysis technique with random intercepts to account for the multilevel structure. Results. Below a reference HDI level of 0.48, HDI was negatively associated with good health (total effect at HDI of 0.23: b = −3.44, 95% CI [−6.39–−0.49] for males and b = −5.16, 95% CI [−9.24,–−1.08] for females) but was positively associated with good health above this reference level (total effect at HDI of 0.75: b = 4.16, 95% CI [−0.33–8.66] for males and b = 6.62, 95% CI [0.85–12.38] for females). We found a small positive effect of HDI on health via education across reference HDI levels (b ranging from 0.24 to 0.29 for males and 0.40 to 0.49 for females) but not via pathways involving BMI only. Submitted 23 August 2016 Accepted 31 January 2017 Published 1 March 2017 Corresponding author Aolin Wang, aolinw@ucla.edu Academic editor Stefan Kuhle Additional Information and Declarations can be found on page 10 DOI 10.7717/peerj.3053 Copyright 2017 Wang and Arah Distributed under Creative Commons CC-BY 4.0 Submitted 23 August 2016 Accepted 31 January 2017 Published 1 March 2017 Corresponding author Aolin Wang, aolinw@ucla.edu Academic editor Stefan Kuhle Additional Information and Declarations can be found on page 10 DOI 10.7717/peerj.3053 Copyright 2017 Wang and Arah Di t ib t d d Conclusion. Human development has a non-linear effect on individual health, but the impact appears to be mainly through pathways other than education and BMI. The impact of human development on individual health: a causal mediation analysis examining pathways through education and body mass index Aolin Wang1,2 and Onyebuchi A. Arah1,2,3 1 Department of Epidemiology, Fielding School of Public Health, University of California, Los Angeles, CA, United States 1 Department of Epidemiology, Fielding School of Public Health, University of California, Los Angeles, CA, United States 2 California Center for Population Research (CCPR), Los Angeles, CA, United States 3 UCLA Center for Health Policy Research, Los Angeles, CA, United States How to cite this article Wang and Arah (2017), The impact of human development on individual health: a causal mediation analysis ex- amining pathways through education and body mass index. PeerJ 5:e3053; DOI 10.7717/peerj.3053 Subjects Epidemiology, Global Health, Public Health, Statistics Keywords Human development, Body mass index, Mediation analysis, General health, Education, Pathways Subjects Epidemiology, Global Health, Public Health, Statistics INTRODUCTION ‘‘Wealthier nations are healthier nations’’ has long been known (Pritchett & Summers, 1996). The relationship between income and health, at both the individual and national levels, is perhaps one of the most documented findings in the population health literature OPEN ACCESS (Preston, 1975; Pritchett & Summers, 1996; Hall, Barnes & Taylor, 2009). In low- and middle-income countries (LMICs), life expectancy has increased dramatically over the past century due to substantial achievements in control of infectious diseases via better sanitation and food safety, vaccines, antibiotics and improved nutrition (Schlipköter & Flahault, 2010). However, in the early 1990s, these countries started to experience rapid changes in lifestyle such as dietary and physical activity behaviors. Energy-dense poor quality diets and sedentary behaviors fueled an obesity epidemic (if not a pandemic), and an increased burden of nutrition-related non-communicable diseases (NCDs) was observed (Popkin, 1998; Hawkes, Chopra & Friel, 2009; Popkin, Adair & Ng, 2012). Though economic growth, increased individual income, and good health tend to go together, these nutritional and lifestyle changes and increased prevalence of NCDs may offset some of the health benefits of economic growth as countries go through economic and social development. Despite being studied using data from several decades ago (Preston, 1975; Pritchett & Summers, 1996), the effect of (macro socioeconomic) development on health will benefit from re-examination in the LMICs using health measures other than mortality and life expectancy, and with a view to explore potential mechanisms such as mediation and interaction. (Preston, 1975; Pritchett & Summers, 1996; Hall, Barnes & Taylor, 2009). In low- and middle-income countries (LMICs), life expectancy has increased dramatically over the past century due to substantial achievements in control of infectious diseases via better sanitation and food safety, vaccines, antibiotics and improved nutrition (Schlipköter & Flahault, 2010). However, in the early 1990s, these countries started to experience rapid changes in lifestyle such as dietary and physical activity behaviors. Energy-dense poor quality diets and sedentary behaviors fueled an obesity epidemic (if not a pandemic), and an increased burden of nutrition-related non-communicable diseases (NCDs) was observed (Popkin, 1998; Hawkes, Chopra & Friel, 2009; Popkin, Adair & Ng, 2012). Though economic growth, increased individual income, and good health tend to go together, these nutritional and lifestyle changes and increased prevalence of NCDs may offset some of the health benefits of economic growth as countries go through economic and social development. Wang and Arah (2017), PeerJ, DOI 10.7717/peerj.3053 Outcome Individuals were asked to report their perceived difficulties based on a 5-point Likert scale question for eight health state domain (two questions per domain): mobility, self-care, pain and discomfort, cognition, interpersonal activities, vision, sleep and energy, and affect (WHO, 2013). The health state measures have been extensively tested (Üstün et al.) and have shown good consistency and reliability (Moussavi et al., 2007). We performed a factor analysis using polychoric correlations to account for the covariance structure of the responses to individual questions. Similar to a previous study (Moussavi et al., 2007), we chose the one-factor solution based on the large eigenvalue of the first factor (8.85, 73% as a cumulative percentage of the variance explained) and the high communalities of the original variables (between 0.36 and 0.69). Then, we used the principal components method for factor extraction and the regression scoring method to obtain the factor scores. The factor score was rescaled to go from 0 (indicating worst health) to 100 (indicating best health). Exposure We chose the Human Development Index (HDI) as a measure of the national socioeco- nomic environment for human development in a country. HDI is a unit-free index between zero and one that is calculated based on life expectancy at birth, adult literacy rate, combined gross enrollment ratio for primary, secondary and tertiary education, and GDP per capita for each country. In this study, we used HDI reported for 1990 (United Nations Development Programme, 2013) and rescaled the score to range from zero to ten. We lagged the HDI for more than ten years to capture its effect on shaping individual education and to minimize reverse causation. We also assumed that HDI from 1990 was a good indicator of the national socioeconomic environment for the period leading up to 1990. Study sample and variables We used the World Health Survey (WHS) data from 49 low- and middle-income countries. Conducted by the WHO from 2002 to 2004, the WHS used a standardized methodology that provided a basis for examining individual health measures across countries (WHO, 2013). Within each country, samples were probabilistically selected with every individual being assigned to a knownnon-zeroselectionprobability. Thesesamples werenationallyrepresen- tative except in China, Comoros, Congo, Côte d’Ivoire, India, and the Russian Federation, where the survey was carried out in geographically limited regions. This study included participants from fourteen countries in the African region, nine in the European region, seven in the Americas, five in the South-East Asia region, five in the Western Pacific region, and two in the Eastern Mediterranean region (Table S2). All respondents were interviewed face-to-face with the standardized WHS survey, which included questions regarding demographic, socioeconomic, and behavioral factors (WHO, 2013; Moussavi et al., 2007). Wang and Arah (2017), PeerJ, DOI 10.7717/peerj.3053 INTRODUCTION Despite being studied using data from several decades ago (Preston, 1975; Pritchett & Summers, 1996), the effect of (macro socioeconomic) development on health will benefit from re-examination in the LMICs using health measures other than mortality and life expectancy, and with a view to explore potential mechanisms such as mediation and interaction. The underlying mechanisms are important for understanding the overall effect of development on health. Many studies on how education affects our health exist (Zimmerman, Woolf & Haley, 2015). Investing in schooling, particularly for girls, is one of the key strategies for developing countries to promote health and economic growth (World Bank, 1993) and could be an important intermediate factor on the pathway. Education also interacts with a country’s level of human development (Van der Kooi et al., 2013) and country context (Rehkopf, Dow & Rosero-Bixby, 2010) in affecting health. Adoption of health behaviors is another potential mediator in the mechanism connecting country development and health. Obesity, a crucial indicator closely related to health behavior, has been linked to various adverse health outcomes (James et al., 2004; Wang, Stronks & Arah, 2014; Wang & Arah, 2015). Moreover, its consequences appear to be nuanced for people with different educational attainments and from countries at various stages of development (Wang, Stronks & Arah, 2014). Indeed, we cannot discuss a person’s health without considering the ‘contextual web’ the person is in Arah (2009). The macro environment we live in projects what we can achieve and how we behave, and in turn, shapes our health in a complex way. Interesting insights can be gained from examining the extent to which the macro environment shapes health via mechanisms involving education, obesity, and other intermediate factors. This study aimed at addressing these mediation (mechanism) questions. More specifically, we used global data to investigate (i) the impact of national human development level as measured by human development index on individual health, and (ii) the mediating roles of both education and body mass index in the relation between human development and health. Wang and Arah (2017), PeerJ, DOI 10.7717/peerj.3053 2/13 METHODS Study sample and variables Confounders Potential confounders we included were WHO region, individual age, and sex. In sensitivity analyses, we further considered potential confounders of the BMI-health relationship that were possibly influenced by education or HDI: living in urban areas, unemployment, marital status, and health behaviors such as smoking, alcohol use, and physical activity. Mediators Individual education was measured by the years of schooling (including higher education). Individual education was measured by the years of schooling (including higher education). Wang and Arah (2017), PeerJ, DOI 10.7717/peerj.3053 3/13 3/13 Figure 1 Graphical representation (solid lines) of pure direct effect of HDI on health (A) and total in- direct effect of HDI via education (B) when education is the mediator of interest (Scenario 1), and nat- ural indirect effect of HDI via BMI only (C) when BMI is the mediator of interest (Scenario 2). In sce- nario 1, the BMI path-specific effect was incorporated in the pure direct effect. Figure 1 Graphical representation (solid lines) of pure direct effect of HDI on health (A) and total in- direct effect of HDI via education (B) when education is the mediator of interest (Scenario 1), and nat- ural indirect effect of HDI via BMI only (C) when BMI is the mediator of interest (Scenario 2). In sce- nario 1, the BMI path-specific effect was incorporated in the pure direct effect. Body mass index (BMI) was defined as an individual’s self-reported weight (kg) divided by self-reported height squared (m2). We excluded participants with height less than 1.22 m (n = 1,174) or greater than 2.11 m (n = 27), and participants with a weight that was 3 SDs above (n = 821) or 2 SDs below (n = 382) the crude sample mean of 63.6 kg. We further excluded individuals with BMI less than 14 kg/m2 (n = 252). Conceptual framework We aimed to examine the path-specific effects under the following two scenarios where education (scenario 1) or BMI (scenario 2) was the mediator of interest, respectively. In scenario 1, the pure direct effect captured the impact of human development on individual health through pathways other than individual-level education (Fig. 1A) whereas the total indirect effect measured such impact through education (Fig. 1B). In examining the mediating role of BMI, a consequence of individual-level education, we further decomposed the pure direct effect of HDI on health into (1) the HDI effect through BMI but not education (i.e., the BMI-path-specific effect as presented in Fig. 1C), and (2) the natural direct effect of HDI on health through neither education nor BMI. Detailed assumptions used for effect identification can be found in Supplemental Information 1. Wang and Arah (2017), PeerJ, DOI 10.7717/peerj.3053 Statistical analysis We used appropriate descriptive statistics to summarize the characteristics of the participants by sex and WHO region. Under the assumptions of general consistency, conditional exchangeability (no-uncontrolled-confounding), and positivity (Vanderweele, Vansteelandt & Robins, 2014), we can estimate different types of effects via regression models. Effect definitions and their empirical expressions are listed in Table S1. We 4/13 Wang and Arah (2017), PeerJ, DOI 10.7717/peerj.3053 included an HDI squared term into the outcome regression model to capture the non- linearity of the effect of HDI on individual health. To account for the within-country clustering, we used PROC NLMIXED procedure to fit multilevel generalized linear models with country-specific random intercept for each model of health score, BMI, and education. The NLMIXED procedure fits nonlinear mixed models by maximizing an approximation to the likelihood integrated over the random effects, using adaptive Gaussian quadrature (default). Users have the flexibility to specify a conditional distribution for the data (given the random effects) to have either a standard form (normal, binomial, Poisson) or a general distribution that you code using SAS programming statements (SAS Institute Inc., 2008). We reported effect estimates and the corresponding 95% confidence intervals (95% CIs) for males and females separately. All analyses were conducted in SAS 9.4 (SAS Institute Inc., Cary, North Carolina, USA). Wang and Arah (2017), PeerJ, DOI 10.7717/peerj.3053 Sensitivity analysis For scenario 1, we relaxed the sample restriction criteria to include individuals with complete information on HDI, education, and health score (N = 148,679) and re-estimated the pure direct effect, total indirect effect and total effect. For scenario 2, we further examined the robustness of our results to the presence of intermediate confounders V (affected by HDI, education or both) of the BMI-health relationship including living in urban areas, marital status, unemployment, smoking, alcohol use, and physical inactivity. We used a g-estimation-like (or a substitution) method to create a confounding-free outcome variable where this new outcome variable was independent of V conditional on HDI, education, BMI, and other covariates (the set Z). Details can be found in the Supplemental Informations 1 and 2. Easte Medi 2,710 0.49 ( 42.9 ( 7.3 (5 23.9 ( 91.3 ( 2,051 0.51 ( 42.2 ( 4.8 (5 24.6 ( 86.9 ( d norm rvey 2 Easte Medi 2,710 0.49 ( 42.9 ( 7.3 (5 23.9 ( 91.3 ( 2,051 0.51 ( 42.2 ( 4.8 (5 24.6 ( 86.9 ( d norm RESULTS Male b (95% CI) Female b (95% CI) Scenario 1a Total effect 1.58 (−0.61, 3.77) 2.61 (−0.09, 5.32) Pure direct effect 1.32 (−0.87, 3.51) 2.18 (−0.52, 4.88) Total indirect effect 0.26 (0.17, 0.35) 0.44 (0.28, 0.59) Scenario 2b Natural indirect effect via BMI only 0.016 (−0.005, 0.037) −0.033 (−0.077, 0.011) Notes. aEducation is the mediator of interest. bBMI path-specific effect, part of the pure direct effect in Scenario 1, was further examined. Table 2 Effect estimate (95% Confidence Interval) for human development level (comparing 0.672– 0.572) on individual health, World Health Survey 2002–2004 (N = 109,448). Table 2 Effect estimate (95% Confidence Interval) for human development level (comparing 0.672– 0.572) on individual health, World Health Survey 2002–2004 (N = 109,448). Sex-specific mean differences in health score associated with a 0.1-unit increase in HDI at the median HDI level (comparing HDI = 0.672 to HDI = 0.572) or at multiple reference HDI levels are presented in Table 2 and Fig. 2 respectively. At the median HDI level of 0.572 in scenario 1, increase in HDI was positively associated with better health in both males (b = 1.58, 95% CI [−0.61–3.77]) and females (b = 2.61, 95% CI [−0.09–5.32]). The impact appeared to be mostly through pathways other than individual-level education (male: b = 1.32, 95% CI [−0.87–3.51]; female: b = 2.18, 95% CI [−0.52–4.88]). A small positive indirect effect of HDI via education was seen in both males (b = 0.26, 95% CI [0.17–0.35]) and females (b = 0.44, 95% CI [0.28–0.59]). The BMI-path-specific effect of HDI was almost null in both sexes (male: b = 0.016, 95% CI [−0.005–0.037]; female: b = −0.033, 95% CI [−0.077–0.011]). All types of effects of HDI on health depended on the reference value of HDI. An increase in HDI below a reference HDI level of 0.483 was negatively associated with good health (total effect at HDI of 0.232: b = −3.44, 95% CI [−6.39–−0.49] for males and b = −5.16, 95% CI [−9.24–−1.08] for females) but was positively associated with good health above this reference level (total effect at HDI of 0.747: b = 4.16, 95% CI [−0.33–8.66] for males and b = 6.62, 95% CI [0.85–12.38] for females). This pattern for the total effect was also seen in pure direct effect. RESULTS Among 195,808 participants aged 25 years or older, 109,448 (55.9%) participants from 42 countries had complete information on all covariates. Country-specific sample size and characteristics are presented in the (Table S2). We excluded participants from Burkina Faso, Chad, Comoros, Ethiopia, Herzegovina, and Georgia (N = 15,770) because of missing country’s HDI measures. Table 1 shows participant characteristics by sex and WHO region. Human development index varied by WHO region, with the European region having the highest mean HDI (0.71) and the South-East Asia region the lowest (0.44). Participants from the Europe region were oldest (mean age: 48.0 for males and 49.0 for females) and most educated (mean years of schooling: 12.4 for males and 12.2 for females), and had the highest mean BMI values (25.7 for males and 25.9 for females) but the lowest health score (86.4 for males and 82.2 for females). Except for the European region and the region of the Americas, participants from other WHO regions had similar sex-specific mean age, education years, BMI, and health scores. Overall, females were less educated and reported poorer health. A descriptive table (Table S3) on participants with complete information on HDI, education, and health score only can be found in Supplemental Information 1; it revealed similar patterns. 5/13 5/13 Wang and Arah (2017), PeerJ, DOI 10.7717/peerj.3053 vey 2002–2004 (N = 109,448). Eastern Mediterranean Europe South-East Asia Western Pacif 2,710 (5.4) 4,562 (9) 8,020 (15.9) 9,273 (18.4) 0.49 (0.08) 0.71 (0.02) 0.44 (0.10) 0.54 (0.09) 42.9 (13.6) 48.0 (15.1) 43.3 (13.4) 43.6 (13.2) 7.3 (5.9) 12.4 (3.4) 6.9 (4.8) 7.8 (4.3) 23.9 (3.9) 25.7 (3.3) 21.2 (3.3) 22.4 (3.4) 91.3 (12.6) 86.4 (13.2) 87.9 (14.1) 88.7 (13.5) 2,051 (3.5) 8,373 (14.2) 7,121 (12.1) 10,277 (17.4) 0.51 (0.08) 0.71 (0.02) 0.44 (0.11) 0.54 (0.09) 42.2 (13.8) 49.0 (15.3) 43.0 (13.6) 42.9 (13.3) 4.8 (5.7) 12.2 (3.5) 5.3 (4.8) 7.1 (4.6) 24.6 (4.5) 25.9 (4.5) 21.2 (3.7) 22.1 (3.9) 86.9 (15.4) 82.2 (14.9) 85.2 (15.8) 87.4 (13.9) normal height and weight status and those whose BMI < 14 kg/m2 (N = 109,448). Paci 8.4) 9) 2) ) 5) 17.4) 9) 3) ) 9) Paci .4) 9) 2) ) 5) 7.4) 9) 3) ) 9) Table 2 Effect estimate (95% Confidence Interval) for human development level (comparing 0.672– 0.572) on individual health, World Health Survey 2002–2004 (N = 109,448). Wang and Arah (2017), PeerJ, DOI 10.7717/peerj.3053 RESULTS S3), effect estimates for the natural indirect effect via BMI Figure 2 Pure direct effect (PDE; A), total indirect effect (TIE; B), and total effect (TE; C) of HDI on health when education is the mediator of interest in Scenario 1, and natural indirect effect via BMI only in Scenario 2 (D), obtained from multilevel regression analysis of the World Health Survey 2002–2004 (N = 109,448). Y axis represents mean difference in health score associated with a 0.1-unit increase in HDI. X axis represents selected reference HDI values within the range of the current sample. Figure 2 Pure direct effect (PDE; A), total indirect effect (TIE; B), and total effect (TE; C) of HDI on health when education is the mediator of interest in Scenario 1, and natural indirect effect via BMI only in Scenario 2 (D), obtained from multilevel regression analysis of the World Health Survey 2002–2004 (N = 109,448). Y axis represents mean difference in health score associated with a 0.1-unit increase in HDI. X axis represents selected reference HDI values within the range of the current sample. intermediate confounders (Fig. S3), effect estimates for the natural indirect effect via BMI only (Fig. S4) were similar to those from the main analyses. intermediate confounders (Fig. S3), effect estimates for the natural indirect effect via BMI only (Fig. S4) were similar to those from the main analyses. Wang and Arah (2017), PeerJ, DOI 10.7717/peerj.3053 RESULTS We found a small positive effect of HDI on health via education across reference HDI levels (b ranging from 0.24 to 0.29 for males and 0.40 to 0.49 for females) but not via pathways involving BMI only. As HDI increased, both mediated effects via education or via BMI only decreased slightly. The effect size among females was larger than that among males. Since the effect of HDI on health can be modeled by a quadratic regression model, in the (Fig. S1) we present an alternative graph depicting the HDI effect when comparing countries with different HDI to Ghana or China (with reference HDI of 0.502). The further the HDI value was from the reference HDI of 0.502, the larger the effect size was. Accordingly, the total effect and the pure direct effect of HDI on health were U-shaped when comparing HDI of different countries to the reference HDI value (of Ghana or China). Sensitivity analyses using a less restricted sample revealed similar point estimates but narrower confidence intervals (Table S4 and Fig. S2). Summary statistics for potential intermediate confounders are presented in the (Table S5). After accounting for the Wang and Arah (2017), PeerJ, DOI 10.7717/peerj.3053 7/13 Figure 2 Pure direct effect (PDE; A), total indirect effect (TIE; B), and total effect (TE; C) of HDI on health when education is the mediator of interest in Scenario 1, and natural indirect effect via BMI only in Scenario 2 (D), obtained from multilevel regression analysis of the World Health Survey 2002–2004 (N = 109,448). Y axis represents mean difference in health score associated with a 0.1-unit increase in HDI. X axis represents selected reference HDI values within the range of the current sample. intermediate confounders (Fig. S3), effect estimates for the natural indirect effect via BMI only (Fig. S4) were similar to those from the main analyses. Figure 2 Pure direct effect (PDE; A), total indirect effect (TIE; B), and total effect (TE; C) of HDI on health when education is the mediator of interest in Scenario 1, and natural indirect effect via BMI only in Scenario 2 (D), obtained from multilevel regression analysis of the World Health Survey 2002–2004 (N = 109,448). Y axis represents mean difference in health score associated with a 0.1-unit increase in HDI. X axis represents selected reference HDI values within the range of the current sample. intermediate confounders (Fig. DISCUSSION This study examined the impact of national human development on individual health and possible pathways via education and BMI, using large population data. We found that the HDI effect on health depended on the reference HDI level: the total effect and pure direct effect of HDI were negative at low HDI level but became positive at the higher level of HDI. The HDI effect on individual-level health was mainly through pathways other than education and BMI. The impact of HDI on health was greater for females than for males. Our study found that the effect of country-level human development on individual health was non-linear. This indicated that at the lower end of the HDI spectrum, people in countries with higher human development level tended to have poorer health whereas, at the upper end of this spectrum, higher human development level predicted better individual health. Around the median level of HDI, people from countries that were 0.1-unit apart in HDI tended to have similar health status. The HDI assessed how well countries are doing in three dimensions: health, education, and living standards (United Nations Development Programme, 2013). The overall health status and educational achievement for residents in a country will tend to grow hand in hand with the country’s adult literacy rate and combined gross enrollment ratio for primary, secondary and tertiary education. Yet improved living standards, as captured by gross national income per capita, may have affected people’s Wang and Arah (2017), PeerJ, DOI 10.7717/peerj.3053 8/13 8/13 health in a complex way: the net health benefit may depend on the interrelationships of various factors including but not limited to improved social infrastructure, public health interventions, technology, and lifestyle changes due to urbanization and globalization. Low- and middle-income countries continue to experience epidemiological transitions from infectious diseases to chronic NCDs. NCDs continue to play increasingly important roles in personal health, especially for older people. In addition, the fast economic growth in some countries may be at the expense of a positive health-supporting environment. All these aspects contributed to the complicated relationship between development and health. Our result may reflect investments in and prioritization of different aspects in overall health improvement as these countries went through various stages of economic and social development. DISCUSSION Such investment may touch all aspects of health and health care: building roads for improving access to care, providing effective treatment for HIV/AIDS, tuberculosis, and malaria, promoting vaccination and proper use of antibiotics, strengthening primary care and preventive interventions and so on. Though all these investments and efforts may have greatly increased life expectancy at birth at the country level, they may differentially correlate with the self-reported health measures that were used in the current study and were heavily dependent on the presence of chronic disease. There can be lessons that we can learn from countries at the lower end of the human development spectrum. People from these countries achieved similar or even higher health status compared to people from countries with median health development level, possibly attributable to their targeted health investments and not being affected by the nutrition transition when the study was conducted. Past work has documented the presence of effect modification of the relationship between education and health by HDI (Van der Kooi et al., 2013), and of the relationship between obesity and health outcomes by education (Schafer & Ferraro, 2011; Wang, Stronks & Arah, 2014). Incorporating such interactions is crucial for the present study. Despite the relatively small effect size here, we found a consistent positive indirect effect of HDI on health through increased education at each reference HDI level. In countries with lower human development levels, the education channel offset part of the negative impact of increased development on health. In countries with higher human development levels, education contributed to the overall positive impact of development on health. However, the pathways through BMI but not education did not appear to play an important role in transmitting the impact of development on health. Possible explanations included that the impact of development via BMI also went through education and other upstream variables, or that the mediating role of BMI depended on HDI and education in a complex way that the current model cannot capture. Future studies could explore pathways through other factors such as neighborhood environment, health care quality, and access to care (Adler & Stewart, 2010). Though women suffer more from ill health than men do (Idler, 2003; Moussavi et al., 2007), our study suggests that women could potentially have more gains in health than men could as a country achieves higher human development. Wang and Arah (2017), PeerJ, DOI 10.7717/peerj.3053 CONCLUSIONS Our study provides an update on the effect of development on health and further examined the underlying pathways through education and BMI. The impact of development on health depended on the level of national development. The effect of national human development on individual health was mainly through pathways other than education and BMI, and it differed by sex. Country-level development may harm or benefit human health, which has future implications for human development (Bloom & Canning, 2003; Jamison, 2006). Characterizing the impact of human development on health can help shed light on how policymakers could translate economic growth into health for all. DISCUSSION At a low human development level, national human development had a more negative impact for women than for men. The Wang and Arah (2017), PeerJ, DOI 10.7717/peerj.3053 9/13 underlying mechanisms for the sex difference are still unclear. Women have been found to be at a higher risk of depression (Patel, 2001). In the current study sample, depression status is highly predictive of poor health (Moussavi et al., 2007). It may be that the level of human development correlates with mental health services and women from countries with good mental health care (usually countries with higher human development) gain more in terms of health compared to their male counterparts. The standardized methodology used in the WHS allowed for pooled analyses from middle- and low-income countries across continents. The use of causal mediation analysis enabled us to incorporate nonlinear relationships such as previously documented interactions between HDI and education (Van der Kooi et al., 2013), and between education and BMI (Wang, Stronks & Arah, 2014) in affecting individual health outcomes. We conducted sensitivity analysis to check the robustness of the BMI-path-specific effect against the presence of confounders of the BMI-health relationship affected by HDI or education. We used linear mixed models to account for the multilevel structure of the data and adjusted for both contextual and individual confounders. Several methodological limitations need to be addressed. We imposed temporality assumptions on the cross-sectional WHS data: individual-level education preceded BMI measurement, which preceded health status at the time of the survey. This is a reasonable assumption since formal education usually happens before age 25. However, there is still a slight chance that middle-age health status affected the cumulative education years. BMI value tends to be stable at middle age, but we cannot rule out the possibility of reverse causation for BMI-health relationship. Despite sensitivity analyses against intermediate confounding, our result for the BMI-path-specific effect of HDI could still be subject to uncontrolled confounding between BMI and health. There could be measurement error in BMI that was created based on self-report height and weight. Wang and Arah (2017), PeerJ, DOI 10.7717/peerj.3053 Competing Interests The authors declare there are no competing interests. The authors declare there are no competing interests. Supplemental Information Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.3053#supplemental-information. Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.3053#supplemental-information. Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ upplemental information for this article can be found online at http://dx.doi.org/10.7717/ eerj.3053#supplemental-information. peerj.3053#supplemental-information. peerj.3053#supplemental-information. Author Contributions • Aolin Wang conceived and designed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper. • Aolin Wang conceived and designed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper. • Onyebuchi A. Arah conceived and designed the experiments, contributed reagents/ma- terials/analysis tools, wrote the paper, reviewed drafts of the paper, supervised AW’s research and training. Funding Funding Aolin Wang was supported by the Dissertation Year Fellowship from the University of California, Los Angeles and a doctoral scholarship from the Chinese Scholarship Council (CSC). Onyebuchi A. Arah was partially supported by grant 1R01HD072296 from the 10/13 Wang and Arah (2017), PeerJ, DOI 10.7717/peerj.3053 Eunice Kennedy Shriver National Institute of Child Health and Human Development. There was no additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Wang and Arah (2017), PeerJ, DOI 10.7717/peerj.3053 Data Availability The following information was supplied regarding data availability: The World Health Survey data are publicly available at http://apps.who.int/healthinfo/ systems/surveydata/index.php/catalog (registration required). The SAS program for the main analyses is available at: https://figshare.com/s/8a03ec6afc06ae81b6cb. Grant Disclosures The following grant information was disclosed by the authors: The following grant information was disclosed by the authors: University of California, Los Angeles. University of California, Los Angeles. Chinese Scholarship Council. Chinese Scholarship Council. Eunice Kennedy Shriver National Institute of Child Health: 1R01HD072296. Eunice Kennedy Shriver National Institute of Child Health: 1R01HD072296. Competing Interests Wang and Arah (2017), PeerJ, DOI 10.7717/peerj.3053 REFERENCES Adler NE, Stewart J. 2010. Health disparities across the lifespan: meaning, methods, and mechanisms. Annals of the New York Academy of Sciences 1186:5–23 DOI 10.1111/j.1749-6632.2009.05337.x. Arah OA. 2009. On the relationship between individual and population health. Medicine, Health Care, and Philosophy 12:235–244 DOI 10.1007/s11019-008-9173-8. Bloom D, Canning D. 2003. Health as human capital and its impact on economic performance. Geneva Papers on Risk and Insurance—Issues and Practice 28:304–315 DOI 10.1111/1468-0440.00225. Hall P, Barnes L, Taylor R. 2009. Why is wealthier healthier? Perspectives on Europe 39(2):4–8. Wang and Arah (2017), PeerJ, DOI 10.7717/peerj.3053 Hawkes C, Chopra M, Friel S. 2009. Globalization, trade, and the nutrition transition. In: Labonté R, Schrecker T, Packer C, Runnels V, eds. Globalization and health: pathways, evidence and policy. New York: Routledge, 235–262. Idler EL. 2003. Discussion: gender differences in self-rated health, in mortality, and in the relationship between the two. The Gerontologist 43:372–375 James WPT, Jackson-leach R, Mhurchu CN, Kalamara E, Shayeghi M, Rigby NJ. 2004. Overweight and obesity (high body mass index). In: Comparative quantification of health risks: global and regional burden of disease attributable to selected major risk factors. Geneva: World Health Organization, 497–596. Jamison DT. 2006. Investing in health. 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Rehkopf DH, Dow WH, Rosero-Bixby L. 2010. Differences in the association of car- diovascular risk factors with education: a comparison of Costa Rica (CRELES) and the USA (NHANES). Journal of Epidemiology and Community Health 64:821–828 DOI 10.1136/jech.2009.086926. SAS Institute Inc. 2008. SAS/STAT(R) 9.3 user’s guide: the NLMIXED procedure. Available at https://support.sas.com/documentation/cdl/en/statug/63962/HTML/ default/viewer.htm#statug_nlmixed_sect002.htm. Schafer MH, Ferraro KF. 2011. Distal and variably proximal causes: education, obesity, and health. Social Science & Medicine(1982) 73:1340–1348 Wang and Arah (2017), PeerJ, DOI 10.7717/peerj.3053 DOI 10.1016/j.socscimed.2011.08.010. Schlipköter U, Flahault A. 2010. Communicable diseases: achievements and challenges for public health. Public Health Reviews 32:90–119. United Nations Development Programme. 2013. Human development report 2013: the rise of the south: human progress in a diverse world. Available at http://hdr.undp. org/sites/default/files/reports/14/hdr2013_en_complete.pdf (accessed on 16 July 2015). Wang and Arah (2017), PeerJ, DOI 10.7717/peerj.3053 12/13 Üstün TB, Chatterji S, Villanueva M, Bendib L, Çelik C, Sadana R, Valentine N, Ortiz J, Tandon A, Salomon J, Cao Y, Jun XW, Özaltin E, Mathers C, Murray CJL. 2003. WHO multi-country survey study on Health responsiveness 2000–2001. In: Health systems performance assessment: debates, methods and empericism. Geneva: World Health Organization. Van der Kooi ALF, Stronks K, Thompson CA, DerSarkissian M, Arah OA. 2013. The modifying influence of country development on the effect of individual educational attainment on self-rated health. American Journal of Public Health 103:e49–e54 DOI 10.2105/AJPH.2013.301593. Vanderweele TJ, Vansteelandt S, Robins JM. 2014. Effect decomposition in the presence of an exposure-induced mediator-outcome confounder. Epidemiology 25:300–306 DOI 10.1097/EDE.0000000000000034. Wang A, Arah OA. 2015. Body mass index and poor self-rated health in 49 low-income and middle-income countries, by sex, 2002–2004. Preventing Chronic Disease 12:150070 DOI 10.5888/pcd12.150070. Wang A, Stronks K, Arah OA. 2014. Global educational disparities in the associations between body mass index and diabetes mellitus in 49 low-income and middle- income countries. Journal of Epidemiology and Community Health 68:705–711 DOI 10.1136/jech-2013-203200. World Bank. 1993. World development report 1993: investing in health. The World Bank DOI 10.1596/0-1952-0890-0. WHO. 2013. World health survey. Available at http://www.who.int/healthinfo/survey/en/ (accessed on 09 June). Zimmerman EB, Woolf SH, Haley A. 2015. Understanding the relationship between education and health: a review of the evidence and an examination of community perspectives. Rockville: Agency for Healthcare Research and Quality. 13/13 13/13
https://openalex.org/W4379233669	https://zenodo.org/record/8001402/files/2.48.pdf	Quechua	null	AYRIM TUR BALIQLARNING YUQUMSIZ KASALLIKLARI-GIPOVITAMINOZLAR	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	1,173	INTERNATIONAL SCIENTIFIC-PRACTICAL CONFERENCE INTERNATIONAL SCIENTIFIC-PRACTICAL CONFERENCE INTERNATIONAL SCIENTIFIC-PRACTICAL CONFERENCE INTERNATIONAL SCIENTIFIC-PRACTICAL CONFERENCE Gipovitaminozning har bir turi o'ziga xos xususiyatlarga ega: A-gipovitaminoz: shox pardaning xiralashishi, ko'z to'qimalarida va suzgichlar tagida qon ketishi, tana rangining o'zgarishi, ko'zlarning shishishi, zaif o'sishi. C-gipovitaminozi: buyrak, jigar, ichak va mushaklarda qon ketishi, ko'zning shikastlanishi, skolyoz (umurtqa pog'onasi egriligi). Terida, kaudal, qorin va ko'krak qanotlari yaqinida o'smalar hosil bo'lib, vaqt o'tishi bilan o'tib ketadi; tendonlar va xaftaga shakllanishining buzilishi umurtqa pog'onasining egriligiga va hatto sinishiga, gill filamentlarining xaftaga filiform bo'linishiga siljishiga olib keladi. B1-gipovitaminoz: tananing muvozanati buziladi, rangi qorayadi, tomchilar kuzatiladi, zaif o'sish, dorsal va ko'krak qanotlarining falajlanishi, mushaklar atrofiyasi; baliq ovqatdan bosh tortadi va konvulsiyalarda (konvulsiyalarda) o'ladi. B2-gipovitaminoz: burun teshigi va gill qopqog'ida qon ketishi, fotofobi, ko'z irislarining anormal ranglanishi, ishtahaning yomonlashishi, terining qorayishi, o'sishni to'liq to'xtatish. B3-gipovitaminoz: ishtahani yo'qotish, yomon o'sish, oshqozon va yo'g'on ichakning shishishi, tartibsiz harakatlar, zaiflik. B4-gipovitaminoz: yomon o'sish, oziq-ovqatning yomon so'rilishi, buyrak va ichaklarda qon ketishi. B4-gipovitaminoz: yomon o'sish, oziq-ovqatning yomon so'rilishi, buyrak va ichaklarda qon ketishi. B4-gipovitaminoz: yomon o'sish, oziq-ovqatning yomon so'rilishi, buyrak va ichaklarda qon ketishi. B5-gipovitaminoz: zaif o'sish, letargiya, gillalarning yopishishi, ishtahaning yo'qolishi, hujayra metabolizmining buzilishi, terida qon ketishi. B5-gipovitaminoz: zaif o'sish, letargiya, gillalarning yopishishi, ishtahaning yo'qolishi, hujayra metabolizmining buzilishi, terida qon ketishi. B6-gipovitaminozi: asabiy buzilishlar, tutilishlar, ishtahaning yo'qolishi, zaif o'sish, tomchilar, konvulsiyalar, gill qopqoqlarining kamayishi, tez va qattiq nafas olish. B6-gipovitaminozi: asabiy buzilishlar, tutilishlar, ishtahaning yo'qolishi, zaif o'sish, tomchilar, konvulsiyalar, gill qopqoqlarining kamayishi, tez va qattiq nafas olish. B6-gipovitaminozi: asabiy buzilishlar, tutilishlar, ishtahaning yo'qolishi, zaif o'sish, tomchilar, konvulsiyalar, gill qopqoqlarining kamayishi, tez va qattiq nafas olish. B8 (inositol) - gipovitaminoz: yomon o'sish, shishiradi, terining shikastlanishi, oshqozon-ichak traktida ovqat hazm qilish vaqtining ko'payishi. B8 (inositol) - gipovitaminoz: yomon o'sish, shishiradi, terining shikastlanishi, oshqozon-ichak traktida ovqat hazm qilish vaqtining ko'payishi. B8 (inositol) - gipovitaminoz: yomon o'sish, shishiradi, terining shikastlanishi, oshqozon-ichak traktida ovqat hazm qilish vaqtining ko'payishi. B12-gipovitaminoz: yomon ishtaha va o'sish, anemiya, tana rangining qorayishi. E-gipovitaminoz: ishtahaning pasayishi, o'sishning sekinlashishi, jigar shikastlanishi belgilari bilan o'limning ko'payishi. O'sishni to'xtatish, jinsiy bezlarning rivojlanishi, A- gipovitaminozning rivojlanishi, anemiya. Zebra baliqlarida bu vitaminning etishmasligi miyaga zarar etkazishi mumkin. E-gipovitaminoz: ishtahaning pasayishi, o'sishning sekinlashishi, jigar shikastlanishi belgilari bilan o'limning ko'payishi. O'sishni to'xtatish, jinsiy bezlarning rivojlanishi, A- gipovitaminozning rivojlanishi, anemiya. Zebra baliqlarida bu vitaminning etishmasligi miyaga zarar etkazishi mumkin. A-gipervitaminoz: ko'zning yallig'lanishi, asab kasalliklari, kaudal finning eroziyasi va o'limi, shuningdek C-gipovitaminozi. A-gipervitaminoz: ko'zning yallig'lanishi, asab kasalliklari, kaudal finning eroziyasi va o'limi, shuningdek C-gipovitaminozi. A-gipervitaminoz: ko'zning yallig'lanishi, asab kasalliklari, kaudal finning eroziyasi va o'limi, shuningdek C-gipovitaminozi. ACTUAL ISSUES OF AGRICULTURAL DEVELOPMENT: PROBLEMS AND SOLUTIONS JUNE 6-7, 2023 Gipovitaminozning har bir turi o'ziga xos xususiyatlarga ega: ACTUAL ISSUES OF AGRICULTURAL DEVELOPMENT: PROBLEMS AND SOLUTIONS JUNE 6-7, 2023 Yaqubov Muzaffar oʻqituvchi Anotatsiya. Respublikamiz fermer xo’jaliklariga ayrim turdagi baliqlarning yuqumsiz kassaliklari tobora oshib bormoqda, baliqchilikda gipovetaminozlar tuli darajada iqtisodiy zararlar keltirib chiqaryapti. A, B, C, D, E Vetemin yetishmaslig oqibatida baliqlar nobit bo’lishini oldini olish uchun qo’shimcha ratsionda oziqlantirish kerak. Vitaminlarning mikdori kunlik extiyoji 1 kg ozuqa xisobida quyidagicha mg xisobida: vitamin A 20-2000 IE, tiamin -0,15 mg, riboflavin 0,2-10 mg, inozitol 200-300 mg, vitamin S-20 mg, vitamin E 70-100 mg, nikotin tislotasi – 0,1-50 mg, xolin – 1500-2000, pirodoksin 5-10mg tashkil qiladi. Ularni saqlash oziqlantirish gigiyenasi va texnologiyasiga bog’liq. Kalit so’zlar: baliq, fermer xo’jaliklari, optimal sharoit, saqlash, oziqlantirish gigiyenasi, texnologik omillar, mikroiqlim. Kirish. Oʻzbekiston Respublikasi Prezidentining «Baliqchilik tormoǵini boshqarish tizimini takomillashtirish chora-tadbirlari tug’risida» qanun 2017-yil 1-aydagi PQ-2939-son qororini ijro etishga qaratilgan bulib, unda “2017 -2021 yillarda baliqchilik tormog’ini jamlanma rivojlantirish chora-tadbirlari dasturi» va «2017-2021 yillarda baliqchilik sohasini rivojlantirishning maqsadli parametrlari» tasdiqlandi. Yukoridagi qarordan kelib chiqib baliqchilik xujaliklarida baliqlardagi kasallik turlari jumladan Gipovitaminozlarni yani yuqimsiz kasalliklarini urganish dolzarb muammolar qatoriga kiradi. Bu bir guruh yuqimsiz kasalliklar bo’lib, turli xil fiziologik holatning izdan chiqishi va patologoanatomik o’zgarishlar bilan xarakterlanib organizmda turli xil vitaminlarning etishmasligi oqibatida kelib chiqadi. Bunda turli xil vitaminlarning organizmga ozuqa orqali etarli mikdorda kelib tushmasligi yoki organizmda etarli mikdorda sintez qilmasligi oqibatida kelib chiqadi. Klinik belgilari. Ko’pchilik gipovitaminoz kasalliklarida ayrim klinik belgilar umumiydir: jumladan, ishtaxani yo’qolishi, xolsizlanish, kam harakatlanish yoki kislorodga bo’lgan extiyojni ortishi, o’sish-rivojlanishdan orqada qolish, turli yuqumli kasalliklarga beriluvchanligini oshishi va baliqlarning ommaviy ravishda nobud bo’lishi. Oldini olish va qarshi ko’rashish tadbirlari. Gipovitaminozlarning oldini olishda universal vosita – bu baliqlarning ratsioniga tirik tabiiy vitaminga boy ozuqalarni kiritish bilan amalga oshiriladi. Baliqchilik tarmog’ini intensifikatsiyalashda bunday imkoniyatlar chegaranganligi sababli ularning ozuqasiga turli xil vitaminli qo’shimchalar, premikslar, drojlar, baliq moyi, ko’k massa, hayvonlarning jigari, ko’k o’t va boshqalar kiritiladi. Baliqlarni sun’iy oziqlantirishda gipovitaminozlarning oldini olishda ularning ratsioni tarkibi, tuyimligi va biologik aktiv moddalar bilan balanslangan bo’lishi kerak. Baliqlarda vitaminlarning mikdori kunlik extiyoji 1 kg ozuqa xisobida quyidagicha mg xisobida: vitamin A 20-2000 IE, tiamin -0,15 mg, riboflavin 0,2-10 mg, inozitol 200-300 mg, vitamin S-20 mg, vitamin E 70-100 mg, nikotin tislotasi – 0,1- 50 mg, xolin – 1500-2000, pirodoksin 5-10mg. Diagnoz. Gipovitaminoz kasalliklarida aniq diagnoz qo’yish juda ham mushkil, chunki ularning klinik belgilari bir-biriga juda o’xshash, Shuning uchun ham ozuqani sifat ko’rsatgichi buyicha, ozuqa ratsionining analiz qilish, klinik belgilar va patanatomik o’zgarishlar asosida diagnoz qo’yiladi. Ayrim yuqumli kasalliklardan farq qilish kerak. 264 INTERNATIONAL SCIENTIFIC-PRACTICAL CONFERENCE INTERNATIONAL SCIENTIFIC PRACTICAL CONFERENCE ACTUAL ISSUES OF AGRICULTURAL DEVELOPMENT: PROBLEMS AND SOLUTIONS JUNE 6-7, 2023 ACTUAL ISSUES OF AGRICULTURAL DEVELOPMENT: PROBLEMS AND SOLUTIONS JUNE 6-7, 2023 Oldini olish: Uy hayvonlari do'konlari hozirda baliq uchun mikroelementlar va vitaminlarni o'z ichiga olgan tayyor tijorat baliqlarining keng assortimentini taklif qiladi. Vitaminlarning etishmasligi monoton va "bo'sh" oziq-ovqat bilan oziqlanadigan baliqlarda kuzatiladi (quritilgan dafniya, sikloplar va gammarus). Gipovitaminozning har bir turi o'ziga xos xususiyatlarga ega: D-gipervitaminozi: suyaklardagi kaltsiy va fosfor miqdorining pasayishi, bu ularning mo'rtligini oshiradi. D-gipervitaminozi: suyaklardagi kaltsiy va fosfor miqdorining pasayishi, bu ularning mo'rtligini oshiradi. H-gipervitaminozi: ishtahaning yo'qolishi, yomon o'sish, anemiya, terining shikastlanishi, mushak distrofiyasi. H-gipervitaminozi: ishtahaning yo'qolishi, yomon o'sish, anemiya, terining shikastlanishi, mushak distrofiyasi. Folik kislota etishmovchiligi: zaif o'sish, qora rang, letargiya, kamqonlik va mo'rt d Yuqoridagi fikr-muloxazalar va xulosalardan kelib chiqqan holda shuni takidlash joizki Respublikada baliqlarini rivojlantirishga barcha shart sharoitlar mavjuddir. Avvalom bor soxani rivojlandirishda baliqlarning oziqlanish ratsioniga zoogigienaliq sharoitiga va veterinariya xizmatlariga etibor qaratish lozim. Yuqoridagi fikr-muloxazalar va xulosalardan kelib chiqqan holda shuni takidlash joizki Respublikada baliqlarini rivojlantirishga barcha shart sharoitlar mavjuddir. Avvalom bor soxani rivojlandirishda baliqlarning oziqlanish ratsioniga zoogigienaliq sharoitiga va veterinariya xizmatlariga etibor qaratish lozim. Dovolash. tubifex, qon qurti, coretra, daphnia, sikloplar, amfipodlar, yomg'ir qurtlari, past baholi baliq, xom jigar. Bu ovqatni ertalab berish yaxshidir, kechqurun. C va B guruhi vitaminlari tanadan tezda chiqariladi. Yog'da eriydigan A, D, E vitaminlari tanadan asta-sekin chiqariladi; dozaga rioya qilmaslik natijasida ular zaharlanishga olib kelishi mumkin - gipervitaminoz. 265 265 265 REFERENCES berdiev P.S. va boshkalar «Balikchilik va balik kasalliklari», Samarkand, 2008 1. Haqberdiev P.S. va boshkalar «Balikchilik va balik kasalliklari», Samarkand, 2008 2. Haqberdiev P.S., Qurbonov F.I., Qarshieva V.SH. «Baliq va asalari kasalliklari» O’quv qullanma, Toshkent, 2016 simchik V,A., E.F. Sadovnikova ”Bolezni rib i pchel” uchebnoe posobie, Minsk, 2017 3. Gerasimchik V,A., E.F. Sadovnikova ”Bolezni rib i pchel” uchebnoe posobie, Minsk, 2017 4. Osetrov V.S. (pod redaksiey) «Bolezni rib». Spravochnik, Moskva Agropromizdat, 1989 4. Osetrov V.S. (pod redaksiey) «Bolezni rib». Spravochnik, Moskva Agropromizdat, 1989 5. Shishkov V.P. «Veterinarniy ensiklopedicheskiy slovar», Moskva, Izdatelstvo «Sovetskaya ensiklopediya», 1981. 266
https://openalex.org/W2000394633	http://www.plosone.org/article/fetchObject.action?representation%3DPDF%26uri%3Dinfo%3Adoi/10.1371/journal.pone.0110040	English	null	Neem Leaf Glycoprotein Prophylaxis Transduces Immune Dependent Stop Signal for Tumor Angiogenic Switch within Tumor Microenvironment	PloS one	2,014	cc-by	11,350	Saptak Banerjee, Tithi Ghosh, Subhasis Barik, Arnab Das, Sarbari Ghosh, Avishek Bhuniya, Anamika Bose., Rathindranath Baral. Department of Immunoregulation and Immunodiagnostics, Chittaranjan National Cancer Institute (CNCI), Kolkata, India Abstract We have reported that prophylactic as well as therapeutic administration of neem leaf glycoprotein (NLGP) induces significant restriction of solid tumor growth in mice. Here, we investigate whether the effect of such pretreatment (25mg/ mice; weekly, 4 times) benefits regulation of tumor angiogenesis, an obligate factor for tumor progression. We show that NLGP pretreatment results in vascular normalization in melanoma and carcinoma bearing mice along with downregulation of CD31, VEGF and VEGFR2. NLGP pretreatment facilitates profound infiltration of CD8+ T cells within tumor parenchyma, which subsequently regulates VEGF-VEGFR2 signaling in CD31+ vascular endothelial cells to prevent aberrant neovascularization. Pericyte stabilization, VEGF dependent inhibition of VEC proliferation and subsequent vascular normalization are also experienced. Studies in immune compromised mice confirmed that these vascular and intratumoral changes in angiogenic profile are dependent upon active adoptive immunity particularly those mediated by CD8+ T cells. Accumulated evidences suggest that NLGP regulated immunomodulation is active in tumor growth restriction and normalization of tumor angiogenesis as well, thereby, signifying its clinical translation. Citation: Banerjee S, Ghosh T, Barik S, Das A, Ghosh S, et al. (2014) Neem Leaf Glycoprotein Prophylaxis Transduces Immune Dependent Stop Signal for Tumor Angiogenic Switch within Tumor Microenvironment. PLoS ONE 9(11): e110040. doi:10.1371/journal.pone.0110040 Editor: Rupesh Chaturvedi, Jawaharlal Nehru University, India Editor: Rupesh Chaturvedi, Jawaharlal Nehru University, India Received June 26, 2014; Accepted September 12, 2014; Published November 12, 2014 Copyright: 2014 Banerjee et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: The work was partially supported by Council of Scientific and Industrial Research, New Delhi (grant no. 09/030(0050)/2008-EMR-I to S. Banerjee, grant no. 09/030(0063)/2011-EMRI to S. Barik and grant SRA, Scientists’ Pool Scheme No: 8463A to A. Bose). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. erests: The authors have declared that no competing interests exis . These authors contributed equally to this work. November 2014 \| Volume 9 \| Issue 11 \| e110040 Results Several recent studies have demonstrated that angiogenesis and suppressed cell-mediated immunity interdependently play central role in the pathogenesis of malignant disease facilitating tumor growth [33,34]. As NLGP prophylaxis reciprocally regulate tumor immune surveillance and angiogenesis to restrict murine tumor growth, next, we used two types of mice models (drug-induced immunosuppressive mice and immunocompromised athymic nude mice) to assess the immune involvement in NLGP mediated angiogenic modulation. As shown in (Figure 3A.1, A.2), mice were divided into three groups and two groups were injected with NLGP prophylactically, while one group was retained as control. Between these two NLGP pretreated mice groups, one group received immunosuppressant cyclosporine before EC tumor challenge as mentioned in ‘Materials and Methods’. Analysis of their angiogenic profile revealed that NLGP pretreatment caused significant normalization of tumor vasculature than control group, as demonstrated in Figure 3A.1 However, in cyclosporine group, NLGP pretreatment failed to normalize angiogenesis, and so they showed prominent with prominent dilated and fragile blood vessels (Figure 3A.1, A.2-D.1, D.2). Observed results clearly indicated that NLGP modulates angiogenesis or vascular normal- ization by activating immune system. Immune Dependent Normalization of Angiogenesis by NLGP Immune Dependent Normalization of Angiogenesis by NLGP Therefore, in the present study, we prophylactically applied NLGP in murine carcinoma and melanoma bearing mice to boost antitumor immune responses and subsequently analyzed the mode of NLGP counteraction on the tumor angiogenesis. We report that NLGP pretreatment associated immune-stimulation, particularly CD8+ T cell activation, regulates the balance between pro- and anti-angiogenic molecules to induce vascular normalization without affecting normal physiological angiogenesis. were found detached from endothelial cells (Figure 2C.2), rendering the blood vessels thick, dilated and leaky. Therefore, obtained results clearly suggest that NLGP mediated alteration of pericytes’ nature and/or that attachment along the vessel wall is intimately associated with observed vascular normalization within tumor. NLGP prophylaxis prevents tumor angiogenesis and normalizes tumor vasculature ‘Dormant’ tumor requires both angiogenic switch and immune escape to proceed towards malignancy [7,9]. As prophylaxis with neem leaf preparation, precursor of NLGP, previously reported to be associated with significant immune-mediated tumor growth restriction [16,29], here, we intended to study how NLGP prophylaxis regulates pathological tumor angiogenesis. Consistent with our previous results prophylactic NLGP administration (46) significantly restricts Ehrlich’s carcinoma and B16 melanoma tumor growth (Figure 1a). Repeated investigations confirmed 4 immunizations with NLGP are required for optimum immune activation [15–18,27–29]. Angiogenic profiles were studied in mice after establishment of tumor (in between day 21 to 32) (Figure 1A.1 and A.2) and visual observations suggested a significant decrease in heavy, very thick, thick blood vessels, while thin blood vessels were retained substantially in NLGP pretreated carcinoma and melanoma tumor bearing mice group compared to PBS controls (Figure 1A.3). Additionally, histological analysis of tumor sections demonstrated less number of blood vessels with more regularized pattern in NLGP pretreated tumors than PBS mice. This regularized pattern of blood vessels is further evidenced by downregulation of CD31, a marker of VECs (Figure 1B). Correlating angiogenic profile with tumor volume revealed that normalized angiogenesis associated with NLGP prophylaxis represents restricted tumor growth, whereas, chaotic angiogenesis is correlated well with bigger tumor volume (Table 1; Figure 1C). Therefore, these data furnish evidences that NLGP can normalize tumor vasculature by decreasing only the thick and ‘tortured’ blood vessels while retaining the more compact thin blood vessels within tumor to maintain the optimum interstitial pressure and vaccine mediated immune benefits. Corroborately, in a separate set of experiments, tumor growth and associated angiogenesis were studied in three groups of NLGP-cyclosporin treated EC bearing mice. Two such groups of mice adoptively received non-adherent immune cells from either NLGP or PBS immunized normal mice (Figure 3A.3). Mice from all the 3 groups were sacrificed after tumor reached a considerable volume (1500 mm3 to 2000 mm3 approximately) and their angiogenic profiles were analyzed (Figure 3B.3–D.3). Enhanced angiogenesis related to the cyclosporine mediated immunosup- pression in NLGP pretreated mice was observed to be almost normalized due to adoptive transfer of splenic immune cells from NLGP treated mice (Figure 3B.3–D.3). These findings might further conclude that NLGP mediated normalization of angio- genesis is immune dependent. November 2014 \| Volume 9 \| Issue 11 \| e110040 Introduction migration of vascular endothelial cells (VECs), thereby, causing neovascularization. These results in aberrant tumor vasculature associated with distorted and enlarged vessels, increased perme- ability, irregular blood flow and micro-hemorrhages [10,12,13]. Therefore, in recent years different works have shown that, for optimum immune-mediated tumor destruction, normalization of tumor vasculature is preferred over complete blockade of tumor angiogenesis [14]. In 2000, Hanahan and Weinberg described angiogenesis as one of the most important hallmark criterion for cancer [1]. In spite of fundamental role of angiogenesis in fetal development and in many physiological conditions like wound healing [2,3] tumors exploit it to promote blood vessel growth and fuel a tumor’s transition from benign to a malignant state [4,5]. Likewise, these malignant transformations need evasion from immune destruction, which has been included recently, in 2011, as another important hallmark of cancer growth [6]. Angiogenesis and immune evasion, these two apparently parallel cancer-intrinsic phenomenon actu- ally possess bidirectional link and convergely promote malignant growth, metastasis and ultimately regulate therapeutic outcome [7]. In cancer, immune system can regulate angiogenesis with both pro- and anti-angiogenic activities [8,9]. Angiogenic molecules by differentially regulating immune system help in the development of sustained immunosuppressive mechanisms within tumor microen- vironment (TME) [10,11]. This immunosuppressive mechanism may promote angiogenesis and tumor growth and inhibits infiltration and homing of activated immune cells within TME. Promoted angiogenesis then deregulates the proliferation and Neem leaf glycoprotein (NLGP), a nontoxic immunomodulator reported previously have significant murine tumor growth restricting potential in prophylactic [15,16] as well as therapeutic [17,18] settings. NLGP facilitates anti-tumor activity by modulat- ing both systemic and local immunity including: i) suppression of regulatory T cells [19], ii) activation of effector NK, NKT and T cells [20,21], iii) modulation of antigen presenting cells by maturating dendritic cells (DCs) towards DC1 phenotype [22,23] and macrophages [24], iv) regulation of cytokine-chemokine balance [25,26] and v) preventing anergy and exhaustion of effector T cells [17,18]. Recently in two consecutive studies, we have reported that therapeutic effectiveness of NLGP is associated with profound tumor infiltration of CD8+ T cells [27] and normalization of tumor-immune-microenvironment [27,28]. November 2014 \| Volume 9 \| Issue 11 \| e110040 PLOS ONE \| www.plosone.org 1 NLGP mediated vascular normalization is associated with down regulation of CD31, VEGF and VEGFR2 To further validate the influence of NLGP-conditioned immune system to restrain tumor angiogenesis, immune-compromised athymic nude mice were pretreated with NLGP before tumor (EC) inoculation. However, NLGP pretreated nude mice failed to normalize tumor angiogenesis (Figure 3A.4–D.4) and adoptive transfer of syngenic non-adherent immune cells or isolated T cells of NLGP immunized normal mice showed tumor growth restriction and vascular normalization or inhibition in angiogenesis in comparison to PBS treated or only NLGP treated group (data not shown). As VEGF-VEGFR2 signaling axis represents the key event in promoting tumor angiogenesis [30–32], expression of these molecules along with other pro-angiogenic molecules were next analyzed in NLGP pretreated carcinoma and melanoma bearing mice. Evidences obtained from RT-PCR (Figure 2A.1 and A.2)and Western Blot analyses demonstrated downregulation of VEGF, VEGFR2, CD31 in tumor from NLGP pretreated mice (Figure 2B.1 and B.2), in comparison to tumor obtained from PBS treated mice. Consistently, immunohistochemical analysis also revealed the significant decrease in expression level of VEGF and its receptor, VEGFR2, along with endothelial cell associated protein CD31 in harvested tumors with NLGP pre-therapy (Figure 2C.1). However, minimal decrease in VEGFR1 and NG2 level (Figure 2C.1 and C.2) was observed with similar treatment. Dual immunofluorescence staining of CD31 with NG2 (Figure 2C.3) indicated optimum and close pericyte coverage over VECs that may help in stabilization of blood vessels in NLGP treated mice group while, in PBS treated mice NG2+ pericytes Tumors harvested from mice with different compromised immune systems with either pretreatment with NLGP or adoptive transfer of immune cells were analyzed for the expression status of VEGF, VEGFR2 and CD31, as we earlier found that NLGP downregulates the elevated expression levels of these molecules during tumor growth. Immunosuppression, either by means of cyclosporine treatment or in nude mice, abrogated the NLGP mediated downregulation of VEGF, VEGFR2 and CD31 PLOS ONE \| www.plosone.org November 2014 \| Volume 9 \| Issue 11 \| e110040 2 Immune Dependent Normalization of Angiogenesis by NLGP Figure 1. NLGP normalizes tumor vasculature. Swiss and C57BL/6 mice were pretreated with NLGP (25mg) once a week for four weeks in total followed by inoculation of EC (16106 cells/mice) and B16 melanoma cells (26105 cells/mice) subcutaneously. A.1. Tumor growth curve till day 27 is presented. p,0.01. A.2. Mice were sacrificed and their angiogenic profile was studied and presented in photographs and bar diagrams (Mean6SD of pixel values). p,0.01. A.3. Mean index of tumor angiogenesis. Mean is presented in Figure 1C November 2014 \| Volume 9 \| Issue 11 \| e110040 Immune Dependent Normalization of Angiogenesis by NLGP Thus, NLGP may normalize angiogenesis by restricting availability of pro-angiogenic molecules. (Figure 3E). Thus, NLGP may normalize angiogenesis by restricting availability of pro-angiogenic molecules. (Figure 3E). Thus, NLGP may normalize angiogenesis by restricting availability of pro-angiogenic molecules. VEGFR2 and CD31 was again upregulated in mice group with CD8+ T cell depletion, as indicated by RT-PCR (Figure 4D.1, D.2) and immunohistochemical (Figure 4D.3) analysis. NLGP mediated vascular normalization is not due to the T cell mediated apoptosis of CD31+ cells rather due to unavailability of VEGF Since, above experiments clearly indicate that immune system has a regulatory role in NLGP driven normalization of tumor angiogenesis, next, we studied the histological sections from carcinoma and melanoma tumors of NLGP and PBS pretreated mice. Prominent infiltration of immune cells was noticed in tumors from NLGP pretreated mice (Figure 4A.1). Flow cytometric analysis of cells from PBMC of either PBS or NLGP treated tumor bearing mice revealed increased CD8+ T cells in NLGP treated mice group (Figure 4A.2 and A.3). To validate the role of CD8+ T cells (if any), such cells were depleted using specific antibody, as work plan is schematically presented in Figure 4B.1. CD8+ T cell depletion was confirmed flow cytometrically (Figure 4B.2). Since CD8+ T cells are found to be responsible for anti- angiogenic effect of NLGP-conditioned immune system (prefer- entially achieved by downregulation of VEGR2+CD31+ endothe- lial cells), initially we analyzed direct cytolytic effect of CD8+ T cells on CD31+ VECs. CD31+ cells were flow sorted from solid B16F10 tumors (Figure S1) and exposed to CD8+ T cells from NLGP pretreated tumor bearing mice. Co-incubation study suggested that CD8+ T cells are unable to exert any direct cytolytic effect in vitro against tumor derived CD31+ endothelial cells (Figure 5A). Furthermore, analysis of pro-apoptotic and apoptotic/necrotic VECs using Annexin V and PI respectively within tumor revealed that NLGP pretreatment has no effect on early apoptosis or late apoptosis/necrosis of CD31+ cells (Figure 5B.1, B.2). As we observed significant enhancement of CD8+ T cells within tumors from NLGP pretreated mice, we wanted to decipher the contributing role of these effector cells in NLGP mediated normalization of angiogenesis by in vivo depletion of CD8+ T cells as described in Materials and Methods and Fig. 4B.1. When mice were sacrificed on day 28 post B16F10 tumor inoculation, analysis of angiogenesis at that time point clearly suggested a predominant role for CD8+ T cells in NLGP driven immune- mediated vascular normalization, since CD8+ T cell depletion completely abolished anti-angiogenic potential of NLGP pretreat- ment (Figure 4C). NLGP mediated downregulation of VEGF, Analysing these two above mentioned results and considering the importance of VEGF as rate limiting factor for uncontrolled VEC proliferation and survival (necessary for neovascularization) next we assessed in situ VEGF concentration and its influence in NLGP mediated vascular normalization. Immune Dependent Normalization of Angiogenesis by NLGP Immune Dependent Normalization of Angiogenesis by NLGP Figure 2. NLGP normalizes tumor microenvironment by downregulating expression of VEGF, VEGFR2 and CD31. B16 melanoma tumors (100 mm3) harvested from either PBS or NLGP pretreated C57BL/6 mice representing tumor microenvironment were used for different analysis. A.1. Representative presentation of mRNA expression levels of vegf, vegfr1, vegfr2, cd31 by RT-PCR analysis (n = 3). A.2. Densitometric analysis of three individual observations is presented with Mean 6 SD. p,0.05; p,0.01. B.1. Another portion (100 mg) of tumors was lysed by freeze-thaw cycles in PBS used for Western Blotting to check the expression of various angiogenic proteins. Representative presentation of expression levels of different molecules as mentioned by Western blot analysis (n = 3) is shown. B.2. Densitometric analysis of three individual observations and Mean 6 SD are presented. p,0.05; *p,0.01. C.1. Immunohistochemistry with monoclonal antibodies, specific for VEGF, VEGFR1, VEGFR2 and CD31 and C.2. NG2 were detected on tumor sections. Arrows showed the pericyte coverage on endothelial cell lining on blood vessels. D. Fluorescence tagged monoclonal antibodies, specific for NG2+ (green) and CD31+ (Red) cells were used to study tumor vasculature. Nuclear staining was performed by DAPI. doi:10 1371/journal pone 0110040 g002 Figure 2. NLGP normalizes tumor microenvironment by downregulating expression of VEGF, VEGFR2 and CD31. B16 melanoma tumors (100 mm3) harvested from either PBS or NLGP pretreated C57BL/6 mice representing tumor microenvironment were used for different analysis. A.1. Representative presentation of mRNA expression levels of vegf, vegfr1, vegfr2, cd31 by RT-PCR analysis (n = 3). A.2. Densitometric analysis of three individual observations is presented with Mean 6 SD. p,0.05; *p,0.01. B.1. Another portion (100 mg) of tumors was lysed by freeze-thaw cycles in PBS used for Western Blotting to check the expression of various angiogenic proteins. Representative presentation of expression levels of different molecules as mentioned by Western blot analysis (n = 3) is shown. B.2. Densitometric analysis of three individual observations and Mean 6 SD are presented. p,0.05; *p,0.01. C.1. Immunohistochemistry with monoclonal antibodies, specific for VEGF, VEGFR1, VEGFR2 and CD31 and C.2. NG2 were detected on tumor sections. Arrows showed the pericyte coverage on endothelial cell lining on blood vessels. D. Fluorescence tagged monoclonal antibodies, specific for NG2+ (green) and CD31+ (Red) cells were used to study tumor vasculature. Nuclear staining was performed by DAPI. d i 10 1371/j l 0110040 002 y doi:10.1371/journal.pone.0110040.g002 (Figure 3E). NLGP mediated vascular normalization is associated with down regulation of CD31, VEGF and VEGFR2 Differentially dilated angiogenic vessels as shown in a representative figure (inset) were counted from NLGP and PBS treated mice and presented in bar diagram. p,0.05; p,0.001. B. Angiogenic blood vessels within tumors were studied by routine histology after H&E staining and CD31+ VECs were studied by immunofluorescence staining. Representative figures in each case are presented. C. Mean index of tumor angiogenesis is presented in bar diagram. p,0.001. doi:10.1371/journal.pone.0110040.g001 Figure 1. NLGP normalizes tumor vasculature. Swiss and C57BL/6 mice were pretreated with NLGP (25mg) once a week for four weeks in total followed by inoculation of EC (16106 cells/mice) and B16 melanoma cells (26105 cells/mice) subcutaneously. A.1. Tumor growth curve till day 27 is presented. p,0.01. A.2. Mice were sacrificed and their angiogenic profile was studied and presented in photographs and bar diagrams (Mean6SD of pixel values). p,0.01. A.3. Differentially dilated angiogenic vessels as shown in a representative figure (inset) were counted from NLGP and PBS treated mice and presented in bar diagram. p,0.05; p,0.001. B. Angiogenic blood vessels within tumors were studied by routine histology after H&E staining and CD31+ VECs were studied by immunofluorescence staining. Representative figures in each case are presented. C. Mean index of tumor angiogenesis is presented in bar diagram. p,0.001. doi:10.1371/journal.pone.0110040.g001 Table 1. MITA relating tumor volume and angiogenesis in NLGP pretreated mice. PBS NLGP Tumor Volume (in mm3) Angiogenesis (in raw score) Index for Tumor Angiogenesis Tumor Volume (in mm3) Angiogenesis (in raw score) Index for Tumor Angiogenesis 220 + (1) 220 126 + (1) 126 600 ++ (2) 1200 245 + (1) 245 907 ++ (2) 1814 665 ++ (2) 1330 2025 ++++ (4) 8100 1267 +++ (3) 3801 1152 +++ (3) 3456 445 + (1) 445 3240 ++++ (4) 12960 1568 ++ (2) 3136 5292 ++++ (4) 21168 1436 +++ (3) 4308 1352 +++ (3) 4056 1008 ++ (2) 2016 Mean 6622 1926 Mean index of tumor angiogenesis. Mean is presented in Figure 1C doi:10.1371/journal.pone.0110040.t001 November 2014 \| Volume 9 \| Issue 11 \| e110040 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 3 November 2014 \| Volume 9 \| Issue 11 \| e110040 NLGP mediated vascular normalization is not due to the T cell mediated apoptosis of CD31+ cells rather due to unavailability of VEGF Evidences obtained from ELISA clearly suggested that availability of VEGF is low in tumor November 2014 \| Volume 9 \| Issue 11 \| e110040 PLOS ONE \| www.plosone.org 4 Immune Dependent Normalization of Angiogenesis by NLGP Figure 3. NLGP mediated normalization of angiogenesis is absent in immunocompromised mice. Schematic presentation of NLGP prophylaxis in A.1. normal mice, A.2. and A.3. cyclosporine treated/immunocompromised and A.4. athymic nude mice. B.1.-B.4. Representative photographs of murine tumors with B.1. PBS, NLGP, B.2. NLGP-Cyclosporin pretreatment, B.3. NLGP pretreatment with adoptive transfer of immune cells and B.4. NLGP and PBS pretreatment (athymic nude mice). C.1.-C.4. Tumor growth curve presenting Mean 6 SD. p,0.001, *p,0.01, in comparison to NLGP group with other above mentioned group and D.1.-D.4. Angiogenic profile of mice with D.1. PBS, NLGP, D.2. NLGP- Cyclosporin pretreatment, D.3. NLGP pretreatment with adoptive transfer of immune cells and D.4. NLGP and PBS pretreatment (athymic nude mice). E. Immunohistochemical detection of VEGF, VEGFR2 and CD31 in tumor sections as mentioned in A.1–A.3. doi:10.1371/journal.pone.0110040.g003 Figure 3. NLGP mediated normalization of angiogenesis is absent in immunocompromised mice. Schematic presentation of NLGP prophylaxis in A.1. normal mice, A.2. and A.3. cyclosporine treated/immunocompromised and A.4. athymic nude mice. B.1.-B.4. Representative photographs of murine tumors with B.1. PBS, NLGP, B.2. NLGP-Cyclosporin pretreatment, B.3. NLGP pretreatment with adoptive transfer of immune cells and B.4. NLGP and PBS pretreatment (athymic nude mice). C.1.-C.4. Tumor growth curve presenting Mean 6 SD. p,0.001, *p,0.01, in comparison to NLGP group with other above mentioned group and D.1.-D.4. Angiogenic profile of mice with D.1. PBS, NLGP, D.2. NLGP- Cyclosporin pretreatment, D.3. NLGP pretreatment with adoptive transfer of immune cells and D.4. NLGP and PBS pretreatment (athymic nude mice). E. Immunohistochemical detection of VEGF, VEGFR2 and CD31 in tumor sections as mentioned in A.1–A.3. doi:10.1371/journal.pone.0110040.g003 comparison to those where PBS-PBMC was used (Figure 5E.1, E.2). Furthermore, flow-sorted CD31+ cells were in vitro exposed to supernatants from NLGP-PBMC-EC cell co-culture and proliferation (monitored by Ki67 staining) of CD31+ endothelial cells was monitored, where significantly less proliferation was noted due to supplementation of supernatant from NLGP-PBMC- EC cell co-culture (Figure 5E.3). Therefore, these results clearly pointed out the prominent role of VEGF downregulation caused after NLGP-instructed CD8+ T cell infiltration in the reciprocal regulation of VEC proliferation and vascular normalization. in situ from NLGP pretreated mice (Figure 5C), which might regulate CD31+ endothelial cell proliferation. NLGP mediated vascular normalization is not due to the T cell mediated apoptosis of CD31+ cells rather due to unavailability of VEGF To further verify this possibility, CD31+Ki67+ proliferating cells were analyzed in B16F10 tumor from NLGP pretreated mice by flow cytometric (Figure 5D.1) and immunofluorescence analysis (Figure 5D.2). Consistently, in vivo BrdU labeling and analysis of CD31+BrdU+ proliferating cells within harvested cells from tumors indicated significant lowering of proliferating cells in tumor bearing mice pretreated with NLGP (Figure 5D.3). The obtained results clearly suggested that the presence of proliferating endothelial cells is significantly less in NLGP pretreated mice than control mice. Again, peripheral blood mononuclear cells (PBMC) from EC bearing PBS and NLGP treated mice were co-cultured with tumor (EC) cells and culture supernatant was analyzed for VEGF and IFNc content. Analysis of such supernatants revealed low content of VEGF and high IFNc (in NLGP-PBMC-EC cell co-culture), in Immune Dependent Normalization of Angiogenesis by NLGP Immune Dependent Normalization of Angiogenesis by NLGP Figure 4. NLGP mediated normalization of tumor vasculature is dependent on CD8+ T cells. B16 melanoma tumors were harvested from both PBS and NLGP pretreated C57BL/6 mice. A.1. Immune infiltration within tumors from PBS and NLGP treated mice were assessed histologically (H&E). A.2. Status of CD8+ T cells in blood was assessed by flow cytometry. A.3. Bar diagram shows the status of CD8+ T cells within carcinoma and melanoma tumors. p,0.01. B.1. Schematic presentation of control and CD8+ T cell depletion in either PBS or NLGP pretreated mice. B.2. Status of CD8+ T cells in all four mice groups (PBS, NLGP, PBS-CD8 dep and NLGP CD8 dep) were presented with representative figures. C. Representative picture of tumors, tumor growth curve and angiogenesis of PBS and NLGP pretreated mice with or without CD8+ T cell depletion. p,0.001. D.1. Total RNA was isolated from tumors of PBS, NLGP, PBS-CD8 depleted group (PBS-CD8 dep) and NLGP-CD8 depleted mice (NLGP-CD8-Dep) group (n = 3 in each case) to analyze genes, like, cd31 and vegf at transcriptional level by RT-PCR and D.2. densitometric analysis of band intensities from 3 individual observations (Mean 6 SD) is presented. p,0.001, p,0.01. D.3. Immunohistochemical analysis of tumors obtained from PBS and NLGP pretreated mice with or without CD8 depletion were performed using monoclonal antibodies, specific for CD31, VEGF and VEGFR2. doi:10.1371/journal.pone.0110040.g004 Figure 4. NLGP mediated normalization of tumor vasculature is dependent on CD8+ T cells. B16 melanoma tumors were harvested from both PBS and NLGP pretreated C57BL/6 mice. A.1. Immune infiltration within tumors from PBS and NLGP treated mice were assessed histologically (H&E). A.2. Status of CD8+ T cells in blood was assessed by flow cytometry. A.3. Bar diagram shows the status of CD8+ T cells within carcinoma and melanoma tumors. p,0.01. B.1. Schematic presentation of control and CD8+ T cell depletion in either PBS or NLGP pretreated mice. B.2. Status of CD8+ T cells in all four mice groups (PBS, NLGP, PBS-CD8 dep and NLGP CD8 dep) were presented with representative figures. C. Representative picture of tumors, tumor growth curve and angiogenesis of PBS and NLGP pretreated mice with or without CD8+ T cell depletion. p,0.001. D.1. Immune Dependent Normalization of Angiogenesis by NLGP Total RNA was isolated from tumors of PBS, NLGP, PBS-CD8 depleted group (PBS-CD8 dep) and NLGP-CD8 depleted mice (NLGP-CD8-Dep) group (n = 3 in each case) to analyze genes, like, cd31 and vegf at transcriptional level by RT-PCR and D.2. densitometric analysis of band intensities from 3 individual observations (Mean 6 SD) is presented. p,0.001, p,0.01. D.3. Immunohistochemical analysis of tumors obtained from PBS and NLGP pretreated mice with or without CD8 depletion were performed using monoclonal antibodies, specific for CD31, VEGF and VEGFR2. doi:10.1371/journal.pone.0110040.g004 NLGP in prophylactic [15,16,29] and therapeutic [17,18] settings. This tumor growth restriction is strictly dependent upon modu- lation of host-tumor immune interaction [20,25], since NLGP is unable to induce direct tumor cell apoptosis [19,35]. Apart from the already discussed immunomodulation by NLGP in cancer [18,20,22], angiogenic normalization property of this molecule is described here for the first time. evaluate the impact of NLGP on cutaneous wound healing process (model of physiological angiogenesis). Mice were treated with NLGP or PBS as described earlier and 4 mm2 wounds were made in the skin of upper back. As shown in (Figure 6A) extent of wound closure in early days (between 4–7) was slightly higher in NLGP treated group, but in later stages (on day 9–14) healing was faster in PBS group and finally all wounds healed fully within a 2-week period. Furthermore, histological analysis revealed no observable differences in skin from both group of mice showing signature of wound healing (rapid epithelialization along with adipose layer were observed and hair follicles were formed) (Figure 6B). Immunofluorescence analysis on CD31+ and NG2+ cells on skin clearly showed no significant changes in wound healed skin from NLGP and PBS pretreated mice (Figure 6C). Therefore, our results clearly suggest that modulatory effect of NLGP on tumor angiogenesis does not hamper the normal physiological angiogenic process. evaluate the impact of NLGP on cutaneous wound healing process (model of physiological angiogenesis). Mice were treated with NLGP or PBS as described earlier and 4 mm2 wounds were made in the skin of upper back. As shown in (Figure 6A) extent of wound closure in early days (between 4–7) was slightly higher in NLGP treated group, but in later stages (on day 9–14) healing was faster in PBS group and finally all wounds healed fully within a 2-week period. Immune Dependent Normalization of Angiogenesis by NLGP Furthermore, histological analysis revealed no observable differences in skin from both group of mice showing signature of wound healing (rapid epithelialization along with adipose layer were observed and hair follicles were formed) (Figure 6B). Immunofluorescence analysis on CD31+ and NG2+ cells on skin clearly showed no significant changes in wound healed skin from NLGP and PBS pretreated mice (Figure 6C). Therefore, our results clearly suggest that modulatory effect of NLGP on tumor angiogenesis does not hamper the normal physiological angiogenic process. As results suggest, prophylactic administration of NLGP (with an interval of 7 days for 4 times) is inhibitory towards neo- vascularization initiated after tumor challenge and the anti- angiogenic effect is indeed associated with the decrease in heavily dilated (thick)/fragile as well as very thin blood vessels. However, the thin and compact vessels were observed to be retained, probably to facilitate the trafficking of immune effector cells. Based on the available data, we reasoned that NLGP pretreatment causes significant reduction in proliferating Ki67+CD31+ VECs within tumor and thereby reduces the tumor micro vessel density (an indicator of tumor angiogenesis). Since proliferation of CD31+ cells corroborates neovascularization [36,37], such reduction plays a great role in angiogenic normalization. Unlike VECs, NLGP do not decrease the number of NG2+ pericytes, but effectively preserve their maturity and coverage of these cells on blood November 2014 \| Volume 9 \| Issue 11 \| e110040 NLGP mediated vascular normalization has no adverse effect on normal wound healing process in mice Given the potential safety concerns of systemic toxicity for strategies targeting tumor angiogenesis, we also intended to November 2014 \| Volume 9 \| Issue 11 \| e110040 November 2014 \| Volume 9 \| Issue 11 \| e110040 PLOS ONE \| www.plosone.org 5 Immune Dependent Normalization of Angiogenesis by NLGP Discussion We have reported significant restriction of murine sarcoma, carcinoma and melanoma growth due to administration of NLP/ November 2014 \| Volume 9 \| Issue 11 \| e110040 PLOS ONE \| www.plosone.org 6 Immune Dependent Normalization of Angiogenesis by NLGP Figure 5. NLGP mediated vascular normalization is not due to CD8+ T cell mediated apoptosis of CD31+ cells. Mice were inoculated with B16 melanoma cells (26105 cells/mice) to grow tumor. After reaching the tumor volume to a considerable size (1372 mm3 approximately), tumor was harvested and CD31+ VECs were isolated by flow sorting. CD8+ T lymphocytes were isolated from NLGP or PBS pretreated (46) mice by MACS purification and CD8+ T cells were co-cultured with the CD31+ VECs. A. Cytotoxicity was measured by LDH release assay. NLGP pretreated C57BL/6 mice were inoculated with B16 melanoma cells as mentioned earlier. B.1. As tumor reached a considerable volume (1372 mm3 approximately), tumors were harvested, single cells prepared and stained with anti-CD31 antibody along with either Annexin V or Propidium Iodide (PI). Representative figures of Annexin-V and PI+ cells from CD31 gated population. B.2. Bar diagram showing % positive cells and MFI. C. Cell lysates prepared from carcinoma and melanoma tumors of PBS and NLGP pretreated mice were used to quantitate the level of VEGF by ELISA. Cytokines were measured as pg/mg of tissue 6 SE and Mean 6 SD of 3 individual observations are presented in bar diagram. p,0.01. D.1. Obtained cells as mentioned in B.1, were stained for CD31, along with Ki67. Gated CD31+ population was assessed for Ki67 staining using Flowjo software and presented in histogram. D.2. Cryo-sections obtained from tumors of NLGP and PBS pretreated mice were stained with fluorescence labeled anti- CD31 (red) and anti-Ki67 (green) antibodies, along with DAPI (blue). Representative figures from 3 separate sets of experiments are presented. D.3. Figure 5. NLGP mediated vascular normalization is not due to CD8+ T cell mediated apoptosis of CD31+ cells. Mice were inoculated with B16 melanoma cells (26105 cells/mice) to grow tumor. After reaching the tumor volume to a considerable size (1372 mm3 approximately), tumor was harvested and CD31+ VECs were isolated by flow sorting. CD8+ T lymphocytes were isolated from NLGP or PBS pretreated (46) mice by MACS purification and CD8+ T cells were co-cultured with the CD31+ VECs. A. Cytotoxicity was measured by LDH release assay. Discussion November 2014 \| Volume 9 \| Issue 11 \| e110040 PLOS ONE \| www.plosone.org 7 Immune Dependent Normalization of Angiogenesis by NLGP Immune Dependent Normalization of Angiogenesis by NLGP PBS and NLGP pretreated tumor bearing mice were injected with BrdU within tumor and sacrificed after 48 hours. Single cells were prepared to check BrdU staining after gating the CD31 population, as shown in a representative figure. E.1, E.2. PBMC were isolated from both PBS and NLGP treated tumor bearing mice and cultured with EC cells (26105 cells) for 24 hours and cell free supernatant were measured in pg/ml for VEGF (E.1) and IFNc (E.2) by ELISA. Cytokines were quantitated as pg/ml 6 SE. p,0.001, p,0.01. E.3. Flow sorted CD31+ ECs isolated from tumor microenvironment were cultured with the above mentioned supernatants (NLGP-PBMC+EC vs PBS-PBMC+EC) for 48 hours and assessed for the EC proliferation flow cytometrically after Ki67 labeling. doi:10.1371/journal.pone.0110040.g005 Accordingly, NLGP selectively targets the VEGF-VEGFR2 signaling in proliferating endothelial cells to create a ‘vascular normalization window’ that might facilitate a decrease in interstitial pressure, enhanced tumor oxygenation and ultimately leads to a better therapeutic response [39,40] in terms of restricted tumor growth [27]. vessels. Under NLGP influence, their tight association with VECs restore the vessel integrity and prevents leakiness. Therefore, by differentially regulating the two important stromal cell features, NLGP controls aberrant tumor vasculature. In context to host- antitumor benefits such results are encouraging as recent preclinical and clinical findings suggest that vascular normaliza- tion, rather than restriction of blood flow, is necessary to maintain the surge of effector immune cells and chemical regimens for cancer therapy [38]. vessels. Under NLGP influence, their tight association with VECs restore the vessel integrity and prevents leakiness. Therefore, by differentially regulating the two important stromal cell features, NLGP controls aberrant tumor vasculature. In context to host- antitumor benefits such results are encouraging as recent preclinical and clinical findings suggest that vascular normaliza- tion, rather than restriction of blood flow, is necessary to maintain the surge of effector immune cells and chemical regimens for cancer therapy [38]. g In view of our consistent observation on central involvement of immune system in NLGP-mediated eradication or prevention of murine tumor growth [17–20], the present study additionally evaluated the involvement of NLGP-instructed immune-modula- tion in controlling tumor-angiogenesis. Discussion Interestingly, we observed a significant abolition of NLGP mediated both anti-angiogenic and anti-tumor effect in cyclosporine [45,46] treated mice having prominent immunosuppression. However, adoptive transfer of immune cells from mice with NLGP therapy again restores both anti-angiogenic and tumor growth restricting effects of NLGP. Analysing these data, we speculated that NLGP-driven immune activation might be involved in anti-angiogenic process. To further validate our hypothesis, we used immunocompromised athymic nude mice and here also NLGP prophylaxis was unable to prevent neovascularization as well as tumor growth. Next, we directly Evidences are accumulated from present study suggesting the interference of NLGP in balancing tumor growth-supportive pro- and anti-angiogenic molecules. Several previous studies show that the VEGF family proteins, which signals through VEGFRs [39– 41] are major factors involved in tumor-induced angiogenesis. Tumor and residing stromal cells secrete several growth factors particularly VEGF [42,43] to stimulate VEGFR+ endothelial cell proliferation and in turn these cells provide the lining of newly formed blood vessels to supply nutrient to growing tumor [39]. Among all VEGFRs, VEGFR2 is mainly found on newly proliferating endothelial cells and targeting of VEGFR2 has been shown in some tumor models to reverse neo-vascularization [44]. Figure 6. NLGP mediated vascular normalization has no adverse effect on normal wound healing process. Swiss mice were pretreate with NLGP (25mg) and PBS once in a week for four weeks and 4 mm3 wound was made on the back of both groups of mice. A. Diameter of wound was measured every two days and percentages of wound closure were calculated and data presented as Mean 6 SD of 6 individual observations. B Histological sections of wound beds were stained with H&E and assessed microscopically. Representative figures are presented. C. Cryo-sections wound beds were stained with fluorescence labeled anti-CD31 (red) and anti-NG2 (green) antibodies along with DAPI. doi:10.1371/journal.pone.0110040.g006 Figure 6. NLGP mediated vascular normalization has no adverse effect on normal wound healing process. Swiss mice were pretreated with NLGP (25mg) and PBS once in a week for four weeks and 4 mm3 wound was made on the back of both groups of mice. A. Diameter of wounds was measured every two days and percentages of wound closure were calculated and data presented as Mean 6 SD of 6 individual observations. B. Histological sections of wound beds were stained with H&E and assessed microscopically. Representative figures are presented. C. Discussion NLGP pretreated C57BL/6 mice were inoculated with B16 melanoma cells as mentioned earlier. B.1. As tumor reached a considerable volume (1372 mm3 approximately), tumors were harvested, single cells prepared and stained with anti-CD31 antibody along with either Annexin V or Propidium Iodide (PI). Representative figures of Annexin-V and PI+ cells from CD31 gated population. B.2. Bar diagram showing % positive cells and MFI. C. Cell lysates prepared from carcinoma and melanoma tumors of PBS and NLGP pretreated mice were used to quantitate the level of VEGF by ELISA. Cytokines were measured as pg/mg of tissue 6 SE and Mean 6 SD of 3 individual observations are presented in bar diagram p,0 01 D 1 Obtained cells as Figure 5. NLGP mediated vascular normalization is not due to CD8+ T cell mediated apoptosis of CD31+ cells. Mice were inoculated with B16 melanoma cells (26105 cells/mice) to grow tumor. After reaching the tumor volume to a considerable size (1372 mm3 approximately), tumor was harvested and CD31+ VECs were isolated by flow sorting. CD8+ T lymphocytes were isolated from NLGP or PBS pretreated (46) mice by MACS purification and CD8+ T cells were co-cultured with the CD31+ VECs. A. Cytotoxicity was measured by LDH release assay. NLGP pretreated C57BL/6 mice were inoculated with B16 melanoma cells as mentioned earlier. B.1. As tumor reached a considerable volume (1372 mm3 approximately), tumors were harvested, single cells prepared and stained with anti-CD31 antibody along with either Annexin V or Propidium Iodide (PI). Representative figures of Annexin-V and PI+ cells from CD31 gated population. B.2. Bar diagram showing % positive cells and MFI. C. Cell lysates prepared from carcinoma and melanoma tumors of PBS and NLGP pretreated mice were used to quantitate the level of VEGF by ELISA. Cytokines were measured as pg/mg of tissue 6 SE and Mean 6 SD of 3 individual observations are presented in bar diagram. *p,0.01. D.1. Obtained cells as mentioned in B.1, were stained for CD31, along with Ki67. Gated CD31+ population was assessed for Ki67 staining using Flowjo software and presented in histogram. D.2. Cryo-sections obtained from tumors of NLGP and PBS pretreated mice were stained with fluorescence labeled anti- CD31 (red) and anti-Ki67 (green) antibodies, along with DAPI (blue). Representative figures from 3 separate sets of experiments are presented. D.3. Mice and tumors p g Considering this important contribution of CD8+ T cells in NLGP mediated anti-angiogenesis, initially we assumed that CD8+ T cells might be directly involved in killing of CD31+ VECs. In several previous studies, it was demonstrated that VEGFR2- specific CTL can be directly involved in the killing of proliferating VECs [48]. Contrary to these reports in our system we do not observe any cytolytic activity of CD8+ T cells isolated from NLGP treated mice towards flow sorted CD31+ VECs. To solve this puzzle, we checked involvement of VEGF, for which sorted CD31+ VECs were incubated with supernatants from co-culture of PBMC from NLGP/PBS EC bearing mice and EC cells. Interestingly, CD31+ VECs proliferated less with NLGP- PBMC+EC cells culture supernatant (having comparatively high level of VEGF), which was again compensated with addition of recombinant VEGF. In vivo BrdU labeling study also suggests less number of CD31+BrdU+ proliferating cells within tumors from NLGP pretreated mice, where VEGF content is significantly less. Therefore, finally, we concluded that unavailability of VEGF might be the predominant rate limiting factor of reduced VEC growth and vascular normalization. Considering the infiltration and essential role of CD8+ T cells, we further assumed that, NLGP treatment may enhance DC migration to lymph node to prime CD8+ T cells, which eventually infiltrates tumor parenchyma to kill tumor cells (that serves as one of the prime source for VEGF). It could also be possible that infiltrated CD8+ T cells produce IFNc and/or infiltrated DC produce IL-12 and either of these immunomodulatory cytokines possess anti-angiogenic effects by altering pro-angiogenic mediators [29,49]. Interestingly, our previous studies suggested the effectiveness of NLGP to influence CD8+ T cells and DC to produce IFNc and IL-12 respectively [20–22]. On the other-hand, in a separate in vitro study, we observed that NLGP can directly modulate B16 melanoma tumor cells by reducing HIF1a and VEGF in normoxic as well in hypoxic condition (unpublished observation). Female C57BL/6 and Swiss mice (Age: 4–6 weeks; Body weight: 24–27 g) were obtained from the National Centre for Laboratory Animal Sciences (NCLAS), Hyderabad and Institu- tional Animal Care and Maintenance Department, Chittaranjan National Cancer Institute (CNCI), Kolkata, India respectively and maintained under standard laboratory conditions. Immuno- compromised athymic nude mice (4–6 weeks old) were purchased from NCLAS, Hyderabad and maintained in a specific pathogen free facility. Immune Dependent Normalization of Angiogenesis by NLGP Immune Dependent Normalization of Angiogenesis by NLGP focussed on the contribution of CD8+ effector T cells, since NLGP selectively increases the trafficking of these effector cells into tumor parenchyma and therapeutic NLGP mediated tumor growth restriction is abrogated completely in CD8+ T cell depleted mice [17,18]. However, infiltrating CD8+ T cells often unable to show cytotoxic effect because, several tumor microenvironmental factors upregulate expression of inhibitory molecules like PD1 and CTLA4 on T cells to attenuate its effector functions and effector cytokine production [47]. In this context, modulatory effect of NLGP on TME is already reported [18,27,28]. More importantly, NLGP minimizes TME-induced anergy and exhaustion of CD8+ T cells, as observed by downregulation of anergy related molecules DGKa, Grail, EGRs etc. [18] and exhaustion related molecules TIM3, LAG3, PD1 and CTLA4 [17,19] to preserve the optimum functional efficacy of infiltrated CD8+ T cells. Likewise, in present study, NLGP administration followed by CD8+ T cell depletion was unable to produce anti-angiogenic effect, as dilated tortuous (thick) blood vessels are seen in these groups of animals. Moreover, NLGP mediated reduction of proliferating CD31+ endothelial cells or VEGF-VEGFR2 expression within tumor is abrogated in CD8+ T cell depleted tumor bearing mice. process, since immune-elimination is not obligate here. However, whether these infiltrated CD8+ T cells affect other VEGF producing cells or any other parallel cascade operational in this NLGP-instructed immune system-mediated anti-angiogenic pro- cess needs further evaluation. Considering the limitation of anti- angiogenic immunotherapy [50,51] combination therapy integrat- ing anti-angiogenic therapy along with immunotherapy or other conventional therapy was proposed by several groups [52,53]. In this context, NLGP treatment would be more promising in the field of cancer management because of its multidirectional fine tuning ability of tumor vasculature as well as of systemic/local immunity without any adverse physiological consequence. Ethics statement on mice experiments For maintenance and experimentation on mice, the relevant guidelines were followed and the Chittaranjan National Cancer Institute animal ethical committee approved the study. Mice and tumors Autoclaved dry pellet diet (Epic Laboratory Animal Feed, West Bengal Govt, Kalyani, India) and water were supplied ad libitum. Ehrlich Carcinoma (EC) was maintained by regular in vivo intraperitoneal passage in Swiss mice. B16F10 melanoma cell line was cultured in vitro in DMEM supplemented with 10% (v/v) FBS, 2 mM L-glutamine and penicillin-streptomycin (100mg/ml) at 37uC humidified conditions. To develop solid tumors in vivo, C57BL/6 and Swiss mice were inoculated subcutaneously (s.c.) in right hind leg quarters with B16F10 melanoma cells (26105) and EC cells (16106) respectively. Discussion Cryo-sections of wound beds were stained with fluorescence labeled anti-CD31 (red) and anti-NG2 (green) antibodies along with DAPI. doi:10.1371/journal.pone.0110040.g006 November 2014 \| Volume 9 \| Issue 11 \| e110040 PLOS ONE \| www.plosone.org 8 Angiogenesis study with blood vessels After attaining considerable size, tumors of either type were harvested from sacrificed mice. Portions of tumors were separately preserved for histology, immunohistochemistry, immunofluores- cence studies, western blot, flow cytometry and RT-PCR analysis. A piece of tumor was cleaned with PBS and chopped into small pieces and treated with mixture of collagenase (2mg/ml) and hyaluronidase (2mg/ml) and passed through the nylon mesh to prepare single cell suspensions. Tumor infiltrating lymphocytes (TILs) were then separated from tumor cells by differential gradient centrifugation at 2000 rpm for 30 minutes to analyze their proportions. To study the role of NLGP on tumor angiogenesis, both groups of NLGP and PBS pre-treated tumor bearing mice were sacrificed and skin were removed carefully from peritoneal region without disturbing the angiogenic vessels adjacent to tumors. These blood vessels were counted macroscopically using convex lens depending on the thickness of the blood vessels and categorized as heavy, very thick, thick, thin and very thin. Area of blood vessels was calculated using Photoshop software (Adobe Systems Incorporat- ed, San Jose, California, USA) and presented in Pixels. Extent of angiogenesis was categorized as 4 (++++), 3 (+++), 2 (++) and 1 (+). Tumor volume (in mm3) and extent of angiogenesis (in raw score) was multiplied to obtain an index for tumor angiogenesis. Mean of score from all mice was presented as Mean Index for Tumor and Angiogenesis (MITA). A piece of tumor was cleaned with PBS and chopped into small pieces and treated with mixture of collagenase (2mg/ml) and hyaluronidase (2mg/ml) and passed through the nylon mesh to prepare single cell suspensions. Tumor infiltrating lymphocytes (TILs) were then separated from tumor cells by differential gradient centrifugation at 2000 rpm for 30 minutes to analyze their proportions. Angiogenesis in immunocompromised mice To study the role of immune system in tumor angiogenesis, Swiss mice were divided into three groups (n = 3) and two groups received NLGP immunization as said before, keeping other group as PBS control. One of these NLGP treated mice group was immune suppressed by three consecutive peritoneal cyclosporine injections (15 mg/Kg) on day 13, 17 and 21. Mice of all groups were inoculated s.c. with EC (16106 cells) on day 24. Again, cyclosporine was injected on day 25 and 28. Similar study was conducted on immune compromised athymic nude mice, where one group received NLGP with another PBS control group. Following completion of immunization all mice received EC (16106 cells) s.c. and tumor growth and survivability were monitored biweekly. Pattern of angiogenesis was noted after sacrificing the mice. Immune Dependent Normalization of Angiogenesis by NLGP Immune Dependent Normalization of Angiogenesis by NLGP from MWG-Biotech AG (Bangalore, India). DAPI was purchased from Sigma, St. Louis, MO, USA. from MWG-Biotech AG (Bangalore, India). DAPI was purchased from Sigma, St. Louis, MO, USA. immunologically suppressed with consecutive peritoneal injection of cyclosporine as mentioned above. Then all three groups of mice were injected with 16106 viable EC cells. Following establishment of tumor (64 mm3 in average), first group was kept as control, second group received splenic immune cells (16107) i.v. through tail vein from PBS treated mice and third group of mice received same number of immune cells from 46NLGP (25mg/100ml/mice) immunized mice. When tumor reached a considerable volume (25 mm) in mice from PBS pretreated group, mice from both groups were sacrificed (on day 45) for comparative monitoring of angiogenesis, as described above. Identical experiment was performed in athymic nude mice with similar cell transfer from either PBS or NLGP injected mice. Histology, immunohistochemistry and immunofluorescence studies Tumors were fixed in 10% formalin for standard histological preparations and embedded in paraffin. Sections (4–5 mm) were prepared and stained with hematoxylin-eosin (H&E) according to standard protocol. Representative tumors were selected for immunohistochemical analysis. Fresh tumor tissues were also frozen for cryo-sectioning. Sections were immunostained for CD31, NG2, VEGF, VEGFR1 and VEGFR2 by the method described [27]. In some cases, tumor or skin sections were snap- frozen in OCT compound. Sections (5 mm) were prepared using cryostat (Leica, Germany), air-dried and fixed in ice-cold methanol for 20-30 min. The sections were blocked with 5% BSA solution and stained with different anti-mouse antibodies (CD31-PE, NG2- FITC, Ki67-FITC) by the method described earlier [27]. Angiogenesis in CD8+ T cell depleted mice Within several immune cells, to study the specific role of CD8+ T cells in the process of angiogenesis, C57BL/6 mice were divided in four groups (n = 4 in each group). Two groups of mice were immunized with NLGP as said before while other two groups of mice were injected with PBS. One NLGP and one PBS treated mice group were peritoneally injected with CD8 depleting antibody (100mg/50ml) on day -1, 6, 13, 20 and 27 as shown in Fig. 4BI. CD8+ T cell depletion status was monitored regularly by analyzing peripheral blood using flow cytometry. On day 24, B16F10 tumors (26105 cells/mice) were inoculated s.c. to the left flank of hind leg. Tumor volumes were monitored biweekly and on reaching a considerable size (25 mm) in mice from PBS pretreated group, mice from both groups (PBS and NLGP) were sacrificed for comparative monitoring in angiogenesis, as described above. NLGP injection and tumor growth restriction assay Two groups (n = 8, in each group) of either C57BL/6 or Swiss mice were immunized once weekly (25mg/100ml PBS/mice s.c.) for 4 weeks in total at left hind leg quarter with NLGP, keeping other group as PBS control. Immunized mice were inoculated with B16F10 and EC tumors respectively as mentioned above to develop solid tumors. Growth of solid tumor (in mm3) was monitored biweekly by caliper measurement using the formula: (width26length)/2. Survival of mice was noted regularly, till tumor size reached to 25 mm in either direction. Neem leaf glycoprotein Mature neem (Azadirachta indica) leaves of identical size and color (indicative of similar age), taken from a standard source were shed-dried and pulverized. Leaf powder was soaked overnight in phosphate buffered saline (PBS), pH 7.4 and supernatant was collected by centrifugation at 1500 rpm, termed neem leaf preparation (NLP) [16,54]. NLP was then extensively dialyzed against PBS and concentrated by Centricon Membrane Filter (Millipore Corporation, Bedford, MA, USA) with 10 KDa molecular weight cut off. Active component of this preparation is a glycoprotein, as characterized earlier [55] and designated as Neem leaf glycoprotein (NLGP). Protein concentration of NLGP solution was measured by Lowry’s method [56] using Folin’s Phenol reagent. Purity of the NLGP was confirmed by HPLC [22] before use. Antibodies and reagents RPMI 1640, DMEM, and FBS were purchased from Invitrogen (NY, USA). Lymphocyte separation media (LSM) was procured from MP Biomedicals, Irvine, CA, USA and HiMedia, Mumbai, India. Fluorescence conjugated different anti-mouse antibodies (CD4, CD8, Ki67) and purified CD31 were procured from either BD-Pharmingen or Biolegends (both in, San Diego, CA, USA). Fluorescence- or peroxidase-labeled secondary antibodies were procured from e-Biosciences (San Diego, CA, USA). Purified anti- mouse Foxp3, VEGF, VEGFR1, VEGFR2, were procured from Santa Cruz Biotech (California, USA). IFNc/IL-10 estimation kits (OptEIA, BD Biosciences, San Jose, CA, USA) 3,39,5,59-tetra- methylbenzidine (TMB) substrate solutions (for ELISA), CytoFix/ CytoPerm kit (for intracellular staining), AnnexinV-Propidium iodide apoptosis detection kit were obtained from BD Pharmin- gen, San Dieago, CA, USA. LDH release assay kit for cytotoxicity and BrdU kit for proliferation were obtained from Roche Diagnostics, Mannheim, Germany. Western lightining chemilu- minescence and immunoperoxidase color detection kit were purchased from Pierce (Rockford, IL, USA) and Vector labora- tories Inc (Burlingame, CA, USA) respectively. Optimal cutting temperature (OCT) compound was purchased from Sakura Finetek, Torrance, CA, USA. RT-PCR primers were procured In summary, our results suggest that NLGP prophylaxis educate whole immune system in such a way that after tumor challenge antigen presenting cells efficiently prime effector CD8+ T cells, which in due course kill tumor cells to reduce tumor promoting growth factor burden within TME. These reduced availability of growth factor especially VEGF subsequently impede the growth of endothelial cells without affecting the vessel integrity to maintain the proper trafficking of immune effector cells within TME. More importantly this strategy does not affect the normal wound healing November 2014 \| Volume 9 \| Issue 11 \| e110040 PLOS ONE \| www.plosone.org 9 Flow cytometric staining Single cell preparation from harvested tumors were labeled with 0.5ml (for 16106 cells) FITC or PE conjugated antibodies, specific for mouse CD8, CD31, Ki67 markers, and surface or intracellular flow cytometry was performed by the method described [17]. Immune Dependent Normalization of Angiogenesis by NLGP different primary antibodies, e.g., CD31, NG2, VEGF, VEGFR1 and VEGFR2, and the procedure followed the method as published [18]. different primary antibodies, e.g., CD31, NG2, VEGF, VEGFR1 and VEGFR2, and the procedure followed the method as published [18]. described [17,18]. Flow sorted CD31+ endothelial cells were co- cultured with CD8+ T cells in 1:10 dilution in serum free media and checked for cytotoxicity by LDH release assay. Cell-free supernatants were used to measure the level of released LDH using the formula: % Cytotoxicity = (Lysis from Effector-Target Mixture – Lysis from Effector only) – Spontaneous Lysis/ (Maximum Lysis – Spontaneous Lysis) 6100. RT-PCR analysis Total RNA was isolated from solid tumors (from PBS and NLGP treated mice) using the TRIZOL Reagent (Ambion, Austin, Texas, USA). The cDNA synthesis was carried out using RevertAid First Strand cDNA Synthesis Kit (Fermentas, K1622) following the manufacturer’s protocol and RT-PCR was carried out using gene-specific primers. The primer sequences of mouse CD31, VEGF, VEGFR1, VEGFR2, NG2 and b-Actin are described in the Table 2. PCR products were identified by image analysis software for gel documentation (Gel Doc XR+ system, BioRad) following electrophoresis on 1.5%–2% agarose gels and staining with ethidium bromide [18,27]. y p y In a separate experiment, splenic cells were purified from EC bearing PBS and NLGP treated mice and co-cultured with EC cells (10:1 ratio) for 24 hrs. Cell free culture supernatants were collected and assessed for VEGF and IFNc content by ELISA. Purified CD31+ endothelial cells were also incubated with such culture supernatants for 48 hrs and their proliferation was assessed by Ki67 staining by the method described [15]. In a parallel experiment NLGP (46) pretreated mice were injected with anti- mouse BrdU antibody injected in tumor as per manufacturer’s manual. After 48 hours of injection both groups of mice were sacrificed to harvest tumors and single cells were prepared as described before. Single cells were stained with anti-CD31 antibody and assessed flow cytometrically as per standard protocol. Flow sorting of CD31+ cells Single cell suspension obtained from harvested tumors was washed with PBS (containing 1% FBS) and passed through cell strainer. This cell pellet was stained with primary anti-mouse- CD31 antibody (30 minutes) and further tagged with appropriate FITC labeled secondary antibody and CD31+ cells were purified by Flow sorting with BD FACS Aria, San Jose, CA. Annexin V-PI staining for apoptosis Harvested tumors from PBS and NLGP treated mice were minced to make single cell suspension as mentioned before. Freshly collected single cells were mixed with 16 binding buffer (100 ml) and kept for 2 min at room temp. Then 5ml of each Annexin-V and PI were added and incubated for 15 mins and then finally analyzed by flow cytometry. Statistical analysis All results represent the average of separate in vivo and in vitro experiments. Number of experiments is mentioned in result section and legends to figures. In each experiment a value represents the mean of three individual observations and presented as mean 6 standard deviation (SD). Statistical significance was established by Student’s t-test using INSTAT 3 Software (GraphPad Software, Inc.), with differences between groups attaining a p value ,0.05 considered as significant. Wound healing assay Mice were pretreated with PBS and NLGP as described earlier. Mice were then anesthetized with peritoneal injection of 0.3 ml of 2-2-2-tribromoethanol (Avertin, Sigma, St. Louis, MO) and back portion was properly shaved to remove all fur and cleaned with 70% alcohol. Subsequently using dual puncher 4 mm2 wound was created on both side of their back and kept in sterile environment. After every 3 days interval wound closure was measured henceforth by a vernier caliper and the wound healing was analyzed. Finally on day 15 mice of both groups were sacrificed and their skins were fixed and sectioned using cryostat. Routine histology and immunofluorescence study was performed in skins. Western blot analysis Tumor lysate or cellular lysate (50 mg) were separated on 6– 20% SDS–polyacrylamide gel and transferred onto a PVDF membrane for Western Blotting. Incubation was performed for To reconfirm the same, NLGP immunized Swiss mice were similarly divided in three groups (n = 3, in each group) and PLOS ONE \| www.plosone.org November 2014 \| Volume 9 \| Issue 11 \| e110040 10 Immune Dependent Normalization of Angiogenesis by NLGP References Trials in Vaccinology, e-pub on Dec 6, 2013. 10. Terme M, Colussi O, Marcheteau E, Tanchot C, Tartour E, et al. (2012) Modulation of Immunity. Antiangiogenic Molecules in Cancer. Clin Dev Immunol 2012:1–8. 29. 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Name Primer sequences (59–39) Product size b-Actin-forward CAACCGTGAAAAGATGACCC 228 bp. b-Actin-reverse ATGAGGTAGTCTGTCAGGTC VEGFR2-forward ACAGACAGTGGGATGGTCC 271 bp VEGFR2-reverse AAACAGGAGGTGAGCGCAG VEGFR1-forward CCAACTACCTCAAGAGCAAAC 315 bp VEGFR1-reverse CCAGGTCCCGATGAATGCAC CD31-forword AGCCCACCAGAGACATGGAA 337 bp CD31-reverse CTGGCTCTGTTGGAGGCTGT VEGF-forward GGACCCTGGCTTTACTGCTG 201 bp VEGF-reverse CACAGGACGGCTTGAAGATG doi:10.1371/journal.pone.0110040.t002 Table 2. Primer sequences of various cytokine genes studied. Table 2. Primer sequences of various cytokine genes studied. PLOS ONE \| www.plosone.org 11 11 Immune Dependent Normalization of Angiogenesis by NLGP Acknowledgments We acknowledge Director, CNCI, Kolkata, India, for providing necessary facilities. Thanks to Dr. Subrata Laskar, Burdwan University, India, for his help in characterization of NLGP. Thanks to Dr. Abhijit Rakshit for providing experimental animals. We also extend our thanks to Dr. P. S. Dasgupta for his help and suggestions in angiogenic study. Figure S1 Purification of CD31+ cells by flow sorting. Solid B16 melanoma tumors were harvested from PBS treated C57BL/6 mice and single cell preparation was made. Cells were labeled with anti-CD31 antibody and positive cells were sorted in flow cytometer (BD FACS Aria). A. FSC/SSC plot of single cell population under study. B. Unstained cell population in FL1 (CD31)/FSC plot. C. CD31+ cells in FL1 (CD31)/FSC plot. D. Purified CD31+ vascular endothelial cells after flow sorting. Author Contributions Conceived and designed the experiments: A. Bose S. Banerjee RB. Performed the experiments: S. Banerjee S. Barik TG SG AD A. Bhuniya. Analyzed the data: S. Banerjee A. Bose TG S. Barik RB. Contributed reagents/materials/analysis tools: RB. Wrote the paper: A. Bose S. Banerjee RB. References 1. Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 7:57–70. T cell efficacy in patients with stage IIIB cervical cancer. 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https://openalex.org/W2781326787	https://uknowledge.uky.edu/cgi/viewcontent.cgi?article=1136&context=internalmedicine_facpub	English	null	A plasma telomeric cell-free DNA level in unaffected women with BRCA1 or/and BRCA2 mutations: a pilot study	Oncotarget	2,017	cc-by	6,656	See next page for additional authors F ll hi d ddi i l k h Part of the Cells Commons, Genetic Processes Commons, Genetic Structures Commons, Medical Genetics Commons, and the Oncology Commons Part of the Cells Commons, Genetic Processes Commons, Genetic Structures Common Genetics Commons, and the Oncology Commons Right click to open a feedback form in a new tab to let us know how this document benefits you. Right click to open a feedback form in a new tab to let us know how this document benefits you. University of Kentucky University of Kentucky UKnowledge UKnowledge University of Kentucky University of Kentucky UKnowledge UKnowledge Internal Medicine Faculty Publications Internal Medicine A Plasma Telomeric Cell-Free DNA Level in Unaffected Women A Plasma Telomeric Cell-Free DNA Level in Unaffected Women with BRCA1 Or/And BRCA2 Mutations: A Pilot Study with BRCA1 Or/And BRCA2 Mutations: A Pilot Study Shatovisha Dey Indiana University Natascia Marino Indiana University Kanokwan Bishop Indiana University Paige N. Dahlgren Indiana University Aditi Shendre Indiana University See next page for additional authors Follow this and additional works at: https://uknowledge.uky.edu/internalmedicine_facpub See next page for additional authors F ll thi d dditi l k t ht © 2018 Dey et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Repository Citation Repository Citation Dey, Shatovisha; Marino, Natascia; Bishop, Kanokwan; Dahlgren, Paige N.; Shendre, Aditi; Storniolo, Anna Maria; He, Chunyan; and Tanaka, Hiromi, "A Plasma Telomeric Cell-Free DNA Level in Unaffected Women with BRCA1 Or/And BRCA2 Mutations: A Pilot Study" (2018). Internal Medicine Faculty Publications. 136. https://uknowledge.uky.edu/internalmedicine_facpub/136 This Article is brought to you for free and open access by the Internal Medicine at UKnowledge. It has been accepted for inclusion in Internal Medicine Faculty Publications by an authorized administrator of UKnowledge. For more information, please contact UKnowledge@lsv.uky.edu. Digital Object Identifier (DOI) https://doi.org/10.18632/oncotarget.23767 Digital Object Identifier (DOI) https://doi.org/10.18632/oncotarget.23767 Notes/Citation Information Notes/Citation Information Published in Oncotarget, v. 9, no. 3, p. 4214-4222. A plasma telomeric cell-free DNA level in unaffected women with BRCA1 or/and BRCA2 mutations: a pilot study Research Paper This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Plasma cell-free DNA (cfDNA) is a small DNA fragment circulating in the bloodstream originating from both non-tumor- and tumor-derived cells. A previous study showed that a plasma telomeric cfDNA level decreases in sporadic breast cancer patients compared to controls. Tumor suppressor gene products including BRCA1 and BRCA2 (BRCA1&2) play an important role in telomere maintenance. In this study, we hypothesized that the plasma telomeric cfDNA level is associated with the mutation status of BRCA1&2 genes. To test this hypothesis, we performed plasma telomeric cfDNA quantitative PCR (qPCR)-based assays to compare 28 women carriers of the BRCA1&2 mutation with age-matched controls of 28 healthy women. The results showed that the plasma telomeric cfDNA level was lower in unaffected BRCA1&2 mutation carriers than in age-matched controls from non-obese women (BMI < 30), while there was no association between unaffected BRCA1&2 mutation carriers and age-matched controls in obese women (BMI > 30). Moreover, the plasma telomeric cfDNA level applied aptly to the Tyrer-Cuzick model in non-obese women. These findings suggest that circulating cfDNA may detect dysfunctional telomeres derived from cells with BRCA1&2 mutations and, therefore, its level is associated with breast cancer susceptibility. This pilot study warrants further investigation to elucidate the implication of plasma telomeric cfDNA levels in relation to cancer and obesity. Authors Authors Shatovisha Dey, Natascia Marino, Kanokwan Bishop, Paige N. Dahlgren, Aditi Shendre, Anna Maria Storniolo, Chunyan He, and Hiromi Tanaka This article is available at UKnowledge: https://uknowledge.uky.edu/internalmedicine_facpub/136 This article is available at UKnowledge: https://uknowledge.uky.edu/internalmedicine_facpub/136 A plasma telomeric cell-free DNA level in unaffected women with BRCA1 or/and BRCA2 mutations: a pilot study Shatovisha Dey1, Natascia Marino2,3, Kanokwan Bishop1, Paige N. Dahlgren1, Aditi Shendre4, Anna Maria Storniolo2,3, Chunyan He5 and Hiromi Tanaka1 1Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA 2Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA 3Susan G. Komen Tissue Bank at IU Simon Cancer Center, Indianapolis, IN, USA 4Department of Epidemiology, Richard M. Fairbanks School of Public Health, Indiana University, Indianapolis, IN, USA 5Department of Internal Medicine, College of Medicine, Markey Cancer Center, University of Kentucky, Lexington, KY, USA Correspondence to: Hiromi Tanaka, email: hirtanak@iu.edu Keywords: BRCA1; BRCA2; telomere; circulating cell-free DNA; qPCR Received: November 03, 2017 Accepted: December 21, 2017 Published: December 29, 2017 www.impactjournals.com/oncotarget/ Oncotarget, 2018, Vol. 9, (No. 3), pp: 4214-4222 Research Paper www.impactjournals.com/oncotarget/ A plasma telomeric cell-free DNA level in unaffected women with BRCA1 or/and BRCA2 mutations: a pilot study Research Paper with BRCA1 or/and BRCA2 mutations: a pilot study Shatovisha Dey1, Natascia Marino2,3, Kanokwan Bishop1, Paige N. Dahlgren1, Aditi Shendre4, Anna Maria Storniolo2,3, Chunyan He5 and Hiromi Tanaka1 1Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA 2Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA 3Susan G. Komen Tissue Bank at IU Simon Cancer Center, Indianapolis, IN, USA 4Department of Epidemiology, Richard M. Fairbanks School of Public Health, Indiana University, Indianapolis, IN, USA 5Department of Internal Medicine, College of Medicine, Markey Cancer Center, University of Kentucky, Lexington, KY, USA Correspondence to: Hiromi Tanaka, email: hirtanak@iu.edu Keywords: BRCA1; BRCA2; telomere; circulating cell-free DNA; qPCR Received: November 03, 2017 Accepted: December 21, 2017 Published: December 29, 2017 Copyright: Dey et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Shatovisha Dey1, Natascia Marino2,3, Kanokwan Bishop1, Paige N. Dahlgren1, Aditi Shendre4, Anna Maria Storniolo2,3, Chunyan He5 and Hiromi Tanaka1 Shatovisha Dey , Natascia Marino , Kanokwan Bishop , Paige N. Dahlgren , Ad Shendre4, Anna Maria Storniolo2,3, Chunyan He5 and Hiromi Tanaka1 1Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA 2Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA 4Department of Epidemiology, Richard M. Fairbanks School of Public Health, Indiana University, Indianapolis, IN, USA 5Department of Internal Medicine, College of Medicine, Markey Cancer Center, University of Kentucky, Lexington, KY, USA Correspondence to: Hiromi Tanaka email: hirtanak@iu edu Department of Epidemiology, Richard M. Fairbanks School of Public Health, Indiana University, Indianapolis, IN, USA 5Department of Internal Medicine, College of Medicine, Markey Cancer Center, University of Kentucky, Lexington, KY, USA Correspondence to: Hiromi Tanaka, email: hirtanak@iu.edu 5Department of Internal Medicine, College of Medicine, Markey Cancer Center, University of Kentucky, Lexington, KY, USA Correspondence to: Hiromi Tanaka, email: hirtanak@iu.edu Keywords: BRCA1; BRCA2; telomere; circulating cell-free DNA; qPCR Published: December 29, 2017 Received: November 03, 2017 Accepted: December 21, 2017 Copyright: Dey et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: Dey et al. INTRODUCTION Our analyses using paired t-test showed that leukocyte telomere length was significantly shorter in BRCA1&2 carriers than those in age-matched controls in the non-obese group (p = 0.0320, d = 0.666) but not in the obese group (p = 0.8203, d = 0.064) (Figure 1C and 1D). However, there was no association between the plasma telomeric cfDNA level (T/L copy ratio) and leukocyte telomere length (T/S ratio) in this study population (Supplementary Figure 1), supporting our previous findings that leukocyte DNA is not a major source of cfDNA production [5]. with no prior treatment, compared to non-cancer controls [5]. While a plasma centromeric cfDNA level was also measured in the study and the difference between breast cancer patients and controls was also statistically significant, the actual effect size was modest between breast cancer patients and controls [5]. These results indicate that changes in telomeric cfDNA levels could be more constructive as a biomarker than changes in centromeric cfDNA levels during breast carcinogenesis. Furthermore, we found that the plasma telomeric cfDNA level in women with advanced breast tumors (stages II–III) was lower than those at earlier stages of breast cancer (stages 0–I) [5]. These findings suggest that telomere dysfunction (e.g., rapid telomere shortening) in body tissues might accumulate during cancer development and be associated with cancer progression, resulting in dynamic changes in the plasma telomeric cfDNA level. Two major factors responsible for telomere length shortening are cellular replicative aging and genetics. BRCA1 and BRCA2 (BRCA1&2) are multifunctional proteins and maintain genomic stability by regulating DNA repair, transcription, and cell cycle in response to DNA damage [6, 7]. Several studies suggest that BRCA1&2 haploinsufficiency could involve defects in telomere maintenance [8–10]. Because BRCA1&2 gene mutation carriers have a high risk of developing early- onset breast and ovarian cancer during their lifetime, the aim of this study is to determine whether plasma telomeric cfDNA level is associated with the BRCA1&2 mutations. This pilot study provides important supportive evidence for ensuring further research to determine the potential value of the plasma telomeric cfDNA level in predicting cancer development and cancer risk. p [ ] To further evaluate our results with other method, we used the Tyrer-Cuzick model that is a well-studied, widely available model for predicting breast cancer risk [12, 13]. INTRODUCTION the assays require prior information on specific genetic or epigenetic alterations present in the original tumor lesion. In order to detect cfDNA alterations associated with breast cancer initiation (where the amount of affected DNA is significantly low), the assays require sufficient improvement in the sensitivity of the assay, making this research area quite challenging [3, 4]. Plasma, serum, and other biofluids contain very low amounts of circulating extracellular cell-free DNA, also called cfDNA. cfDNA has become an increasingly important source for the development of liquid biopsy assays for early cancer detection. Indeed, it has been shown that cancer patients present both normal tissue- and tumor-derived cfDNA in the bloodstream [1]. Current cfDNA assays have mostly focused on monitoring cancer-specific mutations or methylation changes by targeting cell-free tumor DNA (ctDNA) [2]. In this case, We recently devised a qPCR-based cfDNA assay to measure telomeric cfDNA levels (i.e., relative amounts of [TTAGGG]n sequences) in plasma. Using this assay, we reported that plasma telomeric cfDNA levels were significantly decreased in sporadic breast cancer patients www.impactjournals.com/oncotarget Oncotarget 4214 eliminating the confounding factor of age. Overall, there was no difference of the telomeric cfDNA level between BRCA1&2 carriers and controls (p = 0.0774, d = 0.351). However, upon subgrouping the samples by BMI, the plasma telomeric cfDNA level was significantly lower in BRCA1&2 carriers than those in age-matched controls in the non-obese group (n = 14 pairs, p = 0.0253, d = 0.691) (Figure 1A). In contrast, there was no difference in the telomeric cfDNA level between BRCA1&2 carriers and age-matched controls in the obese group (n = 14 pairs, p = 0.8991, d = 0.036) (Figure 1B). Wilcoxon signed-rank test showed that the p-value was also significant when BRCA1&2 carriers were compared with age-matched controls in the non-obese group (p = 0.0257), while the p-value was 0.976 in the obese group (Figure 1B). The effect size (difference) between BRCA1&2 carriers and age-matched controls in the non-obese group was a range from medium to large (d = 0.691). We also measured relative telomere length by qPCR in matched peripheral blood DNA samples as several reports have shown BRCA1&2 carriers having relatively shorter telomeres in leukocytes as compared to healthy controls [10, 11]. INTRODUCTION We hypothesized that either telomeric cfDNA level or telomere length or both fit with the Tyrer-Cuzick model in non-obese women, thus acting as potential biomarker(s) for breast cancer development. When individual risk of developing breast cancer within 10 years was calculated and the plasma telomeric cfDNA level was compared with the 10-year risk, we found that the telomeric cfDNA level was inversely correlated with the 10-year risk (p = 0.00675, r2 = 0.2007), while the telomere length results were not significantly correlated with the 10-year risk (p = 0.1363, r2 = 0.0083) (Figure 2). Only when the data was analyzed in the non-obese controls (the 10-year risk < 5%, n = 22), the telomere length results were correlated with the 10-year risk (p = 0.0161, r2 = 0.2565). This significant correlation is likely attributed to age because the age factor is strongly associated with the 10-year risk score especially when the risk is less than 5% (Supplementary Figure 2).i RESULTS We hypothesized that a circulating telomeric cfDNA level could be affected by BRCA1&2 heterozygous mutations; consequently, the telomeric cfDNA level decreases in BRCA1&2 mutation carriers as compared to healthy controls. In order to avoid reverse causation, we tested this hypothesis only in unaffected women. We analyzed plasma samples from 56 individuals, including 28 unaffected women carrying either a BRCA1 mutation (n = 16), a BRCA2 mutation (n = 9), or both BRCA1 and BRCA2 mutations (n = 3), and 28 age-matched healthy controls. The age range was from 23 to 74. Demographic and clinical characteristics of both cases and controls are provided in Table 1 and Supplementary Table 1, respectively. Telomeric cfDNA qPCR assay was performed as previously described with minor modifications [5]. Both parametric and non-parametric tests were employed to compare the telomeric cfDNA qPCR results collected from the two groups (carriers and controls). We first used paired t-test to compare the results between age- matched BRCA1&2 carriers vs. control women, thereby Our findings indicate that obesity might have an effect on plasma telomere cfDNA level or/and leukocyte telomere length. RESULTS Therefore, we next compared the difference in the www.impactjournals.com/oncotarget Oncotarget 4215 Table 1: Demographic and clinical characteristics, telomeric cfDNA level, and telomere length of BRCA1&2 carrier women women Sample ID Age Race BMI Current smoking status Menstrual Status Mutation Status 10-year risk Telomeric cfDNA level (± S.E.) Relative telomere length (± S.E.) BR02-469 23 White 27.3 No Pre BRCA1 4.9% 0.176 (± 0.0039) 1.97 (± 0.080) BR27-689 23 White 32.1 No Pre BRCA2 5.7% 0.169 (± 0.0044) 2.39 (± 0.093) BR12-497 24 White 28.5 No Pre BRCA1 8.6% 0.114 (± 0.0071) 1.70 (± 0.104) BR31-071 24 White 24.3 No Pre BRCA1 19.0% 0.131 (±0.0009) 2.14 (± 0.084) BR03-560 29 White 27.3 No Pre BRCA1 26.3% 0.146 (± 0.0079) 1.60 (± 0.095) BR05-977 32 White 26.2 No Pre BRCA2 19.4% 0.247 (± 0.0621) 0.97 (± 0.054) BR24-030 34 White 32.0 No Pre BRCA1 66.7% 0.038 (±0.0055) 2.00 (± 0.167) BR07-177 37 White 33.9 No Post BRCA1 & BRCA2 18.8% 0.079 (± 0.0064) 1.70 (± 0.049) BR34-032 37 White 21.1 No Pre BRCA2 16.1% 0.154 (± 0.0067) 2.08 (± 0.151) BR11-468 38 White 27.3 Yes Pre BRCA1 23.8% 0.158 (± 0.0077) 2.24 (± 0.130) BR14-590 39 White 32.8 No Pre BRCA2 18.8% 0.105 (± 0.0091) 1.87 (± 0.136) BR35-034 41 White 31.6 No pre BRCA1 30.1% 0.159 (± 0.0079) 2.33 (± 0.070) BR06-978 42 White 19.1 No Post BRCA2 17.2% 0.042 (± 0.0050) 1.76 (± 0.032) BR13-588 45 White 31.2 No Post BRCA2 22.8% 0.090 (± 0.0071) 1.79 (± 0.067) BR26-158 45 White 39.4 No n/a BRCA1 33.7% 0.214 (± 0.0175) 1.61 (± 0.093) BR01-938 46 White 22.7 No Post BRCA1 32.8% 0.074 (± 0.0048) 1.76 (± 0.110) BR28-925 46 White 32.1 No Pre BRCA2 21.5% 0.147 (± 0.0046) 1.48 (± 0.059) BR16-766 47 White 35.7 No Post BRCA1 34.9% 0.171 (± 0.0185) 1.54 (± 0.110) BR20-204 48 White 25.8 No Post BRCA1 19.9% 0.080 (± 0.0032) 2.14 (± 0.121) BR32-664 49 White 32.6 No Post BRCA1 28.4% 0.126 (± 0.0026) 2.00 (± 0.068) BR04-976 50 White 26.6 No Pre BRCA2 22.8% 0.131 (± 0.0074) 0.81 (± 0.022) BR30-175 51 White 35.4 No Post BRCA1 26.3% 0.090 (± 0.0029) 1.39 (± 0.036) BR29-168 53 White 32.2 No Post BRCA1 26.9% 0.142 (± 0.0086) 1.59 (± 0.039) BR36-383 56 White 29.3 No Post BRCA1 & BRCA2 28.2% 0.108 (± 0.0063) 1.47 (± 0.111) BR19-102 61 White 37.3 No Post BRCA1 & BRCA2 27.9% 0.025 (± 0.0014) 1.46 (± 0.061) BR33-750 66 White 27.1 No Post BRCA1 12.6% 0.195 (± 0.0054) 1.57 (± 0.152) BR21-207 70 White 22.6 No Post BRCA2 29.5% 0.133 (± 0.0163) 1.65 (± 0.142) BR17-822 74 White 33.5 No Post BRCA1 8.5% 0.060 (± 0.0071) 1.58 (± 0.065) *S.E., standard error of the mean. DISCUSSION All p values were shown based on paired Student’s t test. on have a lower plasma telomeric cfDNA level and sho plotted using 14 age-matched pairs from non-obese women (BMI < in controls and 0.135 (± 0.050) in BRCA1&2 carriers. 71.4% ( &2 carries. (B) The telomeric cfDNA level was plotted using 14 he telomeric cfDNA level was 0.115 (± 0.040) in controls and ction in the telomeric cfDNA level in BRCA1&2 carries. (C) Re n (A). Mean (± S.D.) of telomeric cfDNA level was 1.85 (± 0.4 pairs) show a reduction in the telomeric cfDNA level in BRCA f b i (B) M (± S D ) f t l i fDNA Figure 1: Non-obese women with BRCA1&2 gene mutation have a lower plasma telomeric cfDNA level and shorter leukocyte telomere length. (A) The telomeric cfDNA level was plotted using 14 age-matched pairs from non-obese women (BMI < 30). Mean (± S.D.) of the telomeric cfDNA level was 0.191 (± 0.073) in controls and 0.135 (± 0.050) in BRCA1&2 carriers. 71.4% (10 of 14 pairs) show a reduction in the telomeric cfDNA level in BRCA1&2 carries. (B) The telomeric cfDNA level was plotted using 14 age- matched pairs from obese women (BMI > 30). Mean (± S.D.) of the telomeric cfDNA level was 0.115 (± 0.040) in controls and 0.113 (± 0.053) in BRCA1&2 carriers. 50.0% (7 of 14 pairs) show a reduction in the telomeric cfDNA level in BRCA1&2 carries. (C) Relative telomere length was plotted using the same pairs from non-obese in (A). Mean (± S.D.) of telomeric cfDNA level was 1.85 (± 0.45) in controls and 1.69 (± 0.41) in BRCA1&2 carriers. 71.4% (10 of 14 pairs) show a reduction in the telomeric cfDNA level in BRCA1&2 carries. (D) Relative telomere length was plotted using the same pairs from obese women in (B). Mean (± S.D.) of telomeric cfDNA level was 1.79 (± 0.37) in controls and 1.77 (± 0.30) in BRCA1&2 carriers. 64.2% (9 of 14 pairs) show a reduction in the telomeric cfDNA level in BRCA1&2 carries. All p values were shown based on paired Student’s t test. Figure 1: Non-obese women with BRCA1&2 gene mutation have a lower plasma telomeric cfDNA level and shorter leukocyte telomere length. (A) The telomeric cfDNA level was plotted using 14 age-matched pairs from non-obese women (BMI < 30). DISCUSSION plasma telomeric cfDNA level among 19 age-matched low (< 30) and high (> 30) BMI healthy control pairs. Non- parametric Wilcoxon signed-rank test was employed and showed that a lower plasma telomeric cfDNA level was associated with obesity (BMI > 30) in healthy individuals (p = 0.00034, d = 1.0667) (Figure 3A, Supplementary Table 2). In contrast, we did not find the difference in leukocyte telomere length between the obese and the non-obese groups (p = 0.093, d = 0.421) (Figure 3B, Supplementary Table 2), suggesting that obesity (based on BMI) is not a strong modifier of telomere length [14, 15]. This pilot study is the first to determine the association of the plasma telomeric cfDNA level with genetic risk in unaffected women with and without mutations in BRCA1 or/and BRCA2 genes. The findings suggest that circulating plasma telomeric cfDNA levels are affected by BRCA1&2 heterozygous mutations in non- obese women. Until now, conflicting studies report the correlation between shorter telomere length in leukocyte- derived DNA and BRCA1&2 mutation status. Our results www.impactjournals.com/oncotarget Oncotarget 4216 gure 1: Non-obese women with BRCA1&2 gene mutation have a lower plasma telomeric cfDNA level and shor ukocyte telomere length. (A) The telomeric cfDNA level was plotted using 14 age-matched pairs from non-obese women (BMI < 3 ean (± S.D.) of the telomeric cfDNA level was 0.191 (± 0.073) in controls and 0.135 (± 0.050) in BRCA1&2 carriers. 71.4% (10 pairs) show a reduction in the telomeric cfDNA level in BRCA1&2 carries. (B) The telomeric cfDNA level was plotted using 14 a atched pairs from obese women (BMI > 30). Mean (± S.D.) of the telomeric cfDNA level was 0.115 (± 0.040) in controls and 0. 0.053) in BRCA1&2 carriers. 50.0% (7 of 14 pairs) show a reduction in the telomeric cfDNA level in BRCA1&2 carries. (C) Relat omere length was plotted using the same pairs from non-obese in (A). Mean (± S.D.) of telomeric cfDNA level was 1.85 (± 0.45) ntrols and 1.69 (± 0.41) in BRCA1&2 carriers. 71.4% (10 of 14 pairs) show a reduction in the telomeric cfDNA level in BRCA1 rries. (D) Relative telomere length was plotted using the same pairs from obese women in (B). Mean (± S.D.) of telomeric cfDNA le as 1.79 (± 0.37) in controls and 1.77 (± 0.30) in BRCA1&2 carriers. 64.2% (9 of 14 pairs) show a reduction in the telomeric cfDNA le BRCA1&2 carries. DISCUSSION Mean (± S.D.) of the telomeric cfDNA level was 0.191 (± 0.073) in controls and 0.135 (± 0.050) in BRCA1&2 carriers. 71.4% (10 of 14 pairs) show a reduction in the telomeric cfDNA level in BRCA1&2 carries. (B) The telomeric cfDNA level was plotted using 14 age- matched pairs from obese women (BMI > 30). Mean (± S.D.) of the telomeric cfDNA level was 0.115 (± 0.040) in controls and 0.113 (± 0.053) in BRCA1&2 carriers. 50.0% (7 of 14 pairs) show a reduction in the telomeric cfDNA level in BRCA1&2 carries. (C) Relative telomere length was plotted using the same pairs from non-obese in (A). Mean (± S.D.) of telomeric cfDNA level was 1.85 (± 0.45) in controls and 1.69 (± 0.41) in BRCA1&2 carriers. 71.4% (10 of 14 pairs) show a reduction in the telomeric cfDNA level in BRCA1&2 carries. (D) Relative telomere length was plotted using the same pairs from obese women in (B). Mean (± S.D.) of telomeric cfDNA level was 1.79 (± 0.37) in controls and 1.77 (± 0.30) in BRCA1&2 carriers. 64.2% (9 of 14 pairs) show a reduction in the telomeric cfDNA level in BRCA1&2 carries. All p values were shown based on paired Student’s t test. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 4217 possess enhanced proliferative potential in women with BRCA1&2 haploinsufficiency [16–18]. We speculate that after the occurrence of “one-hit” of BRCA1&2 gene, the remaining wild-type allele of BRCA1&2 may not be sufficient to recover the deficiency in DNA repair and/ or replication properly at the telomeres in target somatic tissues. Consequently, a telomere shortening rate in cells of BRCA1&2 carriers could be accelerated during development and aging, compared to controls [19]. Thus, we speculate that the circulating telomeric cfDNA level could reflect the somatic and dynamic changes in telomere dysfunction in target tissue cells; as a result, we confirmed that leukocyte telomere length was affected by BRCA1&2 heterozygous mutations in non-obese women (but not in obese women). Furthermore, it is noteworthy that there is a significant correlation between the telomeric cfDNA level and the Tyrer-Cuzick 10-year risk score of developing breast cancer. These findings support our hypothesis that the telomeric cfDNA level could be a biologically-relevant quantitative biomarker for breast cancer development. DISCUSSION Although the underlying molecular mechanism of alteration in the plasma telomeric cfDNA level remains unclear, several studies reported that epithelial breast cells Oncotarget 4218 mpactjournals.com/oncotarget gure 2: The plasma telomeric cfDNA level is correlated with individual 10-year breast cancer risk in non-obese men. (A) Comparison between the plasma telomeric cfDNA level and the 10-year risk in non-obese women (n = 28). (B) Comparison ween relative leukocyte telomere length and the 10-year risk in non-obese women (n = 28). Figure 2: The plasma telomeric cfDNA level is correlated with individual 10-year breast cancer risk in non-obese women. (A) Comparison between the plasma telomeric cfDNA level and the 10-year risk in non-obese women (n = 28). (B) Comparison between relative leukocyte telomere length and the 10-year risk in non-obese women (n = 28). www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 4218 observed that BRCA1&2 carriers have relatively lower telomeric cfDNA level than controls, even before a total loss of the remaining wild-type allele occurs and cancer appears. Moreover, we demonstrated that a lower plasma telomeric cfDNA level was associated with a higher BMI. The question of why the plasma telomeric cfDNA level is lower in obese women requires further investigation; however, we pose the following two possible explanations. First, adipocyte cells may have relatively shorter telomeres [20], and therefore increased death of the adipocyte cells results in a lower telomeric cfDNA level in plasma. Alternatively, obesity-related metabolic changes may induce higher enzymatic activity (e.g., nuclease) in the bloodstream that in turn facilitate degradation of cfDNA fragments outside-in [21]. Therefore, it is of value to further studying the implications of plasma telomeric cfDNA levels in cancer development and obesity. changes in the plasma telomeric cfDNA level with specific BRCA1&2 mutation sites may provide new insights into risk assessments of breast cancer. In summary, we found that BRCA1&2 heterozygous mutations altered the plasma telomeric cfDNA level in non-obese women. This work highlights that the plasma telomeric cfDNA level may reflect the dynamic changes occurring in the telomeres of the somatic tissue cells as a result of genetic predisposition (i.e., BRCA1&2 mutations). The next steps would be to replicate the findings in a larger study. DISCUSSION It is also critical to examine the prospective associations between the plasma telomeric cfDNA level, predictive risk factors for breast cancer, and breast cancer occurrence, and also to further determine whether the plasma telomeric cfDNA level mediates the association between these risk factors and breast cancer. MATERIALS AND METHODS Because we only included Caucasian women, these findings may not be generalized to men and/or other ethnicities. In addition, because the sample size in this study was limited, we were not able to identify differences in the plasma telomeric cfDNA level between BRCA1 carriers, BRCA2 carriers, and dual carriers. This study also lacks information about the specific mutation site(s) on BRCA1&2 genes. Investigating the correlation of the Human specimens Paired leukocyte DNA and frozen plasma samples were collected from BRCA1&2 mutation carriers (n = 28, 23 ≤ age ≤ 74, mean age = 46) and their age- matched controls (n = 28, ± 1 year) by the Susan G. Komen Figure 3: Healthy obese women have a lower telomeric cfDNA level than healthy non-obese women. (A) The telomeric cfDNA level was sub-grouped by BMI using age-matched control women (n = 19 pairs). Mean (± S.D.) of the telomeric cfDNA level was 0.217 (± 0.084) in the BMI < 30 group and 0.105 (± 0.046) in the BMI > 30 group. 84.2% (16 of 19 pairs) show a reduction in the telomeric cfDNA level in the BMI > 30 group. (B) Relative telomere length was sub-grouped by BMI using age-matched control women (n = 19 pairs). Mean (± S.D.) of telomere length was 1.67 (± 0.35) in the BMI < 30 group and 1.83 (± 0.34) in the BMI > 30 group. There is no difference of telomere length between two groups. All p values were shown based on Wilcoxon signed-rank test. Figure 3: Healthy obese women have a lower telomeric cfDNA level than healthy non-obese women. (A) The telomeric cfDNA level was sub-grouped by BMI using age-matched control women (n = 19 pairs). Mean (± S.D.) of the telomeric cfDNA level was 0.217 (± 0.084) in the BMI < 30 group and 0.105 (± 0.046) in the BMI > 30 group. 84.2% (16 of 19 pairs) show a reduction in the telomeric cfDNA level in the BMI > 30 group. (B) Relative telomere length was sub-grouped by BMI using age-matched control women (n = 19 pairs). Mean (± S.D.) of telomere length was 1.67 (± 0.35) in the BMI < 30 group and 1.83 (± 0.34) in the BMI > 30 group. There is no difference of telomere length between two groups. All p values were shown based on Wilcoxon signed-rank test. www.impactjournals.com/oncotarget Oncotarget 4219 Line copy number) calculated from the average of more than three independent experiments. Tissue Bank at Indiana University Simon Cancer Center (KTB) with an approved Indiana University Institutional Review Board protocol. All samples were collected in accordance with standard operating procedures described on the KTB website and with donor’s written informed consent. All BRCA1&2 carrier cases and healthy controls were unaffected women. Telomeric cfDNA qPCR assay Telomeric cfDNA levels were measured by qPCR as described previously except for some minor modifications [5]. The qPCR was performed on QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific) in 10 μL volume of PCR reaction with 1× SYBR Select (Thermo Fisher Scientific), primers, and cfDNA (~50 pg) as template. The primer sequences are shown in Supplementary Table 3. The thermal cycling profile is Stage 1 for 2 min at 95°C; Stage 2 for 2 cycles of 15 s at 94°C, 15 s at 49°C; and Stage 3 for 40 cycles of 15 s at 94°C, 15 s at 60°C, 20 s at 72°C. The qPCR results were analyzed based on plasmid DNA standard curves. The plasmid DNA contains (TTAGGG)13 and 121 bp of LINE (Long INterspersed Element) nucleotide sequences. Five concentrations of a plasmid DNA sample were prepared by five serial dilutions (3.62 × 109 to 5.79 × 106 copies). To monitor and compensate for inter-plate variations in PCR efficiency, each plate included standard plasmid DNAs as well as reference DNA (Promega, G152A). All experimental DNA samples were repeated at least three times in duplicate. All samples have a standard deviation of less than 0.5 for the threshold cycle (Ct) values. The inter- and intra-assay coefficients of variations (CVs) were 13.0% and 3.9%, respectively. Melting curve analysis was performed on every run to verify specificity and identity of the PCR products. The telomeric cfDNA levels were presented as T/L copy ratios (= telomere copy number/ Leukocyte DNA extraction Paired leukocyte DNAs along with plasma from both case and control women were obtained as a lyophilized power after the Komen Tissue Bank was extracted from whole blood samples using the Flex Star automated system with the AGFStar Fresh WB Extraction Kit (Autogen). The DNA was rehydrated with UltraPure DNase/RNase-free water (Thermo Fisher Scientific, 10977015). The DNA concentration was quantitated by Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). Human specimens Of note, the BRCA1&2 mutation status of controls was based on self-reported information. The demographical and clinical information of the study subjects were collected by the KTB through a baseline questionnaire, and were shown in Table 1 and Supplementary Table 1. Body Mass Index (BMI) was calculated as weight divided by squared height. Breast cancer risk assessment The Tyrer-Cuzick (or International Breast Intervention Study, IBIS) model was used to estimate a woman’s risk of developing breast cancer within the next 10 years using information about BRCA1/2 mutation carrier status and other risk factors including family history of breast and ovarian cancer, age at menarche, parity, age at first childbirth, age at menopause, atypical hyperplasia, lobular carcinoma in situ, height, and body mass index. The risk factors were entered into the model using the freely available software IBIS_RiskEvaluator_ v8b (www.ems-trials.org/riskevaluator) [12, 13]. Relative telomere length measurement Frozen plasma samples were thawed on ice prior to cfDNA isolation and centrifuged for 3 min at ≥ 11,000 × g in order to remove residual cells, cell debris, and particulate matter. A sodium Iodide (NaI)-based method was used for each cfDNA extraction as described previously [5]. cfDNA concentrations were measured using Quant-iT™ PicoGreen™ dsDNA Reagent (Thermo Fisher Scientific). The fluorescence intensity was measured with a Synergy 2 Multi-Mode Reader at emission wavelength of 520 nm and excitation wavelength of 480 nm. Singleplex telomere length qPCR was performed with a QuantStudio 6 Flex Real-Time PCR System and analyzed as described previously [5, 22, 23]. The qPCR was performed on QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific) in 10 μL volume of PCR reaction with 1× SYBR Select (Thermo Fisher Scientific), primers, and DNA as template. The telomere primers (telg and telc) and β-globin gene primers (hbgu and hbgd) were used for this assay. Each DNA standard curve was generated using a pooled female genomic DNA (Promega, G152A) and used for calculation of the T/S ratios (= ratios of “telomere quantities/single copy gene quantities”). All experimental DNA samples were repeated at least three times in duplicate. All replicate samples had a standard deviation of less than 0.5 for the threshold cycle (Ct) values. Statistical analysis Shapiro-Wilk test was used to test for normality in each group. Student’s t test (parametric) and Wilcoxon signed-rank test (non-parametric) were performed to test the association between a telomeric cfDNA level and breast cancer risk factors (BRCA1&2 mutations, BMI). Cohen’s d was used to calculate the effect size for paired www.impactjournals.com/oncotarget Oncotarget 4220 CH), an Indiana University Simon Cancer Center Breast Cancer Signature pilot grant, an Indiana University School of Medicine Biomedical Research grant, the Showalter Foundation, the Friends for an Earlier Breast Cancer Test®, the Susan G. Komen Foundation, and the American Cancer Society. two-sample t test using WebPower (http://webpower. psychstat.org) [24]. Linear regression analysis was used to examine the correlation between the plasma telomeric cfDNA level and leukocyte telomere length, as well as between the 10-year risk and either the plasma telomeric cfDNA level or leukocyte telomere length. R squared (r2) was used for assessing the fit of the regression line. Two- tailed p values of less than 0.05 were considered to be statistically signiﬁcant. Author contributions 4. Volik S, Alcaide M, Morin RD, Collins C. Cell-free DNA (cfDNA): Clinical Significance and Utility in Cancer Shaped By Emerging Technologies. Mol Cancer Res. 2016; 14:898–908. https://doi.org/10.1158/1541-7786. mcr-16-0044. HT, NM, and CH conceived the study. HT, NM and CH designed the experiments. SD, NM, KB, and HT performed the experiments. HT, PND, NM, AS, and CH analyzed and interpreted the data. HT, NM, and CH wrote the manuscript. All authors participated in the discussion and critical revision of the manuscript. All authors read and approved the final manuscript. 5. Wu X, Tanaka H. Aberrant reduction of telomere repetitive sequences in plasma cell-free DNA for early breast cancer detection. Oncotarget. 2015; 6:29795–807. https://doi. org/10.18632/oncotarget.5083. REFERENCES 1. Schwarzenbach H, Hoon DS, Pantel K. Cell-free nucleic acids as biomarkers in cancer patients. Nat Rev Cancer. 2011; 11:426–37. https://doi.org/10.1038/nrc3066. 1. Schwarzenbach H, Hoon DS, Pantel K. Cell-free nucleic acids as biomarkers in cancer patients. Nat Rev Cancer. 2011; 11:426–37. https://doi.org/10.1038/nrc3066. Abbreviations cfDNA: cell-free DNA; ctDNA: cell-free tumor DNA; BMI: Body Mass Index; BRCA1&2: BRCA1 and BRCA2; qPCR: quantitative polymerase chain reaction; Ct: threshold cycle; CV: coefficients of variation; LINE: Long INterspersed Element; IBIS: International Breast Intervention Study. 2. Donaldson J, Park BH. Circulating Tumor DNA: Measurement and Clinical Utility. Annu Rev Med. 2017 Aug 28. https://doi.org/10.1146/annurev-med-041316-085721. [Epub ahead of print]. 3. Aravanis AM, Lee M, Klausner RD. Next-Generation Sequencing of Circulating Tumor DNA for Early Cancer Detection. Cell. 2017; 168:571–4. https://doi.org/10.1016/j. cell.2017.01.030. ACKNOWLEDGMENTS 6. Yoshida K, Miki Y. Role of BRCA1 and BRCA2 as regulators of DNA repair, transcription, and cell cycle in response to DNA damage. Cancer Sci. 2004; 95:866–71. Samples from the Susan G. Komen Tissue Bank at the IU Simon Cancer Center (KTB) were used in this study. We thank contributors, including KTB members who collected samples used in this study, as well as donors and their families, whose help and participation made this work possible. We also thank Rie Matern and Priya Vallanda for technical assistance, Brittney-Shea Herbert for critical reading of the manuscript as well as reagents, and Xi Wu for helping initial experiments at an early stage of this project. KB was a fellow of the Undergraduate Research Opportunity Program at Indiana University- Purdue University at Indianapolis. PND was a fellow of the Student Research Program of Academic Medicine at Indiana University School of Medicine at Indianapolis. 7. O’Donovan PJ, Livingston DM. BRCA1 and BRCA2: breast/ovarian cancer susceptibility gene products and participants in DNA double-strand break repair. Carcinogenesis. 2010; 31:961–7. https://doi.org/10.1093/ carcin/bgq069. 8. Sedic M, Skibinski A, Brown N, Gallardo M, Mulligan P, Martinez P, Keller PJ, Glover E, Richardson AL, Cowan J, Toland AE, Ravichandran K, Riethman H, et al. Haploinsufficiency for BRCA1 leads to cell-type- specific genomic instability and premature senescence. Nat Commun. 2015; 6:7505. https://doi.org/10.1038/ ncomms8505. CONFLICTS OF INTEREST 9. Uziel O, Yerushalmi R, Zuriano L, Naser S, Beery E, Nordenberg J, Lubin I, Adel Y, Shepshelovich D, Yavin H, Ben Aharon I, Pery S, Rizel S, et al. BRCA1/2 mutations perturb telomere biology: characterization of structural and functional abnormalities in vitro and in vivo. Oncotarget. 2016; 7:2433–54. https://doi.org/10.18632/oncotarget.5693. The authors declare no conflicts of interest related to this study. FUNDING 10. Martinez-Delgado B, Yanowsky K, Inglada-Perez L, Domingo S, Urioste M, Osorio A, Benitez J. Genetic anticipation is associated with telomere shortening in This work is supported in part by National Institutes of Health under CA205434 (to HT) and CA194030 (to www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 4221 hereditary breast cancer. PLoS Genet. 2011; 7:e1002182. https://doi.org/10.1371/journal.pgen.1002182. 17. Martins FC, De S, Almendro V, Gonen M, Park SY, Blum JL, Herlihy W, Ethington G, Schnitt SJ, Tung N, Garber JE, Fetten K, Michor F, et al. Evolutionary pathways in BRCA1-associated breast tumors. 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https://openalex.org/W4235272226	https://www.qeios.com/read/54GU0E/pdf	English	null	Complement C2	Definitions	2,020	cc-by	64	Qeios · Definition, February 2, 2020 Open Peer Review on Qeios Complement C2 National Cancer Institute National Cancer Institute Qeios ID: 54GU0E · https://doi.org/10.32388/54GU0E Source National Cancer Institute. Complement C2. NCI Thesaurus. Code C126958. Complement C2 (752 aa, ~83 kDa) is encoded by the human C2 gene. This protein is involved in post-translational protein processing and complement activity. Qeios ID: 54GU0E · https://doi.org/10.32388/54GU0E 1/1
https://openalex.org/W2917388952	https://durham-repository.worktribe.com/file/1303257/1/Published%20Journal%20Article	English	null	BFSP1 C-terminal domains released by post-translational processing events can alter significantly the calcium regulation of AQP0 water permeability	Experimental eye research/Experimental Eye Research	2,019	cc-by	15,563	A B S T R A C T BFSP1 (beaded filament structural protein 1, filensin) is a cytoskeletal protein expressed in the eye lens. It binds AQP0 in vitro and its C-terminal sequences have been suggested to regulate the water channel activity of AQP0. A myristoylated fragment from the C-terminus of BFSP1 was found in AQP0 enriched fractions. Here we identify BFSP1 as a substrate for caspase-mediated cleavage at several C-terminal sites including D433. Cleavage at D433 exposes a cryptic myristoylation sequence (434–440). We confirm that this sequence is an excellent substrate for both NMT1 and 2 (N-myristoyl transferase). Thus caspase cleavage may promote formation of myristoylated fragments derived from the BFSP1 C-terminus (G434-S665). Myristoylation at G434 is not required for membrane association. Biochemical fractio- nation and immunogold labeling confirmed that C-terminal BFSP1 fragments containing the myristoylation sequence colocalized with AQP0 in the same plasma membrane compartments of lens fibre cells. To determine the functional significance of the association of BFSP1 G434-S665 sequences with AQP0, we measured AQP0 water permeability in Xenopus oocytes co-transfected with transcripts expressing both AQP0 and various C-terminal domain fragments of BFSP1 generated by caspase cleavage. We found that different fragments dramatically alter the response of AQP0 to different concentrations of Ca2+. The complete C-terminal fragment (G434-S665) eliminates calcium regulation altogether. Shorter fragments can enhance regulation by elevated calcium or reverse the response, indicative of the regulatory potential of BFSP1 with respect to AQP0. In particular, elimination of the myristoylation site by the mutation G434A reverses the order of water permeability sensitivity to different Ca2+ concentrations. 2003). Beaded filament structural proteins (BFSP1 and BFSP2) co-as- semble to form filaments that closely associate with the protein cha- perone, α-crystallin, (Carter et al., 1995). These filaments are enriched in the plasma-membrane-cytoskeleton complex and are often tightly apposed to the plasma membrane (FitzGerald, 1988; Sandilands et al., 1995a). The precise mechanism of this association with the plasma Contents lists available at ScienceDirect Contents lists available at ScienceDirect BFSP1 C-terminal domains released by post-translational processing events can alter significantly the calcium regulation of AQP0 water permeability Antal Tapodia,1,5, Daniel M. Clemensb,2,5, Alice Uwinezaa, Miguel Jarrina, Martin W. Goldberga, Emmanuelle Thinonc,d,3, William P. Healc,d,4, Edward W. Tatec,d, Karinne Nemeth-Cahalanb, Irene Vorontsovab, James E. Hallb,∗, Roy A. Quinlana,e,∗∗ p g j Received 8 November 2018; Received in revised form 26 January 2019; Accepted 3 February 2019 Available online 18 February 2019 0014-4835/ © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecom m ons.org/licenses/BY/4.0/). 1. Introduction Beaded filaments modulate the optical properties of the lens (Sandilands et al., 2003; Song et al., 2009), adjust its mechanical properties (Fudge et al., 2011) and influence the geometry of lens fiber cells (Gokhin et al., 2012; Maddala et al., 2011; Sandilands et al., Abbreviations: AQP0, Aquaporin zero; BFSP, Beaded filament structural protein; EPPD, Ezrin, Periplakin, Periaxin, and Desmoyokin); BFSP1 HsBFSP1, BFSP2; AQP0, Aquaporin zero; AKAP, AKAP2 A-Kinase Anchor Proteins; PKA, Protein Kinase A; DMEM, Dulbecco's Modified Eagle Medium; EMEM, Eagle's Minimum Essential Medium; MCF7, Breast cancer cell line named after Michigan Cancer Foundation (MCF); FHL124, Fetal Human lens cell line 124; eGFP, enhanced Green Fluorescent Protein; NMT1, N-myristoyltransferase 1; NMT2, N-myristoyltransferase 2; CaNMT, Candida albicans myristoyl-CoA: protein N-myristoyltransferase; EDC, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide, a crosslinker Experim ental Eye Research 185 (2019) 107585 Experim ental Eye Research 185 (2019) 107585 a Department of Biosciences, The University of Durham, South Road, Durham, DH1 3LE, UK b Physiology and Biophysics, UC Irvine, Irvine, CA, USA c Department of Chemistry, Molecular Sciences Research Hub, Imperial College London, Wood Lane, London, W12 0BZ, U d Institute of Chemical Biology, Molecular Sciences Research Hub, Imperial College London, Wood Lane, London, W12 0BZ e Biophysical Sciences Institute, The University of Durham, South Road, Durham, DH1 3LE, UK https://doi.org/10.1016/j.exer.2019.02.001 This interaction of BFSP1 with AQP0 could also be functionally significant. An arbitrarily selected C-terminal portion of BFSP1, not expected to arise from normal post-translational processing modestly increases the water permeability (Pf) of AQP0 (Nakazawa et al., 2011). The ability of calcium to mod- ulate the Pf of AQP0 in the presence of BFSP1 and its fragments was, however, not investigated. The interaction of AQP0 with other mem- brane associated lens proteins, and particularly BFSP1, is likely critical to understanding AQP0 function in the lens (Sindhu Kumari et al., 2015).i In the zebrafish lens, interference with the phosphorylation ma- chinery, which modulates the calcium regulation of AQP0a water per- meability results in a cataractous lens, providing more evidence that calcium regulation of AQP0 water permeability is essential for lens clarity (Clemens et al., 2013). The mounting evidence for both of the importance of calcium regulation of water permeability and the inter- action of BFSP1 with AQP0 impelled us to test the effects of physiolo- gically relevant C-terminal sequences from BFSP1 on the regulation of the water permeability of AQP0 by calcium. In particular, we felt it important to investigate the role of myristoylation and specific caspase cleavage as possible modes of AQP0 Pf regulation by calcium. The principal post-translational modification of BFSP1 is proteolysis during both differentiation and ageing (Su et al., 2011). Peptide-specific antibodies show that fragments derived from the N- and C-terminal regions of BFSP1 localize to different sub-cellular compartments in lens fiber cells (Sandilands et al., 1995b). An internal cleavage was proposed to release the two regions (Sandilands et al., 1995b). The N-terminal region co-assembles with BFSP2 in vitro and forms intermediate fila- ments (Carter et al., 1995). The C-terminal region localizes to the plasma membrane (Sandilands et al., 1995b). Details of the enzymatic activities and the precise site of the proposed cleavage of BFSP1 re- mained elusive until the proteomic analysis of the urea-soluble protein fraction from lens membranes identified a myristoylated fragment de- rived from the C-terminal region of BFSP1 (Wang et al., 2010b). They noticed that a consensus myristoylation site in the sequence DVPDG- GQISK immediately followed a caspase consensus site (Backes et al., 2005; Piippo et al., 2010). Post-translational myristoylation is often associated with the exposure of a cryptic site by caspase cleavage (re- viewed in (Martin et al., 2011)). https://doi.org/10.1016/j.exer.2019.02.001 y 0014-4835/ © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecom m ons.org/licenses/BY/4.0/). Experim ental Eye Research 185 (2019) 107585 A. Tapodi, et al. membranes is not well understood, but both BFSP1 and BFSP2 can bind the C-terminal domain of AQP0 (Lindsey Rose et al., 2006), an integral membrane protein, and both colocalize with AQP0 (Lindsey Rose et al., 2006). It was recently proposed that AQP0 could help anchor the EPPD complex (ezrin, periplakin, periaxin, and desmoyokin), a major site of integration for the lenticular cytoskeleton at the plasma membrane, through its interaction with ezrin (Wang and Schey, 2011). manner in which calcium increases AQP0's water permeability when expressed in Xenopus oocytes (Kalman et al., 2008). If serine 235 is phosphorylated, increasing the external calcium concentration from 1.8 mM to 5 mM increases the water permeability. However if serine 235 is not phosphorylated, lowering the calcium concentration to 0 mM increases the water permeability (Gold et al., 2012). AQP0 phosphor- ylation in vivo is probably controlled by a number of as yet unknown mechanisms, and Gold, Reichow and co-workers (Gold et al., 2012; Kalman et al., 2006; Reichow et al., 2013) have shown that disrupting the interactions between AKAP2 (which acts as a scaffold for PKA) and AQP0 results in a cortical cataract, almost certainly because control of PKA to phosphorylation of AQP0 is disrupted. Moreover AQP0 gene knockout (Shiels et al., 2001) and a number of AQP0 mutations cause cataract (Francis et al., 2000; Hu et al., 2012; Okamura et al., 2003; Senthil Kumar et al., 2013; Wang et al., 2010a). AQP0 is not merely a solo player, but its interaction with other lens proteins (Gold et al., 2012; Lindsey Rose et al., 2006; Wang and Schey, 2011) is essential in lens homeostasis during development, differentiation and ageing (Kalman et al., 2006).i Additionally some fragments of BFSP1 resist alkali extraction (Brunkener and Georgatos, 1992), a biochemical property associated with integral membrane proteins (Milks et al., 1988). In addition to in vitro evidence for the binding of BFSP1 to AQP0 (Lindsey Rose et al., 2006), EDC cross-linking studies of bovine lens membrane fractions found a cross-link between the D246 in AQP0 and K455 in BFSP1 (Wang and Schey, 2017), indicating that the C-terminal domain of BFSP1 participates in an interaction with AQP0. https://doi.org/10.1016/j.exer.2019.02.001 Caspase activity is not always linked to apoptosis, but is required during development and normal cell differ- entiation (Busso et al., 2010; Rudrapatna et al., 2013). Moreover such caspase activity can be readily detected in differentiating lens cells (Weber and Menko, 2005). Therefore whilst the mechanism for the revelation of this cryptic myristoylation motif and its role as a substrate for the N-methyltransferases expressed in the lens (Shrivastav et al., 2004) remain to be formally demonstrated (Wang et al., 2010b), these proteomic data have identified BFSP1 polypeptides derived from the C- terminal domain of BFSP1 that may well be regulators of AQP0. 2.2. Cloning and purification of BFSP1 fragments Full length human BFSP1 was obtained from Source Bioscience (http://www.lifesciences.sourcebioscience.com/IMAGE clones 6154051 and 5406467). The C-terminal region of HsBFSP1 was amplified by PCR, the products cloned into and sequenced in pGEM-T Easy (Promega, UK; www.promega.com). Two caspase-resistant mutants, D433A and D549A BFSP1, a myristoylation defective (G434A) as well as domains within the C-terminal region of BFSP1 were generated by PCR and after sequencing were sub-cloned into pET28a vectors to generate protein by expression in BL21pLysS E. coli with a C-terminal histidine tag. Histidine tagged re- combinant BFSP1 fragments were purified on Affinity Columns following manufacturers instructions (His-Select Nickel Affinity Gel; Sigma-Aldrich UK (www.sigmaaldrich.com)) and with a final purification step using a size exclusion column (Sephadex G10; Sigma-Aldrich). In the lens, calcium and its regulation are critical to lens transpar- ency (Maddala et al., 2013; Truscott et al., 1990). Both AQP0 (Ma et al., 2005) and BFSP1 (Marcantonio, 1992) are substrates for the calcium dependent protease, calpain. BFSP1 becomes a substrate when calcium is added to lens extracts or when lenses in culture were treated with a calcium ionophore to induce opacification by changing the calcium regulation (Marcantonio, 1992). Calcium regulates the water perme- ability of AQP0 in vitro (Reichow et al., 2013) (Nemeth-Cahalan and Hall, 2000; Nemeth-Cahalan et al., 2004) and probably also in the lens (Nemeth-Cahalan et al., 2013) through calmodulin (Reichow et al., 2013; Reichow and Gonen, 2008). Calcium also regulates the binding of A-Kinase Anchor Proteins (AKAP) binding to plasma membranes (Tao et al., 2006) and AQP0 activity is regulated by PKA phosphorylation (Gold et al., 2012; Németh-Cahalan et al., 2004). PKA mediated phos- phorylation of AQP0 (now known to be facilitated by AKAP2) alters the 2.1. Sequence analysis software The programs GrabCAS (Backes et al., 2005), CasCleave2 (Wang et al., 2014) (http://www.structbioinfor.org/cascleave2/) and MYRIS- TOYLATOR (Bologna et al., 2004) (http://web.expasy.org/cgi-bin/ myristoylator/myristoylator.pl) were used to search for putative cas- pase sites and myristoylation motif in BFSP1. We used clustalW2 package (http://www.ebi.ac.uk/Tools/clustalw2/index.html) for se- quence alignments and SeaView4.0 with default settings (Gouy et al., 2010) for its representation. 2.3. In vitro caspase assay Purified human recombinant BFSP1 and its D433A and G434A mutants (50 ng) were incubated with 5 units of purified recombinant activated caspases (1–10) for 3 h at 37 °C in caspase-assay buffer as recommended by the manufacturer (Enzo Life Sciences Technology, 2 A. Tapodi, et al. Experim ental Eye Research 185 (2019) 107585 UK; www.enzolifesciences.com). BFSP1 and its fragments were then visualized with Coomassie Blue R250 staining after SDS-PAGE on 10% (w/v) SDS polyacrylamide gels, with modifications to the original published protocol (Laemmli, 1970), or using antibodies (3241; (Sandilands et al., 2004). His-Tag mouse monoclonal antibodies (Merck-Millipore #70796) were used to detect BFSP1 signal by im- munoblotting after transfer of proteins from the gel to nitrocellulose paper using the semi-dry method (Kyhse-Andersen, 1984). 2.7. Raising peptide antibodies against new neo-epitope sequences flanking the D433 caspase site 2.7. Raising peptide antibodies against new neo-epitope sequences flanking the D433 caspase site The short amino acid sequences (PLTQEGAPEDVPD: NP53 and PDGGQISKGFGKL: NPTail) flanking the D433 caspase site and myr- istoylation motif in HsBFSP1 were encoded into a nanoparticle carrier for immunization to prepare rabbit polyclonal antibodies following the described strategy (Schroeder et al., 2009) specific for caspase cleaved BFSP1 at site D433. Briefly, complimentary oligonucleotides were de- signed, annealed, phosphorylated and then cloned into the nanoparticle vector, and proteins were purified using the histidine tag in preparation for immunization and antibody production via a commercial source (Moravian Biotechnology (http://www.moravian-biotech.com). Pre- immune sera were checked for cross reactivity to recombinant HsBFSP1 before immunization. Sera were harvested after the 3rd boost. 2.4. Myristoylation of BFSP1 at G434 The N-myristoyl transferase (NMT) substrates of the truncated BFSP1 Tail (GGQ-548P-His6) or PfARF1 (Plasmodium falciparum ADP- ribosylation factor 1) as the positive control were co-expressed sepa- rately with Candida albicans NMT (CaNMT) in BL21/pLysS E. coli strain in the presence of azido-myristate. CaNMT was subcloned into pET11c vector (ampicillin resistant) and co-transformed with protein substrates subcloned in pET28a (kanamycin resistant). Azido-myristoylated target proteins were captured and visualized by bioorthogonal labelling (CLICK Chemistry) as described previously (Heal et al., 2008, 2011). Briefly, after cell lysis and centrifugation, the soluble fraction was in- cubated with the appropriate capture reagent (Azido-TAMRA). Myr- istoylated proteins were separated by SDS-PAGE and detected with Cy3-TAMRA using a Typhoon 9400 fluorescence scanner (GE Health- care UK; http://www.gelifesciences.com/). 2.9. Immunogold labeling and scanning electron microscopy Silicon mounts (Agar Scientific, UK) were cleaned with acetone and membrane samples were applied and incubated for 1 h at room tem- perature in a moist chamber. Mounts were fixed for 2 h in 2% (w/v) paraformaldehyde, 0.1 M cacodylate pH7.0, then transferred into PBS for 5min, then 100 mM glycine in PBS for 5min. Samples were blocked for 1 h in 1% (w/v) bovine serum albumin (BSA) in phosphate buffered saline (PBS) and incubated with primary antibody (1:50 dilution) for 60 min at room temperature in a moist chamber, then washed three times for 10min with PBS before incubating for 30 min at room tem- perature with the secondary gold-labelled antibodies (BBI Solutions; diluted 1:50). After washing in PBS, the mounts were left overnight at 4 °C in fix (0.1 M cacodylate buffer pH7.0, 2.5% (v/v) glutaraldehyde), then rinsed with 0.1 M sodium cacodylate pH 7.0 for 2min before post- fixing for 10min in 1% (v/v) osmium tetroxide in 0.1 M cacodylate pH7.0. They were dehydrated with a graded ethanol series before drying in a critical point dryer (Bal-Tech CPD 030) prior to coating with chromium (Cressington Scientific Instruments Ltd), ready for imaging in a JEOL 5400 SEM. Images from the scanning and backscattered 2.8. Preparation of human eye lens extracts Human eyes from donors in the age range 18–79 years were ob- tained from the Bristol Eye Bank with national research ethics com- mittee approval and were used as recommended by the Declaration of Helsinki and following the procedures recommended under the Human Tissue Authority license to the University of Durham. Human lenses were dissected out from the eyes as soon as practically possible.f Lenses were decapsulated and isolation buffer (10 mM Sodium Phosphate pH7.4, 5 mM EDTA and 100 mM NaCl) was added in the ratio of 2ml/lens and stirred on ice for 15–20 min. This buffer to lens ratio was maintained for all subsequent extractions. The lens nucleus was removed and placed into fresh extraction buffer and stirred over- night at 4°C. Samples were homogenized with a Dounce homogenizer and centrifuged at 31,000×g @ rmax at 4°C for 20 min (Beckman SA20 rotor) to produce a pellet (P1) and supernatant (S1) fraction. This step was repeated. The plasma membrane cytoskeleton complex (PMCC) was prepared from S1 and S2 by recentrifugation at 80,000×g @ rav at 4°C for 30min (Beckman SW28 rotor). To prepare the lipid membranes from the cortex and nucleus of the lens, the pellet fraction was then extracted sequentially with a series of buffers designed to remove all but integral membrane proteins. Each extraction was repeated once. The buffer series was as follows: 10 mM Sodium Phosphate pH7.4, 5 mM EDTA and 1.5 M KCl to generate pellets P3 and P4; 100 mM NH4HCO3 1 mM EDTA; isolation buffer; 10 mM Sodium Phosphate pH7.4, 5 mM EDTA and 8 M urea; isolation buffer; 0.1 M NaOH; isola- tion buffer. The final lens membrane pellet was re-suspended in isola- tion buffer at a buffer ratio of 25μl/lens and stored at 4°C until required. Km and Vmax values for human NMT1 and NMT2 using the octa- peptide (GGQISKGF) derived from HsBFSP1 were measured as de- scribed (Goncalves et al., 2012) and compared to values for the sub- strate standard, a pp60src octapeptide, GSNKSKPK which is a demonstrated in vivo substrate (Lacal et al., 1988). Values plotted are the mean from three assays made on the same day with the same proteins. 2.5. Cell culture and transient transfection The mammary carcinoma cell line, MCF7 and the human lens epi- thelial cell line, FHL124 cells (Wormstone et al., 2000) were seeded in DMEM and EMEM respectively supplemented with glutamine, peni- cillin, streptomycin and 10% (v/v) FBS. Cells were transfected with constructs designed to express BFSP1 constructs that had been sub- cloned into pEGFPN3 expression vector (http://www.clontech.com/ UK/Support/Applications/Using_Fluorescent_Proteins/EGFP_Vectors). Full length BFSP1 with or without the D433G and G434A mutations were transfected into MCF7 cells. For transfections into FHL124 cells, the 1–460 BFSP1 sequence fused to the N-terminus of eGFP was used to evidence cleavage at the D433 by caspase. Transfections were achieved using the GeneJuice transfection reagent (Novagen; http://www. emdmillipore.com/life-science-research/novagen/) according to man- ufacturers guidelines. The mammary carcinoma cell line, MCF7 and the human lens epi- thelial cell line, FHL124 cells (Wormstone et al., 2000) were seeded in DMEM and EMEM respectively supplemented with glutamine, peni- cillin, streptomycin and 10% (v/v) FBS. Cells were transfected with constructs designed to express BFSP1 constructs that had been sub- cloned into pEGFPN3 expression vector (http://www.clontech.com/ UK/Support/Applications/Using_Fluorescent_Proteins/EGFP_Vectors). Full length BFSP1 with or without the D433G and G434A mutations were transfected into MCF7 cells. For transfections into FHL124 cells, the 1–460 BFSP1 sequence fused to the N-terminus of eGFP was used to evidence cleavage at the D433 by caspase. Transfections were achieved using the GeneJuice transfection reagent (Novagen; http://www. emdmillipore.com/life-science-research/novagen/) according to man- ufacturers guidelines. A. Tapodi, et al. 2.11. Xenopus oocyte swelling assay Oocytes were obtained in two ways. Female Xenopus laevis were anesthetized, and stage V and VI oocytes prepared and injected with 10 ng cRNA as previously described (Németh-Cahalan et al., 2004, 2007). In some experiments, especially later ones, oocytes were ob- tained from Ecocyte, Austin TX. The oocytes were incubated in 100% ND96 with the desired test Ca2+ concentration or pH for 5min before the swelling assay. Swelling assays were performed at room tempera- ture (20–21 °C) by transferring oocytes from a 200 mOsm (100% ND96: 96 mM NaCl 96, 2 mM KCl, 5 mM HEPES, 1.8 mM CaCl2, 1 mM MgCl2, 2.5 mM sodium pyruvate, pH7.4) to a 70mOsm (30% (v/v) ND96) so- lution adjusted to the desired calcium concentration or pH. Water permeability, Pf, was calculated from optical measurements of the in- crease in cross-sectional area of the oocyte with time in response to diluted ND96 using the formula: We first analyzed whether BFSP1 was a potential caspase substrate in silico using the program CasCleave2 (Fig. 1A). Three potential sites were identified in the C-terminal region of BFSP1 (DVPD 430–433, IEPD, 446–449, and EERD, 566–569). If the site at D433 were cleaved, it would reveal a cryptic myristoylation motif. This was indicated by MYRISTOYLATOR and in agreement with the published proteomic data (Wang et al., 2010b). The C-terminal region of BFSP1 generally shows low sequence conservation between species (Song et al., 2009), but the caspase and associated myristoylation sequences (Fig. 1B) is a region that is highly conserved and adjacent to the K455 (Fig. 1B green label) which was identified by chemical cross-linking to interact with AQP0 (Wang and Schey, 2017). The conservation of the caspase site (DVPD 430–433) and myr- istoylation motif (GGQISK, 434–439) is especially striking as seen by the alignment examples from cartilaginous (whale shark), ray-finned (killifish, sunfish, zebrafish) and lobe-finned fishes (coelacanth) as well as amphibians (Xenopus), birds (chicken, duck) and of course mammals (mouse, rat cow, human; Fig. 1C). This suggests that caspase cleavage and subsequent N-terminal myristoylation may have physiological significance for a very broad range of animals. The observed chemical cross-link between K455 in BFSP1 and D246 in AQP0 (Wang and Schey, 2017) also identifies this a region that interacts directly with AQP0. Pf = [d(V/Vo)/dt][Vo/S]/[ΔosmVw)] where V is the volume as a function of time, V0 is the initial volume, S is the geometric surface area, Δosm is the osmotic gradient, and Vw is the molar volume of water. Error bars are ± SEM. A two-way ANOVA was performed with the software package R (www.r-project.org) to de- termine the statistical significance of the effect of calcium concentra- tion, BFSP1 fragment, and their effect on AQP0 permeability. Each data point is the average of at least nine measurements (three different batches of oocytes, three oocytes from each batch). Error bars are shown as ± SEM. A two-way ANOVA was performed with the software package R (www.r-project.org) to determine the statistical significance of the effect of calcium concentration, BFSP1 fragment, and their in- teraction on AQP0 permeability. Next we established biochemically that purified recombinant BFSP1 is indeed a substrate for activated caspases and identified those cas- pases specific for BFSP1 (Fig. 2A). Caspases 2, 3 and 5–7 all cleaved recombinant BFSP1 as demonstrated by immunoblotting using anti- bodies specific to the N-terminal region of BFSP1 (Fig. 2A; 3241) and to the C-terminal His-tag (Fig. 2A; Anti-His Tag). Notice that the frag- mentation pattern for the two antibodies are different and there are also differences in these respective patterns after cleavage by the various caspases (Fig. 2A). We interpret the band detected by the 3241 anti- bodies as equivalent to a 53 kDa fragment ending at residue D433 (Fig. 1A), which is a major fragment produced after exposure to cas- pases 2, 3 and 7, all of which were proposed to cleave at D433 (Fig. 1A). To confirm this was indeed a caspase site, we disrupted the consensus sequence with the mutation D433A and found that this completely abolished the caspase 2 mediated cleavage of BFSP1 (Fig. 2). Mutating D549, another potential caspase 2 site, also altered the cleavage of BFSP1 (Fig. 2B, asterisks). We conclude that caspase 2 efficiently 3.2. BFSP1 is a substrate for caspase cleavage and myristoylation Proteomic data has suggested that BFSP1 is potentially cleaved by caspases and myristoylated (Wang et al., 2010b), but there are no biochemical data identifying the specificity of the caspases and NMTs for BFSP1.i 3. Results electron modes for the instrument were collected and merged using Adobe Photoshop CS5. electron modes for the instrument were collected and merged using Adobe Photoshop CS5. 2.10. cRNA preparation for Xenopus oocytes Our results all lead to the conclusion that different BFSP1 fragments interact with AQP0 and modulate the regulation of its water perme- ability by calcium. We begin by showing that BFSP1 is a substrate for caspases and that caspase cleavage can generate C-terminal domain fragments of BFSP1 that alter AQP0 Pf regulation. We also demonstrate that myristoylation (which takes place at a cryptic site, G434, revealed by caspase cleavage) plays a critical role in the functional interaction of different BFSP1 fragments with AQP0. We show that AQP0 and BFSP1 fragments colocalize in the lens, and finally we demonstrate in vitro that the different BFSP1 fragments generated by caspase cleavage modulate calcium regulation of AQP0 Pf in different ways. The expression construct for bovine AQP0 (pXßG -AQP0) was a kind gift from Peter Agre (Baltimore MD). Wild type and mutated AQP0 cRNAs were transcribed in vitro using the T3 RNA polymerase (mMACHINE Kit; Ambion Inc.).f Expression constructs for different BFSP fragments were cloned from pET23 vectors with BamHI and BglII and ligating them into the BamHI cloning site in pXßG for expression in Xenopus oocytes. In most experiments 10 ng of cRNA of AQP0 and 10 ng of the experimentally tested BFSP fragment were injected into oocytes for expression. Typically two days elapsed between cRNA injection and measurement of Pf in the oocyte swelling assay. 2.6. Caspase assays in MCF7 and FHL124 cells MCF7 and FHL124 cells were transfected with wild type and caspase mutant BFSP1 fragments fused to the eGFP N-terminus. The caspase cleavage of BFSP1 was determined by immunoblotting to detect BFSP1 fragments using either the BFSP1 antibodies 3241 (Sandilands et al., 2004), or the eGFP antibodies (Cell Signaling Technologies, Rabbit polyclonal #2555). After transfection, MCF7 and FHL-124 cells were treated with 0.15 mM hydrogen peroxide for 3 h as an apoptogen to activate caspases. Cells were collected in lysis buffer (10 mM Tris-HCl pH6.8, 1 mM EDTA, 1X protease cOMPLETE inhibitor cocktail Roche (http://www.roche.co.uk), 1% (w/v) SDS) and then boiled after the addition of 5X sample buffer followed by SDS PAGE and immunoblot- ting. 3 Experim ental Eye Research 185 (2019) 107585 Experim ental Eye Research 185 (2019) 107585 A. Tapodi, et al. 2.11. Xenopus oocyte swelling assay Indeed a C-terminal region of rat BFSP1 distal to D433 has been ob- served to increase AQP0 water channel activity about two fold, but the influence of myristoylation or the flanking sequences (Fig. 1B) on the regulation of AQP0 by BFSP1 was not considered (Nakazawa et al., 2011).i 2.12. Preparation of oocyte membrane protein and western blotting Total membrane protein was isolated from Xenopus laevis oocytes using the ProteoExtract native membrane protein extraction kit (Calbiochem) according to the manufacturer's directions. One oocytes worth of membrane protein in each lane was separated on a 4–12% SDS NuPAGE gel (Invitrogen) and transferred onto a nitrocellulose mem- brane. The blot was blocked in 5% (w/v) skimmed milk in Tris-buf- fered-saline with 0.1% (v/v) Tween (TBS-T), then incubated with anti- BFSP1 antibody (Rabbit polyclonal antibody 3241 (Sandilands et al., 2004);) diluted at 1:200 in blocking solution (1% (w/v) BSA, 2 mM EDTA in TBS-T). The immunoblot was then washed and incubated with goat anti-rabbit HRP conjugated antibody diluted at 1:1000 (Pierce) for 1 h at room temperature and visualized by ECL Prime Blotting Detec- tion reagent (Amersham). 4 Experim ental Eye Research 185 (2019) 107585 A. Tapodi, et al. p , p y Fig. 1. Bioinformatic analysis of the C-terminal sequences of BFSP1. A. The main structural features of human BFSP1 and the location of predicted caspase cleavage sites. The residue numbers for potential cleavage sites are included. B. Sequence alignment of human, rat and bovine BFSP1 using CLUSTALW to identify regions of homology. Caspase sites as predicted for BFSP1 are indicated (red). The caspase site adjacent to the cryptic myristoylation motif (mauve) is in a region of homology between human, rat and cow. The construct used to study the potential functional interaction of rat BFSP1 with AQP0 is written in blue script. The BFSP1 residue identified by crosslinking with AQP0 is indicated (green). Notice the relative positions of the myristoylation sequence and the predicted caspase sites and the variability in the location of these between the three species. C. The caspase site and adjacent cryptic myristoylation sequence in BFSP1 is conserved over a wide range of animals from whale shark to human. Fig. 1. Bioinformatic analysis of the C-terminal sequences of BFSP1. ig. 1. Bioinformatic analysis of the C-terminal sequences of BFSP1. Fig. 1. Bioinformatic analysis of the C-terminal sequences of BFSP1. A. The main structural features of human BFSP1 and the location of predicted caspase cleavage sites. The residue numbers for potential cleavage sites are included. B. Sequence alignment of human, rat and bovine BFSP1 using CLUSTALW to identify regions of homology. Caspase sites as predicted for BFSP1 are indicated (red). 2.12. Preparation of oocyte membrane protein and western blotting Comparing WT, D433A and D549A BFSP1 sensitivity to caspase 2 cleavage resulted in the absence of the characteristic 53 kDa fragment (arrowhead) in the G433A BFSP1 as detected by the 3241 polyclonal antibodies. Using the anti-His tag antibodies, two characteristic fragments (arrowheads) were detected in the wild type and D549A BFSP1 samples, but only the faster migrating band in the D433A BFSP1 sample. Similarly the bands indicated (Asterisks) were present in the WT and D433A BFSP1 samples but missing from the D549A BFSP1 sample. This indicates that the mutations have abolished one site at position D433 and one at D549 for caspase 2. A. In vitro cleavage of BFSP1 by purified caspases 1–10. Full length recombinant BFSP1 containing a C-terminal polyhistidine tag is indicated (chevron). Using the polyclonal antibody 3241 that was raised against the bovine 53 kDa fragment of BFSP1, two major fragments were detected (arrows). One corresponded to the 53 kDa fragment and like the slower migrating band of the two, both were undetected by the His-tag directed antibodies. Therefore both these fragments likely lack different proportions of the C-terminal sequences. Using the anti-His tag antibodies, two prominent bands were detected in the caspase 2 treated BFSP1 sample (arrowheads). Neither of these bands comigrated with bands detected by the 3241 polyclonal antibodies. B. To evidence cleavage of BFSP1 by caspase 2 at the D433 and D549 sites, two BFSP1 mutants were produced, D433A and D549A. Comparing WT, D433A and D549A BFSP1 sensitivity to caspase 2 cleavage resulted in the absence of the characteristic 53 kDa fragment (arrowhead) in the G433A BFSP1 as detected by the 3241 polyclonal antibodies. Using the anti-His tag antibodies, two characteristic fragments (arrowheads) were detected in the wild type and D549A BFSP1 samples, but only the faster migrating band in the D433A BFSP1 sample. Similarly the bands indicated (Asterisks) were present in the WT and D433A BFSP1 samples but missing from the D549A BFSP1 sample. This indicates that the mutations have abolished one site at position D433 and one at D549 for caspase 2. D433A mutation prevented the appearance of a prominent 53 kDa product as identified by the 3241 antibodies (Fig. 3B, arrow) and sev- eral other products too (Fig. 3B arrowheads). The patterns achieved for the WT, D433A and G434A BFSP1 were not identical suggestive of a more complex caspase sensitivity for BFSP1 in MCF7 cells (Fig. 2.12. Preparation of oocyte membrane protein and western blotting The caspase site adjacent to the cryptic myristoylation motif (mauve) is in a region of homology between human, rat and cow. The construct used to study the potential functional interaction of rat BFSP1 with AQP0 is written in blue script. The BFSP1 residue identified by crosslinking with AQP0 is indicated (green). Notice the relative positions of the myristoylation sequence and the predicted caspase sites and the variability in the location of these between the three species. C. The caspase site and adjacent cryptic myristoylation sequence in BFSP1 is conserved over a wide range of animals from whale shark to human. general caspase inhibitor zVAD prevented the detection by im- munoblotting of discrete BFSP1 fragments in MCF7 cells (Fig. 3A). In- troducing the D433A mutation also prevented the appearance of the 53 kDa BFSP1 fragment in transfected MCF7 cells (Fig. 3A, arrow) whilst the G434A mutation had little effect on the formation of the 53 kDa fragment (Fig. 3A, arrow). Similarly, in FHL124 cells, the In order to demonstrate the caspase sensitivity of BFSP1 in vivo, BFSP1 constructs were transfected into two types of cultured cells (Fig. 3A and B). In both MCF7 cells (Fig. 3A) and the lens derived human cell line FHL124 (Fig. 3B), transiently transfected BFSP1 was also cleaved in a caspase dependent manner. The inclusion of the 5 Experim ental Eye Research 185 (2019) 107585 A. Tapodi, et al. Fig. 2. Characterization of the caspase sensitivity of BFSP1.i Fig. 2. Characterization of the caspase sensitivity of BFSP1.i Fig. 2. Characterization of the caspase sensitivity of BFSP1. A. In vitro cleavage of BFSP1 by purified caspases 1–10. Full length recombinant BFSP1 containing a C-terminal polyhistidine tag is indicated (chevron). Using the polyclonal antibody 3241 that was raised against the bovine 53 kDa fragment of BFSP1, two major fragments were detected (arrows). One corresponded to the 53 kDa fragment and like the slower migrating band of the two, both were undetected by the His-tag directed antibodies. Therefore both these fragments likely lack different proportions of the C-terminal sequences. Using the anti-His tag antibodies, two prominent bands were detected in the caspase 2 treated BFSP1 sample (arrowheads). Neither of these bands comigrated with bands detected by the 3241 polyclonal antibodies. B. To evidence cleavage of BFSP1 by caspase 2 at the D433 and D549 sites, two BFSP1 mutants were produced, D433A and D549A. 3.4. Different fragments of BFSP affect AQP0 regulation differently 3.4. Different fragments of BFSP affect AQP0 regulation differently Our caspase sensitivity (Figs. 2 and 3) and immunoblotting (Fig. 5A) data suggest that the BFSP1 C-terminal sequence distal to the caspase- cleavage site at D433 is likely further processed by proteolysis. Evi- dence for such additional caspase-mediated proteolysis is not apparent in the literature, but identifying such sites will require further in- vestigation (Wang and Schey, 2011). We designed a series of constructs (Fig. 6A) to investigate the potential roles of the myristoylation motif in BFSP1 and other C-terminal sequences based on the predicted caspase sites at D433 and D549 (Figs. 1 and 6A). Although we have no evidence as yet to suggest that D549 is a physiological caspase site, it provided a rationale to divide the C-terminal region of BFSP1 into smaller domains in order to probe potential functional differences. Immunoblots showed that BFSP1 was expressed and located in the membrane fraction pre- pared from injected Xenopus oocytes (Fig. 6B). Most of the BFSP1 C-terminal fragments, full length BFSP and full length BFSP containing the D433A mutant and the D549A mutant lock the water permeability of AQP0 high and eliminate the increase of Pf in response to low Ca2+ (Fig. 6A and Table 1). The fragment, A434 – P548, has no effect on the response of AQP0 to calcium. (See Fig. 6A and Table 1). The G434 – S665 fragment blunts the calcium response even though the difference between Pf in 0 mM Ca2+ and 1.8 mM Ca2+ is marginally significant. We then used these antibodies to immunolocalise both BFSP1 C- terminal sequences containing the myristoylation motif and AQP0 on alkali stripped plasma membranes prepared from the 51 year old human lens nucleus (Fig. 5B) and cortex (Fig. 5C) by scanning electron microscopy. Previous immunofluorescence analysis had shown by fluorescence light microscopy that both proteins occupy the same plasma membrane regions in the lens (Lindsey Rose et al., 2006), but immunogold electron microscopy provides greater resolution. Whilst NP53 antibodies (Fig. 5E) and preimmune serum (Fig. 5D) gave very little signal, the NP Tail serum clearly stained the lens membranes (Fig. 5F) Co-staining of plasma membranes with both NPTail and B11 antibodies (Fig. 5B and C; BFSP1, 10 nm gold particles; AQP0, 5 nm i For wild type (WT) AQP0 alone, 0 mM Ca2+ approximately doubles Pf. 2.12. Preparation of oocyte membrane protein and western blotting 3A) and most likely FHL124 cells too (Fig. 3B) involving other BFSP1 caspase sites as predicted by our bioinformatic (Fig. 1A) and biochemical (Fig. 2A) analyses. These transient transfection data also support the conclusion that D433 is a bone fide caspase site in BFSP1 (Wang et al., 2010b). Supplementary Fig. 1) values obtained for the BFSP1 octapeptide were comparable to those obtained for the pp60src octapeptide standard (2.5 ± 0.2 and 0.9 ± 0.1 μM μM for NMT1 and NMT2 respectively) for as reported previously for this substrate in vitro assay (Goncalves et al., 2012). NMT1 and NMT2 are both present in the lens (Shrivastav et al., 2004). These data therefore provide the biochemical evidence to support the previous proteomic data that identified a myristoylated G434 after cleavage at D433 (Bassnett et al., 2009; Wang et al., 2010b, 2013; Wang and Schey, 2015).fi Azido-myristate very efficiently labelled many proteins in MCF7 transfected cells (data not shown) making it difficult to detect cleaved myristoylated BFSP1. We therefore generated two polyclonal antibodies (NP53 and NPtail) to identify the DVPD and GGQSIK containing epi- topes in BFSP1. We demonstrate that these two antibodies identify different immunoreactive banding patterns in samples from human lens plasma membranes (Fig. 4C). Indeed the major fragment detected by the NPtail antibodies in alkali stripped, lens fiber cell plasma mem- branes was less than 25 kDa indicating that the C-terminal region is not only cleaved at position D433, but also very likely at other distal sites as predicted (Fig. 1A). To demonstrate the myristoylation potential of the N-terminal G434 in the caspase-cleaved BFSP1, we took two different approaches. In the first, a bacterial system that co-expressed the Candida albicans NMT with a putative target was analyzed (Fig. 4A). Only when both azido- myristate and the NMT were present in the same bacterial extract was a positive signal detected (Fig. 4A, tracks 10 and 12) for the positive control (PfARF1) and for the BFSP1 C-terminal sequences starting at G434. In the second approach (Fig. 4B), the BFSP1 octapeptide span- ning residues 434–441, GGQISKGF, was analyzed for its substrate po- tential using recombinant human NMT1 and NMT2. The Km (5.7 ± 0.7 μM and 3.4 ± 0.7 μM for NMT1 and NMT2 respectively; 6 Experim ental Eye Research 185 (2019) 107585 A. Tapodi, et al. Fig. 3. Caspase sensitivity of transfected BFSP1 in MCF7 and FHL124 cells. 80 kDa product (chevrons). Notice the major breakdown product (53 kDa) for the construct containing the wild type D433 residue and the very significant reduction when this was replaced by A433. Slower migrating products (arrowheads) could arise by caspase cleavage either within the BFSP1 or eGFP parts of the fusion protein. Marker proteins (m) are indicated (•) and correspond to 250, 130, 100, 70, 55, 35, 25 and 15 kDa as per PageRuler prestained standards. 3.3. Co-immunolocalization of BFSP1 C-terminal sequences and AQP0 in human lens plasma membranes 3.3. Co-immunolocalization of BFSP1 C-terminal sequences and AQP0 in human lens plasma membranes 3.3. Co-immunolocalization of BFSP1 C-terminal sequences and AQP0 in human lens plasma membranes gold particles) showed that AQP0 and the G434 containing fragments of BFSP1were adjacent to each other in the membrane preparations (Fig. 5B and C; arrows and arrowheads). These data indicate that AQP0 and BFSP1 C-terminal fragments containing the myristoylation se- quence are in very close proximity at the plasma membranes of the human lens. These data are in agreement with previous crosslinking (Wang and Schey, 2017) and proteomic (Bassnett et al., 2009; Wang et al., 2010b, 2013; Wang and Schey, 2015) data. Previous biochemical fractionation of the lens identified a ∼51 kDa C-terminal BFSP1 fragment associated with lens plasma membranes even after urea extraction (Brunkener and Georgatos, 1992). It was important to show that the BFSP1 C-terminal region distal to G434 was indeed a resident plasma membrane polypeptide and to confirm recent proteomic analyses (Bassnett et al., 2009; Wang et al., 2010b, 2013; Wang and Schey, 2015), we utilized the NPTail and NP53 polyclonal antibodies to identify both the caspase cleavage site (NP53 PAb) and the myristoylation sequence (NPTail PAb) in samples prepared from a single male 51 year old donor. Both sera detected complex patterns in human lens extracts, which were absent from the pre-immune sera (Fig. 5A). We confirmed that alkali-extracted plasma membranes pur- ified from the cortex and nucleus of human lenses retained only the C- terminal sequences containing the myristoylation sequence in donor lenses aged from 18 to 79 and in both the nuclear and cortical lens membranes (Supplementary Fig. 2). Moreover, these alkali stripped membranes were enriched in BFSP1 fragments in the 20 kDa range immunoreactive with these antibodies indicating that further proteo- lysis had likely occurred (Fig. 5A; Supplementary Fig. 1). Using the NP53 polyclonal antibodies to detect the adjacent caspase-cleavage site to the myristoylation sequence, we found that this N-terminal portion of BFSP1 was lost from the alkali-stripped membranes (Fig. 5A). Im- munoblotting of the same fractions with AQP0 antibodies confirmed that the fractionation enriched for integral membrane proteins as they were retained after alkali stripping (Fig. 5A). 2.12. Preparation of oocyte membrane protein and western blotting MCF7 and FHL124 cells were transiently transfected with constructs encoding eGFP alone, or as a N- terminal fusion with human BFSP1, and the mutants G433A (MCF7 and FHL124) and G434A BFSP1 (MCF7 only). Transfected BFSP1 was detected using the polyclonal antibodies 3241. A. MCF7 cells were either untreated or treated with the generic caspase inhibitor (zVAD) or with 0.15 M H202 in order to induce effector caspases. Full length eGFP-BFSP1 is indicated (chevron). A 53 kDa fragment (arrow) was de- tected by the 3241 antibodies. Note the absence of the 53 kDa signal in cells transfected with D433A, but not the G434A BFSP1 construct. The caspase inhibitor also prevented the formation of this fragment. B. The D433A mutation blocks the caspase sen- sitivity of BFSP1 constructs transiently trans- fected into the human lens epithelial cell line FHL124. The BFSP1 sequence 1–460 was fused to the N-terminus of eGFP and transiently transfected into FHL124 cells resulting in an 80 kDa product (chevrons). Notice the major breakdown product (53 kDa) for the construct containing the wild type D433 residue and the very significant reduction when this was replaced by A433. Slower migrating products (arrowheads) could arise by caspase cleavage either within the BFSP1 or eGFP parts of the fusion protein. Marker proteins (m) are indicated (•) and correspond to 250, 130, 100, 70, 55, 35, 25 and 15 kDa as per PageRuler prestained standards. Fig. 3. Caspase sensitivity of transfected BFSP1 in MCF7 and FHL124 cells. MCF7 and FHL124 cells were transiently transfected with constructs encoding eGFP alone, or as a N- terminal fusion with human BFSP1, and the mutants G433A (MCF7 and FHL124) and G434A BFSP1 (MCF7 only). Transfected BFSP1 was detected using the polyclonal antibodies 3241. Fig. 3. Caspase sensitivity of transfected BFSP1 in MCF7 and FHL124 cells. MCF7 and FHL124 cells were transiently transfected with constructs encoding eGFP alone, or as a N- terminal fusion with human BFSP1, and the mutants G433A (MCF7 and FHL124) and G434A BFSP1 (MCF7 only). Transfected BFSP1 was detected using the polyclonal antibodies 3241. A. MCF7 cells were either untreated or treated with the generic caspase inhibitor (zVAD) or with 0.15 M H202 in order to induce effector caspases. Full length eGFP-BFSP1 is indicated (chevron). A 53 kDa fragment (arrow) was de- tected by the 3241 antibodies. 2.12. Preparation of oocyte membrane protein and western blotting Note the absence of the 53 kDa signal in cells transfected with D433A, but not the G434A BFSP1 construct. The caspase inhibitor also prevented the formation of this fragment. A. MCF7 cells were either untreated or treated with the generic caspase inhibitor (zVAD) or with 0.15 M H202 in order to induce effector caspases. Full length eGFP-BFSP1 is indicated (chevron). A 53 kDa fragment (arrow) was de- tected by the 3241 antibodies. Note the absence of the 53 kDa signal in cells transfected with D433A, but not the G434A BFSP1 construct. The caspase inhibitor also prevented the formation of this fragment. g B. The D433A mutation blocks the caspase sen- sitivity of BFSP1 constructs transiently trans- fected into the human lens epithelial cell line FHL124. The BFSP1 sequence 1–460 was fused to the N-terminus of eGFP and transiently transfected into FHL124 cells resulting in an 80 kDa product (chevrons). Notice the major breakdown product (53 kDa) for the construct containing the wild type D433 residue and the very significant reduction when this was replaced by A433. Slower migrating products (arrowheads) could arise by caspase cleavage either within the BFSP1 or eGFP parts of the fusion protein. Marker proteins (m) are indicated (•) and correspond to 250, 130, 100, 70, 55, 35, 25 and 15 kDa as per PageRuler prestained standards. 3.4. Different fragments of BFSP affect AQP0 regulation differently The soluble protein fraction (S1), the plasma membrane cytoskeleton complex (PMCC) and plasma membrane fraction (P3) were isolated from the human cortex and separated by SDS PAGE and the 10% (w/v) polyacrylamide gel prior to either staining with Coomassie Blue or immunoblotting and probing with the polyclonal antibodies 3241, NP-53 and NP-Tail. Notice that the major im- munoreactive band detected in the P3 sample by the NP-tail antibodies would correspond to a C-terminal fragment of less than 25 kDa. Marker proteins (m) are indicated (•) and correspond to 250, 130, 100, 70, 55, 35, 25 and 15 kDa as per PageRuler prestained standards. Fig. 4. In vivo Myristoylation assay of truncated Tail fragment of BFSP1: Fig. 4. In vivo Myristoylation assay of truncated Tail fragment of BFSP1: A: Truncated Tail fragment of BFSP1 was co-expressed with CaNMT in E. coli in the presence of azido-myristate. CLICK-chemistry was used to detect myristolyated proteins and detected in the gel after fluorescently labelled with Cy3-TAMRA. PfARF1 (Plasmodium falciparum ADP-ribosylation factor-1) was included as a positive control of myristoylation. Track1 and 2, Pre- and post-induction of PfARF expression in E.coli; Tracks 3 and 4, Pre- and post-induction of PfARF with C.albicans NMT expression in E.coli; Tracks 5 and 6: Pre- and post-induction of G434-P548 BFSP1 expression in E.coli; Tracks 7 and 8, Pre- and post-induction of BFSP1 G434-P548 with C.albicans NMT expression in E.coli; Tracks 9 and 10, Pre- and post-induction of PfARF with C.albicans NMT expression in E.coli fed with azido-myristate followed by CLICK chemistry detection; Tracks 11 and 12, Pre- and post-induction of BFSP1 G434-P548 with C.albicans NMT expression in E.coli fed with azido- myristate followed by CLICK chemistry detection. Notice the positive bands in tracks 10 and 12 only. B. Km and Vmax determination for recombinant human NMT1 and 2 for the BFSP1 sequence G434- F441. C Characterization of the polyclonal antibodies NP Tail and NP53 using human lens samples The soluble protein fraction (S1) the plasma membrane cytoskeleton g y y y g A: Truncated Tail fragment of BFSP1 was co-expressed with CaNMT in E. coli in the presence of azido-myristate. CLICK-chemistry was used to detect myristolyated proteins and detected in the gel after fluorescently labelled with Cy3-TAMRA. PfARF1 (Plasmodium falciparum ADP-ribosylation factor-1) was included as a positive control of myristoylation. 4.1. BFSP1 – a substrate for both caspase and NMT activities A myristoylated fragment of BFSP1 was previously identified in the bovine lens (Wang et al., 2010b) and we have demonstrated in vitro that the sequence GGQISK is efficiently myristoylated in vitro by both human NMT1 and 2 and when co-tranfected with a fungal NMT in E. coli. The eye lens contains both NMT1 and 2 (Shrivastav et al., 2004) that ac- tively myristoylate a specific subset of lens proteins (Cenedella and Chandrasekher, 1999). The myristoylation motif in BFSP1 is a cryptic 3.4. Different fragments of BFSP affect AQP0 regulation differently The soluble protein fraction (S1), the plasma membrane cytoskeleton complex (PMCC) and plasma membrane fraction (P3) were isolated from the human cortex and separated by SDS PAGE and the 10% (w/v) polyacrylamide gel prior to either staining with Coomassie Blue or immunoblotting and probing with the polyclonal antibodies 3241, NP-53 and NP-Tail. Notice that the major im- munoreactive band detected in the P3 sample by the NP-tail antibodies would correspond to a C-terminal fragment of less than 25 kDa. Marker proteins (m) are indicated (•) and correspond to 250, 130, 100, 70, 55, 35, 25 and 15 kDa as per PageRuler prestained standards. terminal tail fragments derived from full length BFSP1 by caspase cleavage can affect the calcium regulation of the water permeability of AQP0 using a Xenopus oocyte swelling assay. We also show that blocking the myristoylation potential of such fragments can reverse this potential regulation (Fig. 7). Whilst direct translation to lens fiber cells is speculative, the data suggest important physiological implications for the calcium mediated effects upon AQP0-dependent water transport in lens fiber cells. BFSP1 with a mutation at caspase site 433 or 549 (D433A or D549A; Fig. 6B). Notice the presence of a major immunoreactive band in ad- dition to the full length BFSP1 (Fig. 6B; arrowheads), which was eliminated by the D433A mutation in BFSP1. Notice too that the im- munoreactive banding pattern was affected by co-transfection with AQP0 (Fig. 6B D433A BFSP1 cf AQP0+D433A BFSP1; asterix). A second caspase cleaved product (Fig. 6B D459A BFSP1 and AQP0+D459A BFSP1; dot) was retained in both the D433A and D549A mutants, which was absent from the wild type BFSP1 sample. Full length BFSP1 and full length BFSP1 with either caspase-nulling muta- tion both eliminated Pf regulation by altered Ca2+ concentration (Fig. 6C). 3.4. Different fragments of BFSP affect AQP0 regulation differently But if serine 235 is phosphorylated, 0 mM Ca2+ has no effect on Pf. The data in Fig. 6A and Table 1 establish that fragments which contain a competent myristoylation site at D434 are capable of altering the Ca2+ regulation of Pf and, with the probable exception of the G434 – S665 fragment, of locking the Pf of AQP0 high.f We also evaluated the effects of full length BFSP1 and full length 7 Fig. 4. In vivo Myristoylation assay of truncated Tail fragment of BFSP1: A: Truncated Tail fragment of BFSP1 was co-expressed with CaNMT in E. coli in the presence of azido-myristate. CLICK-chemistry was used to detect myristolyated i d d d i h l f fl l l b ll d i h f ( l di f l i ib l i f ) i l d d i i A. Tapodi, et al. Experim ental Eye Research 185 (2019) 107585 Experim ental Eye Research 185 (2019) 107585 A. Tapodi, et al. Fig. 4. In vivo Myristoylation assay of truncated Tail fragment of BFSP1: A: Truncated Tail fragment of BFSP1 was co-expressed with CaNMT in E. coli in the presence of azido-myristate. CLICK-chemistry was used to detect myristolyated proteins and detected in the gel after fluorescently labelled with Cy3-TAMRA. PfARF1 (Plasmodium falciparum ADP-ribosylation factor-1) was included as a positive control of myristoylation. Track1 and 2, Pre- and post-induction of PfARF expression in E.coli; Tracks 3 and 4, Pre- and post-induction of PfARF with C.albicans NMT expression in E.coli; Tracks 5 and 6: Pre- and post-induction of G434-P548 BFSP1 expression in E.coli; Tracks 7 and 8, Pre- and post-induction of BFSP1 G434-P548 with C.albicans NMT expression in E.coli; Tracks 9 and 10, Pre- and post-induction of PfARF with C.albicans NMT expression in E.coli fed with azido-myristate followed by CLICK chemistry detection; Tracks 11 and 12, Pre- and post-induction of BFSP1 G434-P548 with C.albicans NMT expression in E.coli fed with azido- myristate followed by CLICK chemistry detection. Notice the positive bands in tracks 10 and 12 only. B. Km and Vmax determination for recombinant human NMT1 and 2 for the BFSP1 sequence G434- F441. C. Characterization of the polyclonal antibodies NP-Tail and NP53 using human lens samples. 3.4. Different fragments of BFSP affect AQP0 regulation differently Track1 and 2, Pre- and post-induction of PfARF expression in E.coli; Tracks 3 and 4, Pre- and post-induction of PfARF with C.albicans NMT expression in E.coli; Tracks 5 and 6: Pre- and post-induction of G434-P548 BFSP1 expression in E.coli; Tracks 7 and 8, Pre- and post-induction of BFSP1 G434-P548 with C.albicans NMT expression in E.coli; Tracks 9 and 10, Pre- and post-induction of PfARF with C.albicans NMT expression in E.coli fed with azido-myristate followed by CLICK chemistry detection; Tracks 11 and 12, Pre- and post-induction of BFSP1 G434-P548 with C.albicans NMT expression in E.coli fed with azido- myristate followed by CLICK chemistry detection. Notice the positive bands in tracks 10 and 12 only. B. Km and Vmax determination for recombinant human NMT1 and 2 for the BFSP1 sequence G434- F441. C. Characterization of the polyclonal antibodies NP-Tail and NP53 using human lens samples. The soluble protein fraction (S1), the plasma membrane cytoskeleton complex (PMCC) and plasma membrane fraction (P3) were isolated from the human cortex and separated by SDS PAGE and the 10% (w/v) polyacrylamide gel prior to either staining with Coomassie Blue or immunoblotting and probing with the polyclonal antibodies 3241, NP-53 and NP-Tail. Notice that the major im- munoreactive band detected in the P3 sample by the NP-tail antibodies would correspond to a C-terminal fragment of less than 25 kDa. Marker proteins (m) are indicated (•) and correspond to 250, 130, 100, 70, 55, 35, 25 and 15 kDa as per PageRuler prestained standards. C. Characterization of the polyclonal antibodies NP-Tail and NP53 using human lens samples. The soluble protein fraction (S1), the plasma membrane cytoskeleton complex (PMCC) and plasma membrane fraction (P3) were isolated from the human cortex and separated by SDS PAGE and the 10% (w/v) polyacrylamide gel prior to either staining with Coomassie Blue or immunoblotting and probing with the polyclonal antibodies 3241, NP-53 and NP-Tail. Notice that the major im- munoreactive band detected in the P3 sample by the NP-tail antibodies would correspond to a C-terminal fragment of less than 25 kDa. Marker proteins (m) are indicated (•) and correspond to 250, 130, 100, 70, 55, 35, 25 and 15 kDa as per PageRuler prestained standards. C. Characterization of the polyclonal antibodies NP-Tail and NP53 using human lens samples. 4. Discussion Coomassie blue staining of the proteins in these fractions (CB Stain) revealed a few prominent low molecular weight proteins between the 25 and 15 kDa markers. Immunoblotting of these plasma membrane fractions with the polyclonal antibodies NP-53 identified BFSP1 N-terminal-derived fragments only in the urea extracted membranes. These were removed after alkali extraction. In contrast, the NP Tail antibodies identified bands in both urea and alkali extracted membrane fractions, although the fragments retained in the alkali stripped membrane fraction were those with faster electrophoretic mobility equivalent to approx. 25 kDa. Probing these fractions with AQP0 antibodies demonstrated the enrichment of AQP0 signal in the alkali stripped plasma membrane fractions of both the nuclear and cortical parts of the lens. Marker proteins (m) are indicated (•) and correspond to 250, 130, 100, 70, 55, 35, 25 and 15 kDa as per PageRuler prestained standards. B, C. Co-immuno-gold labeling of alkali stripped plasma membranes from the nucleus (B) and cortex (C) of human lenses using the rabbit polyclonal NP Tail antibodies to detect BFSP1 and mouse monoclonal antibodies to detect AQP0. To visualize the antibodies in the SEM, 5 nm and 10 nm gold labelled secondary antibodies were used to detect AQP0 (arrowheads) and BFSP1(arrows) respectively. D, E and F. Immunogold labeling of alkali stripped membranes from human lens nuclei and labelled with either the preimmune serum for the NP Tail antibodies (D) or the polyclonal antibodies NP-53 (E). These two evidence the background labeling achievable either with preimmune (D) or closely related polyclonal rabbit tib di (E) Th NP53 d t t bl BFSP1 f t h b d b th lk li t ti f th b ti (A) Th i l d t t d b th D, E and F. Immunogold labeling of alkali stripped membranes from human lens nuclei and labelled with either the preimmune serum for the NP Tail antibodies (D) or the polyclonal antibodies NP-53 (E). These two evidence the background labeling achievable either with preimmune (D) or closely related polyclonal rabbit antibodies (E). The NP53 detectable BFSP1 fragments have been removed by the alkali extraction of these membrane preparations (A). The signal detected by the polyclonal NP Tail antibodies (F) is very clearly above background levels. site requiring protein cleavage for it to become available for myr- istoylation. 4. Discussion The plasma membranes were extracted with buffer containing 8 M urea (UREA) and then with 0.1 M NaOH (NaOH) and samples of the pelleted material taken and separated by SDS PAGE. Coomassie blue staining of the proteins in these fractions (CB Stain) revealed a few prominent low molecular weight proteins between the 25 and 15 kDa markers. Immunoblotting of these plasma membrane fractions with the polyclonal antibodies NP-53 identified BFSP1 N-terminal-derived fragments only in the urea extracted membranes. These were removed after alkali extraction. In contrast, the NP Tail antibodies identified bands in both urea and alkali extracted membrane fractions, although the fragments retained in the alkali stripped membrane fraction were those with faster electrophoretic mobility equivalent to approx. 25 kDa. Probing these fractions with AQP0 antibodies demonstrated the enrichment of AQP0 signal in the alkali stripped plasma membrane fractions of both the nuclear and cortical parts of the lens. Marker proteins (m) are indicated (•) and correspond to 250, 130, 100, 70, 55, 35, 25 and 15 kDa as per PageRuler prestained standards. B, C. Co-immuno-gold labeling of alkali stripped plasma membranes from the nucleus (B) and cortex (C) of human lenses using the rabbit polyclonal NP Tail antibodies to detect BFSP1 and mouse monoclonal antibodies to detect AQP0. To visualize the antibodies in the SEM, 5 nm and 10 nm gold labelled secondary antibodies were used to detect AQP0 (arrowheads) and BFSP1(arrows) respectively. D, E and F. Immunogold labeling of alkali stripped membranes from human lens nuclei and labelled with either the preimmune serum for the NP Tail antibodies (D) or the polyclonal antibodies NP-53 (E). These two evidence the background labeling achievable either with preimmune (D) or closely related polyclonal rabbit antibodies (E). The NP53 detectable BFSP1 fragments have been removed by the alkali extraction of these membrane preparations (A). The signal detected by the polyclonal NP Tail antibodies (F) is very clearly above background levels. Fig. 5. Cofractionation and colocalization of BFSP1 and AQP0 to the plasma membrane compartment of lens fibre cells. A. Plasma membranes were prepared from the nucleus (N) and cortical (C) parts of the lens from a 51 year old male donor. The plasma membranes were extracted with buffer containing 8 M urea (UREA) and then with 0.1 M NaOH (NaOH) and samples of the pelleted material taken and separated by SDS PAGE. 4. Discussion In this paper we provide biochemical evidence to support the ob- served myristoylation of BFSP1 (Wang et al., 2010b) at a cryptic C- terminal site (G434) revealed after caspase cleavage. We show that C- 8 5. Cofractionation and colocalization of BFSP1 and AQP0 to the plasma membrane compartment of lens fibre cells. asma membranes were prepared from the nucleus (N) and cortical (C) parts of the lens from a 51 year old male donor. The plasma membranes were buffer containing 8 M urea (UREA) and then with 0.1 M NaOH (NaOH) and samples of the pelleted material taken and separated by SDS PAGE. Coom ng of the proteins in these fractions (CB Stain) revealed a few prominent low molecular weight proteins between the 25 and 15 kDa markers. Immunob plasma membrane fractions with the polyclonal antibodies NP-53 identified BFSP1 N-terminal-derived fragments only in the urea extracted membra removed after alkali extraction. In contrast, the NP Tail antibodies identified bands in both urea and alkali extracted membrane fractions, alth ments retained in the alkali stripped membrane fraction were those with faster electrophoretic mobility equivalent to approx. 25 kDa. Probing these AQP0 antibodies demonstrated the enrichment of AQP0 signal in the alkali stripped plasma membrane fractions of both the nuclear and cortical p Marker proteins (m) are indicated (•) and correspond to 250, 130, 100, 70, 55, 35, 25 and 15 kDa as per PageRuler prestained standards. Co-immuno-gold labeling of alkali stripped plasma membranes from the nucleus (B) and cortex (C) of human lenses using the rabbit polyclona odies to detect BFSP1 and mouse monoclonal antibodies to detect AQP0. To visualize the antibodies in the SEM, 5 nm and 10 nm gold labelled odies were used to detect AQP0 (arrowheads) and BFSP1(arrows) respectively. and F Immunogold labeling of alkali stripped membranes from human lens nuclei and labelled with either the preimmune serum for the NP Tail anti podi, et al. Experim ental Eye Research 185 (20 Experim ental Eye Research 185 (2019) 107585 A. Tapodi, et al. Fig. 5. Cofractionation and colocalization of BFSP1 and AQP0 to the plasma membrane compartment of lens fibre cells. A. Plasma membranes were prepared from the nucleus (N) and cortical (C) parts of the lens from a 51 year old male donor. 4. Discussion Regulation of AQP0 by BFSP1 C-terminal domains. Fig. 6. Regulation of AQP0 by BFSP1 C-terminal domains. A. Xenopus oocytes were injected with AQP0 alone or in combination with the indicated BFSP1 constructs. C-terminal constructs could be divided into two classes: ones which left the calcium response essentially identical to wild type, that is Pf was low in 1.8 mM Ca2+ and high in 0 mM Ca2+; or ones in which Pf did not change with Ca2+ concentration. Results were analyzed by omnibus ANOVA followed by pair wise t-tests for individual 0 mM Ca2+ - 1.8 mM Ca2+ comparisons. In the cases where Pf increased in response to no calcium, pairwise p values were less than 0.05 (). In cases where Pf did not respond to changes in calcium, p values were greater than 0.3 (ns). Conditional p values for 1.8 mM Ca2+ vs no Ca2+: AQP0 alone = 0.0232, +434–665 = 0.0328, +434–548 = 0.6823, +A434-548 = 0.0128, B. Immunoblots showing that BFSP1 was expressed in Xenopus oocytes when appropriate constructs were injected. Two major immunoreactive bands were detected (arrows), the faster migrating band a fragment derived by proteolytic cleavage because the D433E mutation in BFSP1 altered the pattern achieved. Notice too that the presence of AQP0 altered the banding pattern achieved with the D433A mutant (). BFSP1 immunoreactive bands are found in the pellet fraction prepared from injected oocytes. C. Full length BFSP1 and full length BFSP1 with mutations at putative caspase sites eliminated Pf regulation by calcium. Conditional p values for 1.8 mM Ca2+ vs no Ca2+: AQP0 alone = 0.001, +BFSP1 = 0.617, +BFSP1 D433A = 0.3913, +BFSP1 D549A = 0.6445. y ull length BFSP1 and full length BFSP1 with mutations at putative caspase sites eliminated Pf regulation by calcium. Condition : AQP0 alone = 0.001, +BFSP1 = 0.617, +BFSP1 D433A = 0.3913, +BFSP1 D549A = 0.6445. with mutations at putative caspase sites eliminated Pf regulation by calcium. Conditional p values for 1.8 mM Ca2+ vs no 0.617, +BFSP1 D433A = 0.3913, +BFSP1 D549A = 0.6445. multiple caspases (Fig. 2) and we have demonstrated that BFSP1 is cleaved at D433 (Figs. 2 and 3) in tissue culture cells, including the lens epithelial cell line FHL124 (Fig. 3). 4. Discussion Whilst these data do not identify which caspase(s) might be responsible, the lens clearly has caspase and caspase-like activities (Foley et al., 2004; Weber and Menko, 2005; Zandy and Bassnett, 2007) that would expose the cryptic myristoylation site in BFSP1. Indeed, the site (DxxDG) in BFSP1 conforms to the most favored to effect caspase cleavage (Mahrus et al., 2008). It is worth noting that the aspartate residue D433 in the caspase recognition se- quence, undergoes isomerization (Wang et al., 2010b). This post- translational modification is residue specific as other potential DG sites remain unchanged in BFSP1. Moreover the modification is found only in uncleaved peptides (Wang et al., 2010b), suggesting this might modulate caspase cleavage at D433 in BFSP1 and introducing a further mechanism to alter its interaction with AQP0. There are also many examples where post-translational myr- istoylation is associated with the exposure of a cryptic site after caspase cleavage of the protein (reviewed in (Martin et al., 2011)). We suggest that BFSP1, like actin (Utsumi et al., 2003), gelsolin (Sakurai and Utsumi, 2006) and cytoplasmic dynein intermediate chain 2A (Martin Table 1 Effects of BFSP1 Fragments on AQP0 calcium regulation. 4. Discussion Caspase activity is readily detected in the absence of apoptotic signals in the lens (Foley et al., 2004; Weber and Menko, 2005). The BFSP1 myristoylation motif is preceded immediately by a conventional caspase consensus site (Fig. 1) that is readily cleaved in vitro and in MCF7 cells (Fig. 2) and in lens derived human FHL124 cells (Fig. 3B). We suggest that the caspase-mediated cleavage to expose a cryptic myristoylation site is a credible mechanism to ex- plain the observed myristoylation of BFSP1 in lens membrane fractions (Wang et al., 2010b). Our data confirm that BFSP1 is a substrate for 9 Experim ental Eye Research 185 (2019) 107585 A. Tapodi, et al. Fig. 6. Regulation of AQP0 by BFSP1 C-terminal domains. A. Xenopus oocytes were injected with AQP0 alone or in combination with the indicated BFSP1 constructs. C-terminal constructs could be divided into two classes: ones which left the calcium response essentially identical to wild type, that is Pf was low in 1.8 mM Ca2+ and high in 0 mM Ca2+; or ones in which Pf did not change with Ca2+ concentration. Results were analyzed by omnibus ANOVA followed by pair wise t-tests for individual 0 mM Ca2+ - 1.8 mM Ca2+ comparisons. In the cases where Pf increased in response to no calcium, pairwise p values were less than 0.05 (). In cases where Pf did not respond to changes in calcium, p values were greater than 0.3 (ns). Conditional p values for 1.8 mM Ca2+ vs no Ca2+: AQP0 alone = 0.0232, +434–665 = 0.0328, +434–548 = 0.6823, +A434-548 = 0.0128, B. Immunoblots showing that BFSP1 was expressed in Xenopus oocytes when appropriate constructs were injected. Two major immunoreactive bands were detected (arrows), the faster migrating band a fragment derived by proteolytic cleavage because the D433E mutation in BFSP1 altered the pattern achieved. Notice too that the presence of AQP0 altered the banding pattern achieved with the D433A mutant (). BFSP1 immunoreactive bands are found in the pellet fraction prepared from injected oocytes. C. Full length BFSP1 and full length BFSP1 with mutations at putative caspase sites eliminated Pf regulation by calcium. Conditional p values for 1.8 mM Ca2+ vs no Ca2+: AQP0 alone = 0.001, +BFSP1 = 0.617, +BFSP1 D433A = 0.3913, +BFSP1 D549A = 0.6445. A. Tapodi, et al. Experim ental Eye Research 185 (2019) 107585 Fig. 6. Regulation of AQP0 by BFSP1 C-terminal domains. Fig. 6. 4.2. Functional role of BFSP1 myristoylation Myristoylation is insufficient to tether a protein to a membrane (Peitzsch and McLaughlin, 1993), but requires other proteins or poly- basic sequences to effect membrane targeting (Resh, 2004). For this reason myristoylation is believed to be a regulatory post-translational modification (Martin et al., 2011; Resh, 2006; Zha et al., 2000). Ana- lysis of the C-terminal sequences of BFSP1 (Fig. 1A) revealed that the myristoylation site was indeed conserved from whale shark to mam- mals (Fig. 1C) and was adjacent to a lysine/arginine-rich region. Fol- lowing BFSP1 cleavage by caspase activities, the fragments derived from this C-terminal region of BFSP1 resisted urea and alkali extraction (Figs. 4C and 5A) in agreement with previous proteomic analysis of such fractions from human lens fibre cell plasma membranes (Wang et al., 2010b, 2013; Wang and Schey, 2015). This evidences that they are strongly bound to the plasma membrane with properties more si- milar to integral membrane proteins like eg gap junction proteins (Milks et al., 1988). AQP0 bound BFSP1 in an affinity chromatography approach (Lindsey Rose et al., 2006) and recently a chemically induced cross-link between the D246 in AQP0 and K455 in BFSP1 was also re- ported (Wang and Schey, 2017). Our immunoelectron microscopy data (Fig. 5) confirm that AQP0 and the C-terminal fragment of BFSP1 containing the myristoylation sequence are in very close proximity to each other in alkali extracted membrane preparations of human lenses. An arbitrarily selected C-terminal fragment was reported to potentially regulate AQP0 water channel activity (Nakazawa et al., 2011). Our data now assess whether AQP0 can potentially be regulated by caspase generated BFSP1 fragments in a myristoylation-dependent manner. A previous study arbitrarily selected a BFSP1 C-terminal sequence to investigate potential effects upon AQP0 water permeability (Nakazawa et al., 2011) without considering potential caspase site location or subsequent myristoylation (Fig. 1A). The present study shows that the presence of caspase sites and the availability of a cryptic myristoylation site can all influence the water permeability of AQP0 by altering the sensitivity of AQP0 Pf to calcium (Fig. 6). All the experimental condi- tions except the test osmotic solution are identical, so neither change in expression nor changes in transport to the membrane can account for the altered regulation we have observed. Table 1f et al., 2012) and regulators of the cytoskeleton (Block et al., 2012; Han et al., 2009; Martin et al., 2012; Ott et al., 2013; Vilas et al., 2006) is another instance where the exposure of a cryptic myristoylation site after protein cleavage is a regulatory mechanism. Whilst many of these mechanisms are linked to apoptosis some have been found to be linked to differentiation such as PAK2 (Cathelin et al., 2006). In the case of gelsolin (Sakurai and Utsumi, 2006) and ctPKCε (Martin et al., 2012), caspase cleavage and myristoylation protects against apoptosis. Caspase activity is also required during development (Busso et al., 2010) and metastatic cell invasion (Rudrapatna et al., 2013). et al., 2012) and regulators of the cytoskeleton (Block et al., 2012; Han et al., 2009; Martin et al., 2012; Ott et al., 2013; Vilas et al., 2006) is another instance where the exposure of a cryptic myristoylation site after protein cleavage is a regulatory mechanism. Whilst many of these mechanisms are linked to apoptosis some have been found to be linked to differentiation such as PAK2 (Cathelin et al., 2006). In the case of gelsolin (Sakurai and Utsumi, 2006) and ctPKCε (Martin et al., 2012), caspase cleavage and myristoylation protects against apoptosis. Caspase activity is also required during development (Busso et al., 2010) and metastatic cell invasion (Rudrapatna et al., 2013). Our data also indicate that the 434–665 sequences of human BFSP1 potentially have multiple caspase sites capable of producing BFSP1 fragments to AQP0 water channel activity, but further investigations are needed to identify those that are physiologically important in the lens. The simplest interpretation of our data (Fig. 6B,C) is that caspase 2, present in Xenopus oocytes (Nutt et al., 2009), generates BFSP1 C- terminal fragments that interact directly with the coinjected AQP0 (Wang and Schey, 2017). We know from measuring the effects of par- ticular fragments on AQP0 Pf regulation that some will alter the calcium sensitivity of AQP0 Pf in a myristoylation dependent manner. One or more of these fragments would be generated from full length BFSP1 if caspases were present, and this accounts for the ability of full length BFSP1 containing the mutants D433A and D549A to alter the calcium sensitivity of AQP0 in the Xenopus oocyte assay. 4.2. Functional role of BFSP1 myristoylation This is contrast to a previous study (Nakazawa et al., 2011) where the magnitude of the Pf is the sole quantity measured and where the potential role of myristoylation was not considered. Table 1f Table 1 Effects of BFSP1 Fragments on AQP0 calcium regulation. BFSP1 fragments no effect on AQP0 calcium regulation Locked high AQP0 calcium regulation Full length X Full length/D433A disrupts myristoylation site X Full length/D549A disrupts one caspase site X 434–548 myristoylation site, two LBDs X A434-548 no myristoylation site X 434–460 one LBD X 461–548 one LBD X 550–665 no myristoylation site, two LBDs X no effect on AQP0 calcium regulation Locked high AQP0 calcium regulation et al., 2012) and regulators of the cytoskeleton (Block et al., 2012; Han et al., 2009; Martin et al., 2012; Ott et al., 2013; Vilas et al., 2006) is another instance where the exposure of a cryptic myristoylation site after protein cleavage is a regulatory mechanism. Whilst many of these mechanisms are linked to apoptosis some have been found to be linked to differentiation such as PAK2 (Cathelin et al., 2006). In the case of gelsolin (Sakurai and Utsumi, 2006) and ctPKCε (Martin et al., 2012), caspase cleavage and myristoylation protects against apoptosis. Caspase activity is also required during development (Busso et al., 2010) and metastatic cell invasion (Rudrapatna et al., 2013). Our data also indi potentially have mul fragments to AQP0 w are needed to identif lens. The simplest inter present in Xenopus o terminal fragments t (Wang and Schey, 20 ticular fragments on A sensitivity of AQP0 P A. Tapodi, et al. Experim ental Eye Research 185 (2019) 107585 A. Tapodi, et al. A. Tapodi, et al. Fig. 7. AQP0 activity is differentially modu- lated by BFSP1 and its post-translationally modified fragments. Full length human BFSP1 is cleaved by caspases at sites 433 and 549. The 433 site reveals a cryptic myristoylation site. The lens expresses both NMT1 and NMT2. The 434–549 and 434–665 fragments, once myristoylated, can ef- fectively eliminate the regulation of AQP0 Pf by Ca2+. Preventing myristoylation restores the Ca2+ regulation of AQP0 Pf (right hand side of the figure). Fig. 7. AQP0 activity is differentially modu- lated by BFSP1 and its post-translationally modified fragments. i Full length human BFSP1 is cleaved by caspases at sites 433 and 549. The 433 site reveals a cryptic myristoylation site. The lens expresses both NMT1 and NMT2. The 434–549 and 434–665 fragments, once myristoylated, can ef- fectively eliminate the regulation of AQP0 Pf by Ca2+. Preventing myristoylation restores the Ca2+ regulation of AQP0 Pf (right hand side of the figure). 4.4. Summary Gokhin, D.S., Nowak, R.B., Kim, N.E., Arnett, E.E., Chen, A.C., Sah, R.L., Clark, J.I., Fowler, V.M., 2012. Tmod1 and CP49 synergize to control the fiber cell geometry, transparency, and mechanical stiffness of the mouse lens. PLoS One 7, e48734. ld i h ' ill i b d b Even though we do not know the detailed mechanism of AQP0 Pf regulation, our data clearly show that it can be modulated by C-term- inal domain fragments of BFSP1 generated by caspase cleavage (Summarized in Fig. 7). Moreover, the revelation of a cryptic myr- istoylation site generates a fragment (G434-548) with the ability to modulate AQP0 Pf in the presence of calcium while a nearly identical fragment (G434A-548) lacks this ability. AQP0 is essential for lens clarity and proper development. Mutant Aqp0a lacking Pf regulation by Ca2+ cannot rescue knockdown of wild type Aqp0a in zebrafish (Clemens et al., 2013). We conjecture the modulation of AQP0 Pf is essential for proper lens development and for proper generation of the appropriate gradient of index of refraction (Vorontsova, I., Gehring, I., Hall, J. E., & Schilling, T. F. (2018). Aqp0a Regulates Suture Stability in the Zebrafish Lens. Investigative Ophthalmology & Visual Science, 59(7), 2869–2879. http://doi.org/10.1167/iovs.18-24044). Clearly BFSP1 plays a role in this modulation. 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The latter could be by, for instance, an interaction of BFSP1 fragments with another protein that in turn in- teracts with AQP0, such as AKAP2 (Gold et al., 2012). There is no evidence to date that BFSP1 fragments interact with calmodulin or AKAPs, although another intermediate filament protein, synemin, is reported to be a cardiac AKAP (Russell et al., 2006). PKA is an im- portant regulator of AQP0 via phosphorylation (Gold et al., 2012) and the plasma membrane, urea/alkali resistant BFSP1 is also phosphory- lated at S488 as reported in the recent proteome of lens membrane proteins (Wang et al., 2013), but this is not a consensus PKA site (http://kinasephos2.mbc.nctu.edu.tw/predict.php) that could suggest an avenue for future investigation given the report of a chemical cross- link between D246 in AQP0 and K455 in BFSP1 (Wang and Schey, 2017). The myristoyl group (G434-548 versus G434A-548) could po- tentially attenuate the regulation of AQP0 in the lens after caspase cleavage at D433 via the lipid environment of AQP0 itself (Tong et al., 2013) and interestingly AQP0 is also acylated at lysine 238 with oleic acid (Schey et al., 2010). Coincidentally K238 is near the BFSP1 binding region and its myristoylation sequence. We report here that the myristoylation competent fragment G434- S665 eliminates the calcium regulation of AQP0 (Fig. 6; Table 1). Our attempt to inhibit myristoylation pharmacologically in the Xenopus oocyte system was unsuccessful as AQP0 activity was completely in- hibited by the inhibitor (data not shown (Rackham et al., 2013);). However, comparison of the effects of two nearly identical fragments, A434 – P548 and G434 – P548, clearly demonstrates that myristoyla- tion could play a critical regulatory role. 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https://openalex.org/W4297810544	https://zenodo.org/record/1302083/files/Evaluation%20of%20Bone%20Mineral%20Density%20and%20Biochemical%20Parameters%20of%20Bone%20Metabolism%20after%20Treatment%20with%20Biofield%20Energy%20Treated%20Vitamin%20D3%20in%20MG-63%20Cells.pdf	English	null	Evaluation of Bone Mineral Density and Biochemical Parameters of Bone Metabolism after Treatment with Biofield Energy Treated Vitamin D3 in MG-63 Cells	Zenodo (CERN European Organization for Nuclear Research)	2,018	cc-by	6,860	Abstract The aim of this study was to examine the potential of Consciousness Energy Healing based vitamin D3 and DMEM medium on bone health parameters. The test items (viz. vitamin D3 and DMEM), were divided into two parts. One part of each test item was received the Biofield Treatment by Jagdish Singh and those samples were labeled as Biofield Treated (BT) samples, while other parts of each sample were denoted as Untreated Test Items (UT). The test samples were found as safe in tested concentrations by MTT assay. ALP was significantly increased by 130.51%, 84.39%, and 67.51% in UT-DMEM + BT-Test item, BT-DMEM + UT-Test item, and BT-DMEM + BT-Test item, respectively at 1 µg/mL than UT-DMEM + UT-Test item group. Moreover, level of collagen was also significantly enhanced by 117.37% in BT-DMEM + UT-Test item at 100 µg/mL than untreated. Other parameter like collagen was significantly increased by 171.30%, 168.51%, and 110.48% in UT-DMEM + BT-Test item, BT-DMEM + UT-Test item, and BT-DMEM + BT-Test item groups, respectively at 50 µg/mL as compared to untreated. Additionally, level of collagen also elevated by 88.49%, 280.08%, and 63.28% in UT-DMEM + BT-Test item, BT-DMEM + UT-Test item, and BT-DMEM + BT-Test item groups, respectively, at 100 µg/mL compared to untreated. Besides, percent of bone mineralization was distinctly increased by 79.34%, 146.74%, and 162.76% in UT-DMEM + BT-Test item, BT-DMEM + UT-Test item, and BT-DMEM + BT-Test item groups, respectively at 100µg/mL compared to untreated. Overall, Biofield Treated vitamin D3 was significantly improved the bone health parameters and it could be a powerful alternative nutraceutical supplement to combat vitamin D3 deficiency and fight against various bone related problems including rickets, osteomalacia, osteoporosis, osteogenesis imperfect, bone fractures, osteoma, chondrodystrophia fetalis, stress management and prevention, autoimmune and inflammatory diseases, and anti- aging by improving overall health. anti-inflammatory, anti-aging, anti-stress, anti-arthritic, anti- osteoporosis, anti-apoptotic, wound healing, anti-cancer, anti- psychotic and anti-fibrotic actions [1]. Vitamin D receptors are widely distributed in most of the body organs viz. brain, liver, heart, lungs, kidney, pancreas, small and large intestines, muscles, reproductive, nervous system, etc. Vitamin D receptors influence cell-to-cell communication, normal cell growth, cell differentiation, cell cycling and proliferation, hormonal balance, neurotransmission process, skin health, immune and cardiovascular functions. In any living vertebrates, vitamin D plays an important role in maintaining a healthy skeletal structure and is essential for bone health. J Nutri Health May 2018 Vol.:4, Issue:1 © All rights are reserved by Singh, et al. J Nutri Health May 2018 Vol.:4, Issue:1 © All rights are reserved by Singh, et al. J Nutri Health May 2018 Vol.:4, Issue:1 © All rights are reserved by Singh, et al. Research Article Open Access Abstract Naturally, it is synthesized in the presence of sunlight in the skin [2]. Most foods do not contain any vitamin D, additionally now-a-days due to aging, use of sunscreen, and change of zenith angle of sun the production of vitamin D3 has reduced [3]. Increasing age is not only related to a decrease in bone marrow depression and muscle strength but is also associated with marked changes in the immune and inflammatory responses [4]. Deficiency of vitamin D3 causes metabolic bone diseases like osteomalacia and exacerbates osteoporosis, etc. [5]. The quality of life for menopausal women is one of the most critical health problems in the today world. Metabolic bone disorders like osteoporosis are mainly prevalent in post-menopausal women. Hormonal factors and rapid bone loss in post-menopausal women leads to an increased risk of fractures [6]. Hence, the serum calcium and Alkaline Phosphatase (ALP) levels in post-menopausal women are the main two vital biochemical markers of bone metabolism. However, bone-specific ALP is the most important marker for osteoblast differentiation [7]. Further, it is generally accepted that an increased calcium intake along with an adequate source of vitamin D is important for maintaining good bone health. Vitamin D also plays an important role in maintaining an adequate level of serum calcium and phosphorus. Therefore, vitamin D has a great impact in forming and maintaining strong bones [8,9]. Bone strength depends on the quality, geometry, shape, micro architecture, turnover, mineral content, and the collagen content. Collagen is the major structural protein responsible for bone calcification. In the aging state, the mechanical properties of the bones become impaired and the bones Abbreviations MG-63: Human Bone Osteosarcoma Cells; ALP: Alkaline phosphatase; CAM: Complementary and alternative medicine; NHIS: National Health Interview Survey; NCCIH: National Center of Complementary and Integrative Health; DMEM: Dulbecco’s Modified Eagle’s Medium; FBS: Fetal Bovine Serum; ATCC: American Type Culture Collection; UT: Untreated; BT: Biofield Energy Treated; TI: Test Item Evaluation of Bone Mineral Density and Biochemical Parameters of Bone Metabolism after Treatment with Biofield Energy Treated Vitamin D3 in MG-63 Cells Singh J1, Trivedi MK1, Branton A1, Trivedi D1, Nayak G1, Mondal SC2 and Jana S2* 1Trivedi Global Inc, USA 2Trivedi Science Research Laboratory Pvt. Ltd, India Address for Correspondence Snehasis Jana, Trivedi Science Research Laboratory Pvt. Ltd., Bhopal, India, Email: publication@trivedisrl.com Submission: 23 April 2018 Accepted: 29 May 2018 Published: 31 May 2018 Copyright: © 2018 Singh J, 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. Singh J1, Trivedi MK1, Branton A1, Trivedi D1, Nayak G1, Mondal SC2 and Jana S2 1Trivedi Global Inc, USA 2Trivedi Science Research Laboratory Pvt. Ltd, India Address for Correspondence Snehasis Jana, Trivedi Science Research Laboratory Pvt. Ltd., Bhopal, India, Email: publication@trivedisrl.com Submission: 23 April 2018 Accepted: 29 May 2018 Published: 31 May 2018 Keywords: The Trivedi Effect; Biofield energy healing treatment; Osteosarcoma cells (MG-63); Collagen; Bone mineralization; Vitamin D3 deficiency Copyright: © 2018 Singh J, 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. Journal of Nutrition and Health Evaluation of Bone Mineral Density and Biochemical Parameters of Bone Metabolism after Treatment with Biofield Energy Treated Vitamin D3 in MG-63 Cells Citation: Singh J, Trivedi MK, Branton A, Trivedi D, Nayak G, et al. Evaluation of Bone Mineral Density and Biochemical Parameters of Bone Metabolism after Treatment with Biofield Energy Treated Vitamin D3 in MG-63 Cells. J Nutri Health. 2018;4(1): 6. Cell culture The human bone osteosarcoma cells (MG-63) were used as the test system in the current study. The MG-63 cells were maintained under the DMEM growth medium for routine culture and supplemented with 10% FBS. Growth conditions were maintained as 37 °C, 5% CO2 and 95% humidity and sub-cultured by trypsinisation followed by splitting the cell suspension into fresh flasks and supplementing with fresh cell growth medium. Three days before the start of the study (i.e., day -3), the growth medium of near-confluent cells was replaced with fresh phenol-free DMEM, supplemented with 10% charcoal dextran stripped FBS (CD-FBS) and 1% penicillin-streptomycin [38]. E i t l d i In recent years, several scientific reports and clinical trials have revealed the useful effects of Biofield Energy Treatments, which have shown to enhance immune function in cases of cervical cancer patients via therapeutic touch [12], massage therapy [13], etc. Complementary and Alternative Medicine (CAM) therapies are now rising as preferred models of treatment, among which Biofield Therapy (or Healing Modalities) is one approach that has been reported to have several benefits to enhance physical, mental and emotional human wellness. However, as per the data of 2012 from the National Health Interview Survey (NHIS), which indicated that the highest percentage (17.7%) of the Americans used dietary supplements as a complementary health approach as compared with other practices in past years. The National Center of Complementary and Integrative Health (NCCIH) has recognized and accepted Biofield Energy Healing as a CAM health care approach in addition to other therapies, medicines and practices such as natural products, deep breathing, yoga, Tai Chi, Qi Gong, chiropractic/osteopathic manipulation, meditation, massage, special diets, homeopathy, progressive relaxation, guided imagery, acupressure, acupuncture, relaxation techniques, hypnotherapy, healing touch, movement therapy, pilates, Rolfing structural integration, mindfulness, Ayurvedic medicine, traditional Chinese herbs and medicines, naturopathy, essential oils, aromatherapy, Reiki, and cranial sacral therapy. Human Biofield Energy has subtle energy that has the capacity to work in an effective manner [14]. CAM therapies have been practiced worldwide with reported clinical benefits in different health disease profiles [15]. This energy can be harnessed and transmitted by the experts into living and non-living things via the process of Biofield Energy Healing. ISSN: 2469-4185 get fragile, that causes various clinical disorders associated with bone collagen abnormalities and bone fragility, such as osteogenesis imperfect a and osteoporosis [10,11]. Consciousness energy healing treatment strategies The test item (vitamin D3) and DMEM were divided into two parts. One part each of the test item and DMEM were treated with the Biofield Energy (also known as The Trivedi Effect®) and coded as the Biofield Energy Treated items, while the second part did not receive any sort of treatment and was defined as the untreated samples. This Biofield Energy Healing Treatment was provided by Jagdish Singh, who participated in this study and performed the Biofield Energy Healing Treatment remotely for ~5 minutes. Biofield Energy Healer was remotely located in the USA, while the test samples were located in the research laboratory of Dabur Research Foundation, New Delhi, India. The Biofield Energy Treatment was administered for 5 minutes through the healer’s unique Energy Transmission process remotely to the test samples under laboratory conditions. Biofield Energy Healer (Jagdish Singh) in this study never visited the laboratory in person, nor had any contact with the Test item and medium. Further, the control group was treated with a sham healer for comparative purposes. The sham healer did not have any knowledge about the Biofield Energy Treatment. After that, the Biofield Energy Treated and untreated samples were kept in similar sealed conditions for experimental study. Based on the literature information and importance of vitamin D3 on bone health, the authors sought to evaluate the impact of the Biofield Energy Treatment (The Trivedi Effect®) on the test samples (vitamin D3 and DMEM medium) for bone health activity with respect to the assessment of different bone health parameters like ALP, collagen content, and bone mineralization using standard assays in MG-63 cells. J Nutri Health 4(1): 6 (2018) Cell culture Biofield Energy Treatment (The Trivedi Effect®) has been published in numerous peer-reviewed science journals with significant outcomes in many scientific fields such as cancer research [16,17], microbiology [18-21], biotechnology [22,23], pharmaceutical science [24-27], agricultural science [28-31], materials science [32-35], nutraceuticals [36,37], skin health, human health and wellness. Citation: Singh J, Trivedi MK, Branton A, Trivedi D, Nayak G, et al. Evaluation of Bone Mineral Density and Biochemical Parameters of Bone Metabolism after Treatment with Biofield Energy Treated Vitamin D3 in MG-63 Cells. J Nutri Health. 2018;4(1): 6. Introduction Vitamin D has multiple effects, which regulate the functions in different organs viz. brain, liver, lungs, heart, kidneys, skeletal, immune and reproductive systems. Moreover, it has significant Experimental design The experimental groups consisted of cells in baseline control (untreated cells), vehicle control groups (0.05% DMSO with Biofield Energy Treated and untreated DMEM), a positive control group (rutin hydrate) and experimental test groups. The experimental groups included the combination of the Biofield Energy Treated and untreated vitamin D3/DMEM. It consisted of four major treatment groups on specified cells with UT-DMEM + UT-Test item, UT- DMEM + Biofield Energy Treated test item (BT-Test item), BT- DMEM + UT-Test item, and BT-DMEM + BT-Test item. Assessment of bone mineralization by alizarin red s Staining The cells were counted using a hemocytometer and plated in a 24- well plate at the density corresponding 1 x 104 cells/well in phenol free DMEM supplemented with 10% CD-FBS. Following the respective treatments, the cells in the above plate were incubated for 48 hours in CO2 incubator at 37 °C, 5% CO2 and 95% humidity. After 48 hours of incubation, the plate was taken out and processed for the measurement of ALP enzyme activity. The cells were washed with 1X PBS and lysed by freeze thaw method i.e., incubation at -80 °C for 20 minutes followed by incubation at 37 °C for 10 minutes . To the lysed cells, 50 µL of substrate solution i.e., 5 mM of p-Nitrophenyl Phosphate (pNPP) in 1M diethanolamine and 0.24 mM magnesium chloride (MgCl2) solution (pH 10.4) was added to all the wells followed by incubation for 1 hour at 37 °C. The absorbance of the above solution was read at 405 nm using Synergy HT micro plate reader (Biotek, USA). The absorbance values obtained were normalized with substrate blank (pNPP solution alone) absorbance values. The percentage increase in ALP enzyme activity with respect to the untreated cells (baseline group) was calculated using Equation 3: The MG-63 cells were counted using a hemocytometer and plated in 24-well plate at the density corresponding to 10 x103 cells/well in phenol free DMEM supplemented with 10% CD-FBS. Following the respective treatments, the cells in the above plate were incubated for 48 hours in CO2 incubator at 37 °C, 5% CO2 and 95% humidity to allow cell recovery and exponential growth. Following overnight incubation, the above cells were subjected to serum stripping for 24 hours. The cells were then treated with non-cytotoxic concentrations of the test samples and positive control. After 3 to 7 days of incubation with the test samples and positive control, the plates were taken out, cell layers processed further by staining with Alizarin Red S dye. The cells were fixed in 70% ethanol for 1 hour, after which Alizarin Red solution (40 µm; pH 4.2) was added to the samples for 20 minutes with shaking. The cells were washed with distilled water to remove unbound dye. For quantitative analysis by absorbance evaluation, nodules were solubilized with 10% cetylpyridinium chloride for 15 minutes with shaking. Absorbance was measured at 562 nm using Biotek Synergy HT micro plate reader. ISSN: 2469-4185 Temperature (RT). After 1 hour of incubation, the above wells were washed with mille Q water and air dried. The cells were then stained with Sirius red dye solution for 1 hour at RT followed by washing in 0.01 N HCl to remove unbound dye. The collagen dye complex obtained in the above step was dissolved in 0.1 N NaOH and absorbance was read at 540 nm using Biotek Synergy HT micro plate reader. The level of collagen was extrapolated using standard curve obtained from purified Calf Collagen Bornstein and Traub Type I (Sigma Type III). The percentage increase in collagen level with respect to the untreated cells (baseline group) was calculated using Equation 4: Temperature (RT). After 1 hour of incubation, the above wells were washed with mille Q water and air dried. The cells were then stained with Sirius red dye solution for 1 hour at RT followed by washing in 0.01 N HCl to remove unbound dye. The collagen dye complex obtained in the above step was dissolved in 0.1 N NaOH and absorbance was read at 540 nm using Biotek Synergy HT micro plate reader. The level of collagen was extrapolated using standard curve obtained from purified Calf Collagen Bornstein and Traub Type I (Sigma Type III). The percentage increase in collagen level with respect to the untreated cells (baseline group) was calculated using Equation 4: taken out and 20 µL of 5 mg/mL of MTT solution was added to all the wells followed by an additional incubation for 3 hours at 37 °C. The supernatant was aspirated and 150 µL of DMSO and was added to each well to dissolve formazan crystals. The absorbance of each well was read at 540 nm using a Synergy HT micro plate reader, BioTek, USA. Chemicals and reagents The cell viability test was performed by MTT assay in the human bone osteosarcoma cell line (MG-63). The cells were counted and plated in 96-well plates at the density corresponding to 5 X 103 to 10 X 103 cells/well/180 µL of cell growth medium. The above cells were incubated overnight under growth conditions and allowed cell recovery and exponential growth, and then they were subjected to serum stripping or starvation. The cells were treated with the testitem, DMEM, and the positive control. The untreated cells served as baseline control. The cells in the above plate (s) were incubated for a time point ranging from 24 to 72 hours in CO2 incubator at 37 °C, 5% CO2 and 95% humidity. Following incubation, the plates were Antibiotic solution (penicillin-streptomycin) was procured from HiMedia, India, while 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H- tetrazolium) (MTT), Direct Red 80, and Ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma, USA. Fetal Bovine Serum (FBS) and Dulbecco’s Modified Eagle’s Medium (DMEM) were purchased from Life Technology, USA. Rutin hydrate was purchased from TCI, Japan, while vitamin D3 (denoted as test item) and L-ascorbic acid were obtained from Sigma-Aldrich, USA. All the other chemicals used in this experiment were analytical grade procured from India. J Nutri Health 4(1): 6 (2018) Page - 02 Statistical analysis The MG-63 cells were counted using a hemocytometer and plated in 24-well plate at the density corresponding to 10 x 103 cells/well in phenol-free DMEM supplemented with 10% CD-FBS. Following the respective treatments, the cells in the above plate were incubated for 48 hours in CO2 incubator at 37 °C, 5% CO2 and 95% humidity. After 48 hours of incubation, the plate was taken out and the amount of collagen accumulated in MG-63 cells corresponding to each treatment was measured by Direct Sirius red dye binding assay. In brief, the cell layers were washed with PBS and fixed in Bouin’s solution (5% acetic acid, 9% formaldehyde and 0.9% picric acid) for 1 hour at Room All the values were represented as percentage of the respective parameters. For statistical analysis Sigma-Plot (version 11.0) was used as a statistical tool. Statistically significant values were set at the level of p≤0.05. Assessment of bone mineralization by alizarin red s Staining The percentage increase in bone mineralization with respect to the untreated cells (baseline group) was calculated using the following Equation 5: % Increase in ALP = {(X-R)/R}100----------------------- (3) (3) Where, % Increase = {(X-R)/R}100----------------------- (5) Where, X = Absorbance of cells corresponding to positive control and test groups Where, X = Absorbance in cells corresponding to positive control or test groups; R = Absorbance in cells corresponding to baseline (untreated) group. R = Absorbance of cells corresponding to baseline group (untreated cells) Citation: Singh J, Trivedi MK, Branton A, Trivedi D, Nayak G, et al. Evaluation of Bone Mineral Density and Biochemical Parameters of Bone Metabolism after Treatment with Biofield Energy Treated Vitamin D3 in MG-63 Cells. J Nutri Health. 2018;4(1): 6. J Nutri Health 4(1): 6 (2018) ISSN: 2469-4185 The percentage cytotoxicity at each tested concentration of the test substance was calculated using the following Equation 1: % Cytotoxicity = {(1-X)/R}100----------------------- (1) (1) Where, X = Absorbance of treated cells; R = Absorbance of untreated cells % Increase in collagen levels = {(X-R)/R}100-------------------(4) Where, % Increase in collagen levels = {(X-R)/R}100-------------------(4) Where, -(4) The percentage cell viability corresponding to each treatment was then being obtained using the following Equation 2: Where, X = Collagen levels in cells corresponding to positive control and test groups % Cell Viability = 100 - % Cytotoxicity----------------------- (2) The concentrations exhibiting ≥70% Cell viability was considered as non-cytotoxic [39]. R = Collagen levels in cells corresponding to baseline group (untreated cells) ISSN: 2469-4185 DMEM + BT-Test item, BT-DMEM + UT-Test item, and BT-DMEM + BT-Test item groups, respectively compared to the UT-DMEM + UT-Test item group. Additionally, the level of ALP was significantly increased by 13.37%, 17.81%, and 42.01% in the UT-DMEM + BT- Test item, BT-DMEM + UT-Test item, and BT-DMEM + BT-Test item groups, respectively at 50 µg/mL compared to the UT-DMEM + UT-Test item group. At higher concentration (100 µg/mL), the level of ALP was also significantly increased by117.37% and 12.29% in the BT-DMEM + UT-Test item and BT-DMEM + BT-Test item groups, respectively than untreated group (Figure 2). Overall, the Consciousness Energy Healing Treated (The Trivedi Effect®) test item group (i.e., vitamin D3) showed an improved synthesis of ALP level in the human osteosarcoma cells with respect to the untreated item items group at 50 µg/mL. The ALP activity is essential for the bone mineralization and considered a useful biochemical marker for bone formation [43]. Here, it was revealed that the Consciousness Energy Healing Treated vitamin D3 significantly increased the level of ALP expression, which might be very helpful to the patients suffering from various bone-related disorders. DMEM + BT-Test item, BT-DMEM + UT-Test item, and BT-DMEM + BT-Test item groups, respectively compared to the UT-DMEM + UT-Test item group. Additionally, the level of ALP was significantly increased by 13.37%, 17.81%, and 42.01% in the UT-DMEM + BT- Test item, BT-DMEM + UT-Test item, and BT-DMEM + BT-Test item groups, respectively at 50 µg/mL compared to the UT-DMEM + UT-Test item group. At higher concentration (100 µg/mL), the level of ALP was also significantly increased by117.37% and 12.29% in the BT-DMEM + UT-Test item and BT-DMEM + BT-Test item groups, respectively than untreated group (Figure 2). Overall, the Consciousness Energy Healing Treated (The Trivedi Effect®) test item group (i.e., vitamin D3) showed an improved synthesis of ALP level in the human osteosarcoma cells with respect to the untreated item items group at 50 µg/mL. The ALP activity is essential for the bone mineralization and considered a useful biochemical marker for bone formation [43]. Here, it was revealed that the Consciousness Energy Healing Treated vitamin D3 significantly increased the level of ALP expression, which might be very helpful to the patients suffering from various bone-related disorders. used for the screening of viable cells [42]. MTT assay Cell-based assays are used for the assessment of cell proliferation or cytotoxicity [40,41]. The MTT tetrazolium assay has been widely Page - 03 Page - 03 ISSN: 2469-4185 Hence, in this experiment authors used MTT cell viability assay as a systemic tool for the assessment of viable cells count of the test samples in MG-63 cell line. The results of cell viability after treatment with the test samples in MG-63 cells are shown in Figure 1. The data were expressed as percentage, did not show any cytotoxicity (as evidence of cell viability approximately greater than 73%) across all the tested concentrations as maximum of 100 µg/mL. Therefore, the safe concentrations were used in this experiment to study the effect of the test samples on the levels of Alkaline Phosphatase (ALP) activity, collagen synthesis, and bone mineralization inMG-63 cells. Alkaline phosphatase (ALP) activity The effect of the test substances on ALP activity in MG-63 cells is shown in Figure 2. The level of ALP was increased by 20.50% in the Vehicle Control (VC) group compared to the untreated cells group. The ALP activity was increased by 38.78%, 43.61%, and 80.92% in the positive control group at the concentration of 0.01, 0.1, and 1 µg/mL, respectively in a dose-dependent manner compared to the untreated cells group. The level of ALP was significantly increased by 26.44%, 52.83%, and 84.93% in the UT-DMEM + BT-Test item, BT-DMEM + UT-Test item, and BT-DMEM + BT-Test item group, at the concentration of 0.1 µg/mL compared to the UT-DMEM + UT-Test item group. ALP level was further significantly increased by 130.51%, 84.39%, and 67.51% in the UT-DMEM + BT-Test item, BT-DMEM + UT-Test item, and BT-DMEM + BT-Test item group, at the concentration of 1 µg/mL compared to the UT-DMEM + UT-Test item group. Moreover, at 10 µg/mL the level of ALP was significantly enhanced by 19.29%, 59.29%, and 37.08% in the UT- Citation: Singh J, Trivedi MK, Branton A, Trivedi D, Nayak G, et al. Evaluation of Bone Mineral Density and Biochemical Parameters of Bone Metabolism after Treatment with Biofield Energy Treated Vitamin D3 in MG-63 Cells. J Nutri Health. 2018;4(1): 6. Assessment of collagen activity The effect of the test samples on the collagen activity in MG-63 cell line is shown in Figure 3. The Vehicle Control group (VC) showed the level of collagen activity as 2.2% compared to the untreated cells group. The synthesis of collagen was significantly increased by 24%, 50.29%, and 47.71% at 0.01, 0.1, and 1 µg/mL, respectively in the positive control (rutin) group compared to the untreated cells group. The collagen synthesis was significantly increased by 95.03% and 43.97% in the BT-DMEM + UT-Test item and BT-DMEM + BT-Test item groups, respectively at 10 µg/mL, compared to the UT-DMEM + UT-Test item group. Moreover, the level of collagen was significantly increased by 171.30%, 168.51%, and 110.48% in the UT-DMEM + BT-Test item, BT-DMEM + UT-Test item, and BT- DMEM + BT-Test item groups, respectively at 50 µg/mL, compared to the UT-DMEM + UT-Test item group. Additionally, at 100 µg/mL collagen concentration was significantly elevated by 88.49%, 280.08%, and 63.28% in the in the UT-DMEM + BT-Test item, BT-DMEM + UT-Test item, and BT-DMEM + BT-Test item groups, respectively, compared to the UT-DMEM + UT-Test item group (Figure 3). significantly enhanced by 19.29%, 59.29%, and 37.08% in the UT- Figure 1: The effect of the test items on cell viability in MG-63 cells after 72 hours of treatment. VC: Vehicle Control (0.05% DMSO); UT: Untreated; BT: Biofield Energy Treated, TI: Test item Figure 2: The effect of the test samples on Alkaline Phosphatase (ALP) enzyme activity in human bone osteosarcoma cell. VC: Vehicle control (0.05% DMSO), UT: Untreated; BT: Biofield Energy Treated, TI: Test item. Figure 1: The effect of the test items on cell viability in MG-63 cells after 72 hours of treatment. VC: Vehicle Control (0.05% DMSO); UT: Untreated; BT: Biofield Energy Treated, TI: Test item Figure 1: The effect of the test items on cell viability in MG-63 cells after 72 hours of treatment. VC: Vehicle Control (0.05% DMSO); UT: Untreated; BT: Biofield Energy Treated, TI: Test item Altogether, the Consciousness Energy Healing based test item group (i.e., vitamin D3) showed an improved synthesis of collagen content in the human osteosarcoma cells with respect to all the treatment groups. Type I collagen is the major structural protein in extra cellular matrix component responsible for bone calcification Figure 1: The effect of the test items on cell viability in MG-63 cells after 72 hours of treatment. Assessment of collagen activity VC: Vehicle Control (0.05% DMSO); UT: Untreated; BT: Biofield Energy Treated, TI: Test item Figure 3: The effect of the test samples on collagen activity in human bone osteosarcoma cells. VC: Vehicle Control (0.05% DMSO), UT: Untreated; BT: Biofield Energy Treated, TI: Test Item. Figure 2: The effect of the test samples on Alkaline Phosphatase (ALP) enzyme activity in human bone osteosarcoma cell. VC: Vehicle control (0.05% DMSO), UT: Untreated; BT: Biofield Energy Treated, TI: Test item. Figure 2: The effect of the test samples on Alkaline Phosphatase (ALP) enzyme activity in human bone osteosarcoma cell. VC: Vehicle control (0.05% DMSO), UT: Untreated; BT: Biofield Energy Treated, TI: Test item. Figure 3: The effect of the test samples on collagen activity in human bone osteosarcoma cells. VC: Vehicle Control (0.05% DMSO), UT: Untreated; BT: Biofield Energy Treated, TI: Test Item. Page - 04 J Nutri Health 4(1): 6 (2018) Page - 04 ISSN: 2469-4185 group. Further, the level of collagen was also significantly enhanced by 117.37% in the BT-DMEM + UT-Test item at 100 µg/mL than the untreated group. Collagen was significantly increased by 171.30%, 168.51%, and 110.48% in the UT-DMEM + BT-Test item, BT-DMEM + UT-Test item, and BT-DMEM + BT-Test item groups, respectively at 50 µg/mL compared to the untreated group. Additionally, the level of collagen was also elevated by 88.49%, 280.08%, and 63.28% in the UT- DMEM + BT-Test item, BT-DMEM + UT-Test item, and BT-DMEM + BT-Test item groups, respectively, at 100 µg/mL compared to the untreated group. Altogether, the Biofield Energy Treated test samples (The Trivedi Effect®) demonstrated a significant impact on bone health parameters. Therefore, the Consciousness Energy Healing based vitamin D3 might be suitable for the development of an alternative and more effective supplement for vitamin D3 deficiency, which could be useful for the management of various bone related disorders viz. low bone density and osteoporosis, osteogenesis imperfect a, Paget’s disease of bone, rickets, osteomalacia, bone and joint pain, bone fractures, deformed bones, osteoma, chondrodystrophia fetal is, etc. Besides, it can also be utilized in organ transplants (for example kidney transplants, liver transplants and heart transplants), various autoimmune disorders such as Lupus, Addison Disease, Celiac Disease (gluten-sensitive enteropathy), Dermatomyositis, Graves’ Disease, Hashimoto Thyroiditis, Multiple Sclerosis, Myasthenia Gravis, Pernicious Anemia, Aplastic Anemia, Reactive Arthritis, Rheumatoid Arthritis, Sjogren Syndrome, Systemic Lupus Erythematosus, Type 1 Diabetes, Alopecia Areata, Crohn’s Disease, Fibromyalgia, Vitiligo, Psoriasis, Scleroderma, Chronic Fatigue Syndrome and Vasculitis. Further it also is useful in various inflammatory disorders such as Asthma, Ulcerative Colitis, Alzheimer’s disease, Atherosclerosis, Dermatitis, Diverticulitis, Hepatitis, and Irritable Bowel Syndrome. Additionally, Trivedi’s propritory Therapy would be very effective as an anti-stress, anti-arthritic, anti-osteoporosis, anti-apoptotic, wound healing, anti-cancer, anti-psychotic and anti-fibrotic actions stress management and prevention, and anti-aging by improving overall health, Parkinson’s Disease and stress etc. to modulate the immune system by improving overall health. and also promoting osteoblast differentiation [44]. Here, the Biofield Energy Treated vitamin D3 significantly improved the level of collagen which could be beneficial to maintain a health bone. Overall, The Trivedi Effect® - Consciousness Energy Healing Treatment modality showed a significant improvement of the collagen level in human osteosarcoma cells. Thus, it is assumed that The Trivedi effect® has the potential to improve the bone health in various skeletal disorders. Bone mineralization Reduction of bone properties and an increased the risk of bone fracture is due to deficiency of vitamin-D and calcium [45]. Moreover, vitamin D co-regulates calcium homeostasis by influencing intestinal calcium absorption, renal calcium reabsorption and bone resorption by osteoclasts [46,47]. The effect of test items on bone mineralization in MG-63 cells is shown in Figure 4. The vehicle control group showed 19.4% increased the bone mineralization as compared to the untreated cells group. The positive control (rutin) showed 50.46%, 86.16%, and 130.60% increased of percent bone mineralization at 5,10 and 25 µg/mL, respectively compared to the untreated cells group in a concentration- dependent manner. The percent bone mineralization was significantly raised by 31.62%, 19.89%, and 16.32% in the UT-DMEM + BT-Test item, BT-DMEM + UT-Test item, and BT-DMEM + BT-Test item group, respectively at 10 µg/mL compared to the UT-DMEM + UT- Test item group. Further, a noticeably increased the percentage of bone mineralization was observed by 30.07%, 27.95%, and 6.78% in the UT- DMEM + BT-Test item, BT-DMEM + UT-Test item, and BT-DMEM + BT-Test item groups, respectively at 50 µg/mL with respect to the UT-DMEM + UT-Test item group. Further, at 100 µg/mL the percent of bone mineralization was remarkably increased by 79.34%, 146.74%, and 162.76% in the UT-DMEM + BT-Test item, BT-DMEM + UT-Test item, and BT-DMEM + BT-Test item groups, respectively with respect to the UT-DMEM + UT-Test item group (Figure 4). Thus, based on the above findings it is hypothesized that the Consciousness Energy Healing Treatment (The Trivedi Effect®) based test item groups (i.e., vitamin D3) showed a remarkable improvement of bone mineralization content assessed by in vitro in the human osteosarcoma cells (MG-63) with respect to the all others treatment groups. Citation: Singh J, Trivedi MK, Branton A, Trivedi D, Nayak G, et al. Evaluation of Bone Mineral Density and Biochemical Parameters of Bone Metabolism after Treatment with Biofield Energy Treated Vitamin D3 in MG-63 Cells. J Nutri Health. 2018;4(1): 6. ISSN: 2469-4185 J Integr Oncol 4: 141. 36. Trivedi MK, Nayak G, Patil S, Tallapragada RM, Jana S, et al. (2015) Bio-field treatment: An effective strategy to improve the quality of beef extract and meat infusion powder. J Nutr Food Sci 5: 389. 17. Trivedi MK, Patil S, Shettigar H, Gangwar M, Jana S (2015) In vitro evaluation of biofield treatment on cancer biomarkers involved in endometrial and prostate cancer cell lines. J Cancer Sci Ther 7: 253-257. 37. Trivedi MK, Tallapragada RM, Branton A, Trivedi D, Nayak G, et al. (2015) Biofield treatment: A potential strategy for modification of physical and thermal properties of gluten hydrolysate and ipomoea macroelements. J Nutr Food Sci 5: 414. 18. Trivedi MK, Branton A, Trivedi D, Nayak G, Mondal SC, et al. (2015) Antibiogram, biochemical reactions and biotyping of biofield treated Providencia rettgeri. Am J Health Res 3: 344-351. 38. Czekanska EM, Stoddart MJ, Richards RG, Hayes JS (2012) In search of an osteoblast cell model for in vitro research. Eur Cells Mater 24: 1-17. 19. Trivedi MK, Branton A, Trivedi D, Nayak G, Mondal SC, et al. (2015) Antimicrobial sensitivity, biochemical characteristics and biotyping of Staphylococcus saprophyticus: An impact of biofield energy treatment. J Women’s Health Care 4: 271. 39. Biological evaluation of medical devices - Part 5: Tests for in vitro cytotoxicity (ISO 10993-5:2009), I.S.EN ISO, 10993-5: 20093. 20. Trivedi MK, Branton A, Trivedi D, Nayak G, Shettigar H, et al. (2015) Antimicrobial susceptibility pattern, biochemical characteristics and biotyping of Salmonella paratyphi A: An impact of biofield treatment. Clin Microbiol 4: 215. 40. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Meth 65: 55-63. 41. Marshall NJ, Goodwin CJ, Holt SJ (1995) A critical assessment of the use of microculture tetrazolium assays to measure cell growth and function. Growth Regul 5: 69-84. 21. Trivedi MK, Branton A, Trivedi D, Nayak G, Mondal SC, et al. (2015) Antibiogram of biofield-treated Shigella boydii: Global burden of infections. Scie J Clin Med 4: 121-126. 42. Riss TL, Moravec RA, Niles AL, Duellman S, Benink HA, et al. (2016) Cell Viability Assays. In: Sittampalam GS, Coussens NP, Brimacombe K, et al., editors. Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004. 22. Trivedi MK, Branton A, Trivedi D, Nayak G, Mondal SC, et al. ISSN: 2469-4185 9. DeLuca HF (2004) Overview of general physiologic features and functions of vitamin D. Am J Clin Nutr 80: S1689-S1696. of genetic diversity using simple sequence repeat (SSR) markers and growth regulator response in biofield treated cotton (Gossypium hirsutum L.). Am J Agricul Fores 3: 216-221. 10. Viguet-Carrin S, Garnero P, Delmas PD (2006) The role of collagen in bone strength. Osteoporos Int 17: 319-336. 31. Trivedi MK, Branton A, Trivedi D, Nayak G, Gangwar M, et al. (2015) Evaluation of vegetative growth parameters in biofield treated bottle gourd (Lagenaria siceraria) and okra (Abelmoschus esculentus). Int J Nutr Food Sci 4: 688-694. 11. Sroga GE, Vashishth D (2012) Effects of bone matrix proteins on fracture and fragility in osteoporosis. Curr Osteoporos Rep 10: 141-150. 12. Lutgendorf SK, Mullen-Houser E, Russell D, Degeest K, Jacobson G, et al. (2010) Preservation of immune function in cervical cancer patients during chemoradiation using a novel integrative approach. Brain Behav Immun 24: 1231-1240. 32. Trivedi MK, Tallapragada RM, Branton A, Trivedi D, Nayak G, et al. (2015) Evaluation of atomic, physical, and thermal properties of bismuth oxide powder: An impact of biofield energy treatment. Am J Nano Res Appl 3: 94-98. 13. Ironson G, Field T, Scafidi F, Hashimoto M, Kumar M, et al. (1996) Massage therapy is associated with enhancement of the immune system’s cytotoxic capacity. Int J Neurosci 84: 205-217. 33. Trivedi MK, Patil S, Nayak G, Jana S, Latiyal O (2015) Influence of biofield treatment on physical, structural and spectral properties of boron nitride. J Material Sci Eng 4: 181. 14. Jain S, Hammerschlag R, Mills P, Cohen L, Krieger R, et al. (2015) Clinical studies of biofield therapies: Summary, methodological challenges, and recommendations. Glob Adv Health Med 4: 58-66. 34. Trivedi MK, Nayak G, Patil S, Tallapragada RM, Latiyal O, et al. (2015) Characterization of physical and structural properties of brass powder after biofield treatment. J Powder Metall Min 4: 134. 15. Rubik B (2002) The biofield hypothesis: Its biophysical basis and role in medicine. J Altern Complement Med 8: 703-717. 35. Trivedi MK, Nayak G, Patil S, Tallapragada RM, Latiyal O, et al. (2015) Evaluation of biofield treatment on physical and structural properties of bronze powder. Adv Automob Eng 4: 119. 16. Trivedi MK, Patil S, Shettigar H, Mondal SC, Jana S (2015) The potential impact of biofield treatment on human brain tumor cells: A time-lapse video microscopy. Conclusion 1. Holick MF (2004) Sunlight and vitamin D for bone health and prevention of autoimmune diseases cancers, and cardiovascular disease. Am J Clin Nut 80: 1678S-1688S. The MTT cell viability assay data showed more than 73% cells were viable, which indicated that the test samples were safe and nontoxic in all the tested concentrations.ALP was significantly increased by 130.51%, 84.39%, and 67.51% in the UT-DMEM + BT- Test item, BT-DMEM + UT-Test item, and BT-DMEM + BT-Test item, respectively at 1 µg/mL than the UT-DMEM + UT-Test item 2. Holick MF (1996) Vitamin D and bone health. J Nutr 126: 1159S-1164S. 3. Matsuoka LY, Ide L, Wortsman J, MacLaughlin JA, Holick MF (1987) Sunscreens suppress vitamin D3 synthesis. J Clin Endocrinol Metab 64: 1165-1168. 4. Barnes MS, Robson JP, Bonham MP, Strain J, Wallace J (2006) Vitamin D: Status, supplementation and immunomodulation. Cur Nut Food Sci 2: 315- 336. Figure 4: The effect of the test samples on human bone osteosarcoma cells for the assessment of bone mineralization activity. VC: Vehicle control (0.05% DMSO), UT: Untreated; BT: Biofield Energy Treated, TI: Test Item. 5. Laird E, Ward M, McSorley E, Strain JJ, Wallace J (2010) Vitamin D and bone health; Potential mechanisms. Nutrients 2: 693-724. 6. Bhattarai T, Bhattacharya K, Chaudhuri P, Sengupta P (2014) Correlation of common biochemical markers for bone turnover, serum calcium, and alkaline phosphatase in post-menopausal women. Malays J Med Sci 21: 58-61. 7. Iba K, Takada J, Yamashita T (2004) The serum level of bone-specific alkaline phosphatase activity is associated with aortic calcification in osteoporosis patients. J Bone Miner Metab 22: 594-596. 8. Holick MF, Garabedian M (2006) Vitamin D: Photobiology, metabolism, mechanism of action, and clinical applications. Primer on the metabolic bone diseases and disorders of mineral metabolism. Edited by: Favus MJ, Washington, DC pp: 129-137. Page - 05 J Nutri Health 4(1): 6 (2018) Citation: Singh J, Trivedi MK, Branton A, Trivedi D, Nayak G, et al. Evaluation of Bone Mineral Density and Biochemical Parameters of Bone Metabolism after Treatment with Biofield Energy Treated Vitamin D3 in MG-63 Cells. J Nutri Health. 2018;4(1): 6. ISSN: 2469-4185 (2015) Evaluation of antibiogram, genotype and phylogenetic analysis of biofield treated Nocardia otitidis. Biol Syst Open Access 4: 143. 23. Trivedi MK, Branton A, Trivedi D, Nayak G, Charan S, et al. (2015) Phenotyping and 16S rDNA analysis after biofield treatment on Citrobacter braakii: A urinary pathogen. J Clin Med Genom 3: 129. 43. Gerald J. Atkins, David M. Findlay, Paul H (2011) Anderson, Howard A. Morris. Vitamin D (3rd Eds), Vitamin D 1: 411-424. 44. Oishi Y, Fu ZW, Ohnuki Y, Kato H, Noguchi T (2002) Molecular basis of the alteration in skin collagen metabolism in response to in vivo dexamethasone treatment: Effects on the synthesis of collagen type I and III, collagenase, and tissue inhibitors of metalloproteinases. Br J Dermatol 147: 859-868. 24. Trivedi MK, Patil S, Shettigar H, Bairwa K, Jana S (2015) Spectroscopic characterization of chloramphenicol and tetracycline: An impact of biofield. Pharm Anal Acta 6: 395. 25. Trivedi MK, Patil S, Shettigar H, Bairwa K, Jana S (2015) Spectroscopic characterization of biofield treated metronidazole and tinidazole. Med Chem 5: 340-344. 45. Lips P, van Schoor NM (2011) The effect of vitamin D on bone and osteoporosis. Best Pract Res Clin Endocrinol Metab 25: 585-591. 46. Priemel M, von Domarus C, Klatte TO, Kessler S, Schlie J, et al. (2010) Bone mineralization defects and vitamin D defciency: histomorphometric analysis of iliac crest bone biopsies and circulating 25-hydroxyvitamin D in 675 patients. J Bone Miner Res 25: 305-312. 26. Trivedi MK, Patil S, Shettigar H, Bairwa K, Jana S (2015) Effect of biofield treatment on spectral properties of paracetamol and piroxicam. Chem Sci J 6: 98. 27. Trivedi MK, Branton A, Trivedi D, Shettigar H, Bairwa K, et al. (2015) Fourier transform infrared and ultraviolet-visible spectroscopic characterization of biofield treated salicylic acid and sparfloxacin. Nat Prod Chem Res 3: 186. 47. Hossein-Nezhad A, Holick MF (2013) Vitamin D for health: A global perspective. Mayo Clin Proc 88: 720-755. 28. Trivedi MK, Branton A, Trivedi D, Nayak G, Mondal SC, et al. (2015) Morphological characterization, quality, yield and DNA fingerprinting of biofield energy treated alphonso mango (Mangifera indica L.). J Food Nutr Sci 3: 245-250. Acknowledgements 29. Trivedi MK, Branton A, Trivedi D, Nayak G, Gangwar M, et al. (2015) Agronomic characteristics, growth analysis, and yield response of biofield treated mustard, cowpea, horse gram, and groundnuts. Int J Genetics and Genomics 3: 74-80. Authors are grateful to Dabur Research Foundation, Trivedi Global, Inc., Trivedi Science, Trivedi Testimonials and Trivedi Master Wellness for their support throughout the work. 30. Trivedi MK, Branton A, Trivedi D, Nayak G, Gangwar M, et al. (2015) Analysis J Nutri Health 4(1): 6 (2018) Page - 06 Page - 06
https://openalex.org/W2560124912	https://europepmc.org/articles/pmc5302719?pdf=render	English	null	Antibacterial Properties of Visible-Light-Responsive Carbon-Containing Titanium Dioxide Photocatalytic Nanoparticles against Anthrax	Nanomaterials	2,016	cc-by	11,384	Received: 31 August 2016; Accepted: 6 December 2016; Published: 9 December 2016 Received: 31 August 2016; Accepted: 6 December 2016; Published: 9 December 2016 Abstract: The bactericidal activity of conventional titanium dioxide (TiO2) photocatalyst is effective only on irradiation by ultraviolet light, which restricts the applications of TiO2 for use in living environments. Recently, carbon-containing TiO2 nanoparticles [TiO2(C) NP] were found to be a visible-light-responsive photocatalyst (VLRP), which displayed signiﬁcantly enhanced antibacterial properties under visible light illumination. However, whether TiO2(C) NPs exert antibacterial properties against Bacillus anthracis remains elusive. Here, we evaluated these VLRP NPs in the reduction of anthrax-induced pathogenesis. Bacteria-killing experiments indicated that a signiﬁcantly higher proportion (40%–60%) of all tested Bacillus species, including B. subtilis, B. cereus, B. thuringiensis, and B. anthracis, were considerably eliminated by TiO2(C) NPs. Toxin inactivation analysis further suggested that the TiO2(C) NPs efﬁciently detoxify approximately 90% of tested anthrax lethal toxin, a major virulence factor of anthrax. Notably, macrophage clearance experiments further suggested that, even under suboptimal conditions without considerable bacterial killing, the TiO2(C) NP-mediated photocatalysis still exhibited antibacterial properties through the reduction of bacterial resistance against macrophage killing. Our results collectively suggested that TiO2(C) NP is a conceptually feasible anti-anthrax material, and the relevant technologies described herein may be useful in the development of new strategies against anthrax. Keywords: anthrax spore; antibacterial agents; TiO2; carbon-containing TiO2; visible light responsive photocatalyst nanomaterials nanomaterials nanomaterials nanomaterials Article Antibacterial Properties of Visible-Light-Responsive Carbon-Containing Titanium Dioxide Photocatalytic Nanoparticles against Anthrax Der-Shan Sun 1, Jyh-Hwa Kau 2,3, Hsin-Hsien Huang 3, Yao-Hsuan Tseng 4, Wen-Shiang Wu 1 and Hsin-Hou Chang 1,* Der-Shan Sun 1, Jyh-Hwa Kau 2,3, Hsin-Hsien Huang 3, Yao-Hsuan Tseng 4, Wen-Shiang Wu 1 and Hsin-Hou Chang 1,* 1 Department of Molecular Biology and Human Genetics, Tzu-Chi University, Hualien 97004, Taiwan; dssun@mail.tcu.edu.tw (D.-S.S.); englishbiology@yahoo.com.tw (W.-S.W.) 1 Department of Molecular Biology and Human Genetics, Tzu-Chi University, Hualien 97004, Taiwan; dssun@mail.tcu.edu.tw (D.-S.S.); englishbiology@yahoo.com.tw (W.-S.W.) 2 Institute of Microbiology and Immunology, National Defense Medical Center, Taipei 11490, Taiwan jhkau@ndmctsgh.edu.tw 2 Institute of Microbiology and Immunology, National Defense Medical Center, Taipei 11490, Taiwan jhkau@ndmctsgh.edu.tw 3 Institute of Preventive Medicine, National Defense Medical Center, Taipei 23742, Taiwan; jhhhuang@ndmctsgh.edu.tw 4 Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 10607, Taiwan; tyh@mail.ntust.edu.tw * Correspondence: hhchang@mail.tcu.edu.tw; Tel.: +886-3-8565301 (ext. 2667) Academic Editor: Thomas Nann www.mdpi.com/journal/nanomaterials 1. Introduction Anthrax is a life-threatening infectious disease that spreads through contact with spores of the Gram-positive bacterium Bacillus anthracis through skin contact (generally with infected animal products), inhalation, or ingestion [1]. Approximately 2000 to 20,000 cases occur worldwide annually [2], mostly in Africa and central and south Asia [3]. Anthrax spores have been developed as a biological weapon by several countries [4–6]. The 2001 US anthrax letter attacks further evidenced an emerging terrorist threat, leading to renewed attention to the importance of prophylaxis, prevention, and handling procedures for anthrax [7]. Agents commonly cited to inactivate anthrax spores www.mdpi.com/journal/nanomaterials Nanomaterials 2016, 6, 237; doi:10.3390/nano6120237 2 of 12 Nanomaterials 2016, 6, 237 Nanomaterials 2016, 6, 237 include formaldehyde, hypochlorite solutions, chlorine dioxide, and radiation [8]. However, most of these agents are harmful to humans, limiting their use in public environments. Therefore, a safer disinfection technique that can exert a sustainable antimicrobial effect in human living environments is highly desirable. Photocatalytic titanium dioxide (TiO2) substrates have been demonstrated to eliminate organic compounds and to function as disinfectants [9]. On stimulation by ultraviolet (UV) light irradiation, the photon energy excites valance electrons and generates pairs of electrons and holes (electron vacancy in the valence band) that diffuse and become trapped on the TiO2 surfaces. These excited electrons and holes have strong reducing and oxidizing activities and react with atmospheric water and oxygen to yield reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), hydroxyl radicals (•OH), and superoxide anions (O2−) [10], which are extremely reactive on contact with organic compounds, and have been shown to operate in concert to attack polyunsaturated phospholipids and DNA in bacteria [9,11]. The oxidation of bacterial cell components such as lipids and DNA might therefore result in subsequent bacterial cell death [9]. Consequently, the TiO2 photocatalytic process is a conceptually feasible disinfectant technology. p y gy The TiO2 photocatalyst, however, is effective only on irradiation with UV light at the necessary levels, which can induce severe damage to human eyes and skin [12–15]. This greatly restricts the potential applications of the photocatalyst for use in human living environments. To solve this problem, impurity doping of TiO2 with different elements has been used, including carbon, sulfur, nitrogen, and silver, resulting in excitation wavelength shifts from the UV to visible-light [16–25]. Simultaneously, the proper amount of impurity doping of TiO2 may also reduce the recombination rates of electron and hole pairs. 1. Introduction Previously, we reported visible-light-responsive photocatalyst (VLRP) ﬁlms, which offered a complementary and possibly alternative approach for meeting this need to control the spread of anthrax [24]. However, these VLRP ﬁlms must be precoated on the surfaces of particular objects, whereas photocatalytic NPs do not, and as such may have broader applications. To solve this problem, the anti-anthrax properties of VLRP carbon-containing titanium dioxide [TiO2(C)] nanoparticles [TiO2(C) NPs; C200 NPs] [17] were evaluated in this study. The visible-light-responsive photocatalytic activity of C200 NPs has been respectively validated by degradation of methylene blue in liquid phase, oxidation of NO in gas phase, and sterilization in these works under visible light illumination [17,19,26–28]. The existence of carbonaceous species on TiO2 surface was analyzed by X-ray photoelectron spectroscopy (XPS) and diffuse reﬂectance infrared Fourier transform spectra. The effect of carbonaceous species on physical properties was observed on UV-visible absorption spectra, photoluminescence spectroscopy, and Raman spectroscopy as shown in our previous works [26–28]. In addition, we have further demonstrated that C200 NPs exert superior Escherichia coli killing properties under visible light illumination when compared to anatase TiO2 NPs [17,19]. These results collectively suggested that the C200 NPs exhibit a photocatalytic property under visible light illumination. However, whether C200 NPs can eliminate spore-forming bacteria such as Bacillus species has remained uncertain. Therefore, the visible-light-responsive C200 NP-mediated anti-anthrax property was evaluated. The potential applications are discussed herein. 2.1. Analyses of TiO2 NPs Scanning electron microscopy (A, B), X-ray photoelectron spectroscopy (XPS) analysis for the 1s atomic orbital of carbon (C) and UV-Vis absorption spectra (D) of UV100 TiO2 and C200 NPs used in this study. The C200 sample absorbed light extending into the visible (>380 nm) region. Figure 1. Scanning electron microscopy and ultraviolet-visible (UV-Vis) absorption spectrum analyses. Scanning electron microscopy (A,B), X-ray photoelectron spectroscopy (XPS) analysis for the 1s atomic orbital of carbon (C) and UV-Vis absorption spectra (D) of UV100 TiO2 and C200 NPs used in this study. The C200 sample absorbed light extending into the visible (>380 nm) region. Figure 1. Scanning electron microscopy and ultraviolet-visible (UV-Vis) absorption spectrum a alyses Sca i g elect o ic oscopy (A B) X ay photoelect o spect oscopy (XPS) a alysis fo 2.2. Dose-Dependent and Kinetic Analyses of Photocatalytic Inactivation of B. Subtilis 2.2. Dose-Dependent and Kinetic Analyses of Photocatalytic Inactivation of B. Subtilis y g py ( , ), y p p py ( ) the 1s atomic orbital of carbon (C) and UV-Vis absorption spectra (D) of UV100 TiO2 and d i thi t d Th C200 l b b d li ht t di i t th i ibl (>380 ) The antibacterial properties of C200 NPs have been demonstrated [17]; however, whether C200 can also functionally eliminate spore-forming bacteria such as B. anthracis and B. subtilis remains to be further elucidated. Because B. anthracis is hazardous to humans, before analysis using B. anthracis, we employed B. subtilis as a surrogate. To obtain dose-dependent and kinetic data for B. subtilis with C200 NPs, we further analyzed the effects of illumination by visible light at various time points and at various distances (5 cm, 15 cm, and with different illumination intensities of 3 × 104 and 5 × 102 lux (lumen/m2); Figure 2A). The results indicated that C200 substrates can inactivate B. subtilis in half an hour when exposed to various degrees of illumination by visible light (Figure 2B). The bacteria-killing efficiency in the C200 groups was significantly higher than in the respective UV100 TiO2 groups (Figure 2A,B; ** P < 0.01, * P < 0.05). The antibacterial properties of C200 NPs have been demonstrated [17]; however, whether C200 can also functionally eliminate spore-forming bacteria such as B. anthracis and B. subtilis remains to be further elucidated. Because B. anthracis is hazardous to humans, before analysis using B. 2.1. Analyses of TiO2 NPs D d d t (A) d ki ti (B) l f th b t i id l ti it f UV100 TiO d C200 NP Figure 2. Dose-dependent and kinetic analyses of bactericidal activity of C200 NPs against B. subtilis. Dose dependent (A) and kinetic (B) analyses of the bactericidal activity of UV100 TiO and C200 NPs Figure 2. Dose-dependent and kinetic analyses of bactericidal activity of C200 NPs against B. subtilis. Dose-dependent (A) and kinetic (B) analyses of the bactericidal activity of UV100 TiO2 and C200 NPs against B. subtilis after visible-light illumination. Illumination was carried out either at different light densities (at distances of 5 cm, 10 cm and 15 cm with respective illumination intensities of 3 × 104, 1.2 × 103 and 3 × 102 lux) for 30 min (A) or at a light density of 3 × 104 lux (90 mW/cm2) for different periods (B). Under each illumination condition, the surviving bacteria in the UV100 TiO2 groups were normalized to 100%. * P < 0.05 and P < 0.01 compared with the respective UV100 TiO2 groups. n = 6, three experiments with two replicates. 2.3. Antibacterial and Antispore Activities of C200 against Bacillus Species Photocatalyst-mediated killing was performed to determine the antibacterial and antispore effects of photocatalysis on B. cereus, B. thuringiensis, and B. anthracis. Compared with UV100 TiO2, (Figure 2A,B; P < 0.01, * P < 0.05). Figure 2. Dose-dependent and kinetic analyses of bactericidal activity of C200 NPs against B. subtilis. Dose-dependent (A) and kinetic (B) analyses of the bactericidal activity of UV100 TiO2 and C200 NPs against B. subtilis after visible-light illumination. Illumination was carried out either at different light densities (at distances of 5 cm, 10 cm and 15 cm with respective illumination intensities of 3 × 104, 1.2 × 103 and 3 × 102 lux) for 30 min (A) or at a light density of 3 × 104 lux (90 mW/cm2) for different periods (B). Under each illumination condition, the surviving bacteria in the UV100 TiO2 groups were normalized to 100%. * P < 0.05 and ** P < 0.01 compared with the respective UV100 TiO2 groups. n = 6, three experiments with two replicates. 2 3 Antibacterial and Antispore Activities of C200 against Bacillus Species Figure 2. Dose-dependent and kinetic analyses of bactericidal activity of C200 NPs against B. subtilis. 2.1. Analyses of TiO2 NPs Detailed physical properties of UV-responsive pure TiO2 (TiO2; UV100 TiO2) and carbon- containing TiO2 (C200) NPs have been characterized in our previous work [17,26,27]. In the present study, scanning electron microscopy and UV-Vis absorption analyses of the newly prepared C200 NPs were performed (Figure 1). We found that both TiO2 and C200 displayed nanoscale structures (Figure 1A,B), and that an increased content of carbon (Figure 1C) and C200 displayed considerable redshift absorbance compared with TiO2 NPs (Figure 1D), indicating absorbance in the visible light range (wavelength > 380 nm). The UV-Visible diffuse reﬂectance spectra were converted by instrument 3 of 12 Nanomaterials 2016, 6, 237 software to absorbance values, F(R), based on the Kubelka-Munk theory. In the C200 sample, a sharp edge extending to approximately 438 nm and corresponding to a band gap of approximately 2.83 eV was observed, as indicated in one of our previous reports [27]. Nanomaterials 2016, 6, 237 3 of 12 sample, a sharp edge extending to approximately 438 nm and corresponding to a band gap of approximately 2.83 eV was observed, as indicated in one of our previous reports [27]. Nanomaterials 2016 6 237 3 of 12 Figure 1. Scanning electron microscopy and ultraviolet-visible (UV-Vis) absorption spectrum analyses. Scanning electron microscopy (A, B), X-ray photoelectron spectroscopy (XPS) analysis for the 1s atomic orbital of carbon (C) and UV-Vis absorption spectra (D) of UV100 TiO2 and C200 NPs used in this study. The C200 sample absorbed light extending into the visible (>380 nm) region. Figure 1. Scanning electron microscopy and ultraviolet-visible (UV-Vis) absorption spectrum analyses. Scanning electron microscopy (A,B), X-ray photoelectron spectroscopy (XPS) analysis for the 1s atomic orbital of carbon (C) and UV-Vis absorption spectra (D) of UV100 TiO2 and C200 NPs used in this study. The C200 sample absorbed light extending into the visible (>380 nm) region. sample, a sharp edge extending to approximately 438 nm and corresponding to a band gap of approximately 2.83 eV was observed, as indicated in one of our previous reports [27]. Figure 1. Scanning electron microscopy and ultraviolet-visible (UV-Vis) absorption spectrum analyses. Scanning electron microscopy (A, B), X-ray photoelectron spectroscopy (XPS) analysis for edge extending to approximatel 83 eV was observed, as indicated i Figure 1. Scanning electron microscopy and ultraviolet-visible (UV-Vis) absorption spectrum analyses. 2.1. Analyses of TiO2 NPs Antibacterial and Antispore Activities of C200 against Bacillus Species Photocatalyst-mediated killing was performed to determine the antibacterial and antispore effects of photocatalysis on B cereus B thuringiensis and B anthracis Compared with UV100 TiO2 Figure 2. Dose-dependent and kinetic analyses of bactericidal activity of C200 NPs against B. subtilis. Dose-dependent (A) and kinetic (B) analyses of the bactericidal activity of UV100 TiO2 and C200 NPs against B. subtilis after visible-light illumination. Illumination was carried out either at different light densities (at distances of 5 cm, 10 cm and 15 cm with respective illumination intensities of 3 × 104, 1.2 × 103 and 3 × 102 lux) for 30 min (A) or at a light density of 3 × 104 lux (90 mW/cm2) for different periods (B). Under each illumination condition, the surviving bacteria in the UV100 TiO2 groups were normalized to 100%. * P < 0.05 and ** P < 0.01 compared with the respective UV100 TiO2 groups. n = 6, three experiments with two replicates. Figure 2. Dose-dependent and kinetic analyses of bactericidal activity of C200 NPs against B. subtilis. Dose-dependent (A) and kinetic (B) analyses of the bactericidal activity of UV100 TiO2 and C200 NPs against B. subtilis after visible-light illumination. Illumination was carried out either at different light densities (at distances of 5 cm, 10 cm and 15 cm with respective illumination intensities of 3 × 104, 1.2 × 103 and 3 × 102 lux) for 30 min (A) or at a light density of 3 × 104 lux (90 mW/cm2) for different periods (B). Under each illumination condition, the surviving bacteria in the UV100 TiO2 groups were normalized to 100%. * P < 0.05 and P < 0.01 compared with the respective UV100 TiO2 groups. n = 6, three experiments with two replicates. 2.1. Analyses of TiO2 NPs anthracis, we employed B. subtilis as a surrogate. To obtain dose-dependent and kinetic data for B. subtilis with C200 NPs, we further analyzed the effects of illumination by visible light at various time points and at various distances (5 cm, 15 cm, and with different illumination intensities of 3 × 104 and 5 × 102 lux (lumen/m2); Figure 2A). The results indicated that C200 substrates can inactivate B. subtilis in half an hour when exposed to various degrees of illumination by visible light (Figure 2B). The bacteria-killing efﬁciency in the C200 groups was signiﬁcantly higher than in the respective UV100 TiO2 groups (Figure 2A,B; P < 0.01, * P < 0.05). used in this study. The C200 sample absorbed light extending into the visible (>380 nm) region. 2.2. Dose-Dependent and Kinetic Analyses of Photocatalytic Inactivation of B. Subtilis The antibacterial properties of C200 NPs have been demonstrated [17]; however, whether C200 can also functionally eliminate spore-forming bacteria such as B. anthracis and B. subtilis remains to be further elucidated. Because B. anthracis is hazardous to humans, before analysis using B. anthracis, we employed B. subtilis as a surrogate. To obtain dose-dependent and kinetic data for B. subtilis with C200 NPs, we further analyzed the effects of illumination by visible light at various time points and at various distances (5 cm, 15 cm, and with different illumination intensities of 3 × 104 and 5 × 102 lux (lumen/m2); Figure 2A). The results indicated that C200 substrates can inactivate B. subtilis in half an hour when exposed to various degrees of illumination by visible light (Figure 2B). The bacteria-killing efficiency in the C200 groups was significantly higher than in the respective UV100 TiO2 groups Figure 2. Dose-dependent and kinetic analyses of bactericidal activity of C200 NPs against B. subtilis. Dose-dependent (A) and kinetic (B) analyses of the bactericidal activity of UV100 TiO2 and C200 NPs against B. subtilis after visible-light illumination. Illumination was carried out either at different light densities (at distances of 5 cm, 10 cm and 15 cm with respective illumination intensities of 3 × 104, 1.2 103 d 3 102 l ) f 30 i (A) t li ht d it f 3 104 l (90 W/ 2) f diff t i d (Figure 2A,B; ** P < 0.01, * P < 0.05). Figure 2. Dose-dependent and kinetic analyses of bactericidal activity of C200 NPs against B. subtilis. 2.1. Analyses of TiO2 NPs Dose-dependent (A) and kinetic (B) analyses of the bactericidal activity of UV100 TiO2 and C200 NPs against B. subtilis after visible-light illumination. Illumination was carried out either at different light densities (at distances of 5 cm, 10 cm and 15 cm with respective illumination intensities of 3 × 104, 1.2 × 103 and 3 × 102 lux) for 30 min (A) or at a light density of 3 × 104 lux (90 mW/cm2) for different periods (B). Under each illumination condition, the surviving bacteria in the UV100 TiO2 groups were normalized to 100%. * P < 0.05 and ** P < 0.01 compared with the respective UV100 TiO2 groups. n = 6, three experiments with two replicates. re 2. Dose-dependent and kinetic analyses of bactericidal activity of C200 NPs agai -dependent (A) and kinetic (B) analyses of the bactericidal activity of UV100 TiO2 a nst B subtilis after visible light illumination Illumination was carried out either at d B; P < 0.01, P < 0.05). against B. subtilis after visible-light illumination. Illumination was carried out either at different light densities (at distances of 5 cm 10 cm and 15 cm with respective illumination intensities of 3 × 104 1 2 Figure 2 Dose-dependent and kinetic analyses of bactericidal activity of C200 NPs against B subtilis Figure 2. Dose-dependent and kinetic analyses of bactericidal activity of C200 NPs against B. subtilis. against B. subtilis after visible light illumination. Illumination was carried out either at different light densities (at distances of 5 cm, 10 cm and 15 cm with respective illumination intensities of 3 × 104, 1.2 Figure 2. Dose-dependent and kinetic analyses of bactericidal activity of C200 NPs against B. subtilis. Figure 2. Dose-dependent and kinetic analyses of bactericidal activity of C200 NPs against B. subtilis. densities (at distances of 5 cm, 10 cm and 15 cm with respective illumination intensities of 3 × 104, 1.2 × 103 and 3 × 102 lux) for 30 min (A) or at a light density of 3 × 104 lux (90 mW/cm2) for different periods (B). Under each illumination condition, the surviving bacteria in the UV100 TiO2 groups were normalized to 100%. * P < 0.05 and ** P < 0.01 compared with the respective UV100 TiO2 groups. n = 6, three experiments with two replicates. 2.3. y f y Anthrax lethal toxin (LT), which is composed of two 2.4. Photocatalytic Inactivation of Anthrax LT by C200 NPs are shown. The illumination intensity was 3 × 104 lux (90 m * P < 0.05 and ** P < 0.01 compared with respective UV100 T (PA; cell receptor binding) and a lethal factor (LF; metalloprotease of cellular mitogen-activated protein kinase kinase (MAPKKs, MEKs))—is one of the major virulence factors of B. anthracis [29]. Anthrax LT can be detected in culture media and spores of B. anthracis. Treatments of LT can lead to the death of macrophage cells in vitro [30] and lead to mortality in rodents [24,31–35]. To investigate whether photocatalysis can inactivate the protein components and detoxicate the anthrax toxin, LT was subjected to visible-light-activated photocatalysis on UV100 TiO2 and C200 NPs. As expected, C200 NP-mediated photocatalysis markedly reduced the potency of LT to induce cell death of macrophage J774A.1 cells, when compared with UV100 TiO2 groups (Figure 4). These results suggested that treatment with visible-light-responsive C200-mediated photocatalysis of particular objects not only eliminates the bacteria but also reduces the toxicity of LT. Anthrax lethal toxin (LT), which is composed of two protein components—a protective antigen (PA; cell receptor binding) and a lethal factor (LF; metalloprotease of cellular mitogen-activated protein kinase kinase (MAPKKs, MEKs))—is one of the major virulence factors of B. anthracis [29]. Anthrax LT can be detected in culture media and spores of B. anthracis. Treatments of LT can lead to the death of macrophage cells in vitro [30] and lead to mortality in rodents [24,31–35]. To investigate whether photocatalysis can inactivate the protein components and detoxicate the anthrax toxin, LT was subjected to visible-light-activated photocatalysis on UV100 TiO2 and C200 NPs. As expected, C200 NP-mediated photocatalysis markedly reduced the potency of LT to induce cell death of macrophage J774A.1 cells, when compared with UV100 TiO2 groups (Figure 4). These results suggested that treatment with visible-light-responsive C200-mediated photocatalysis of particular objects not only eliminates the bacteria but also reduces the toxicity of LT. two replicates. 2.4. Photocatalytic Inactivation of Anthrax LT by C200 NPs Anthrax lethal toxin (LT), which is composed of two protein components—a protective antigen (PA; cell receptor binding) and a lethal factor (LF; metalloprotease of cellular mitogen-activated protein kinase kinase (MAPKKs, MEKs))—is one of the major virulence factors of B. anthracis [29]. Anthrax LT can be detected in culture media and spores of B. anthracis. Treatments of LT can lead to the death of macrophage cells in vitro [30] and lead to mortality in rodents [24,31–35]. we found that C200 NPs exhibited higher visible-light-responsive anti 2.3. Antibacterial and Antispore Activities of C200 against Bacillus Species Photocatalyst-mediated killing was performed to determine the an 2.3. Antibacterial and Antispore Activities of C200 against Bacillus Species y g p p effects of photocatalysis on B. cereus, B. thuringiensis, and B. anthracis. Compared with UV100 TiO2, we found that C200 NPs exhibited higher visible-light-responsive antibacterial properties to kill B. Photocatalyst-mediated killing was performed to determine the antibacterial and antispore effects of photocatalysis on B. cereus, B. thuringiensis, and B. anthracis. Compared with UV100 TiO2, we found that C200 NPs exhibited higher visible-light-responsive antibacterial properties to kill 4 of 12 Nanomaterials 2016, 6, 237 B. cereus, B. thuringiensis, and B. anthracis vegetative bacteria (Figure 3A, * P < 0.05, ** P < 0.01). Consistent with the antibacterial experiment, in the spore analysis, C200 also exhibited a superior visible-light-responsive antispore activity compared with the UV100 TiO2 NPs, although this activity was less efﬁcient (20%–30% killing/inactivation) (Figure 3B, * P < 0.05) compared with the results obtained in vegetative bacteria experiments (Figure 3A). we found that C200 NPs exhibited higher visible-light-responsive antibacterial properties to kill B. cereus, B. thuringiensis, and B. anthracis vegetative bacteria (Figure 3A, * P < 0.05, ** P < 0.01). Consistent with the antibacterial experiment, in the spore analysis, C200 also exhibited a superior visible-light-responsive antispore activity compared with the UV100 TiO2 NPs, although this activity was less efficient (20%–30% killing/inactivation) (Figure 3B, * P < 0.05) compared with the results obtained in vegetative bacteria experiments (Figure 3A). Nanomaterials 2016, 6, 237 4 of 12 Figure 3. Antibacterial properties of C200 NPs against vegetative bacteria and spores of Bacillus species. Bacteria B. subtilis, B. thuringiensis, B. cereus, and B. anthracis were photocatalyzed using UV100 TiO2 and C200 NPs, respectively. All vegetative bacteria (A) or spores (B) in the UV100 TiO2 groups were normalized to 100%. The relative percentages of surviving pathogens in the C200 groups are shown. The illumination intensity was 3 × 104 lux (90 mW/cm2), and the reaction time was 30 min. * P < 0.05 and ** P < 0.01 compared with respective UV100 TiO2 groups. n = 6, three experiments with two replicates. 2.4. Photocatalytic Inactivation of Anthrax LT by C200 NPs Figure 3. Antibacterial properties of C200 NPs against vegetative bacteria and spores of Bacillus species. Bacteria B. subtilis, B. thuringiensis, B. cereus, and B. anthracis were photocatalyzed using UV100 TiO2 and C200 NPs, respectively. All vegetative bacteria (A) or spores (B) in the UV100 TiO2 groups were normalized to 100%. we found that C200 NPs exhibited higher visible-light-responsive anti 2.3. Antibacterial and Antispore Activities of C200 against Bacillus Species Photocatalyst-mediated killing was performed to determine the an 2.3. Antibacterial and Antispore Activities of C200 against Bacillus Species anthracis were photocatalyzed using UV100 TiO2 and C200 NPs, respectively. All vegetative bacteria (A) or spores (B) in the UV100 TiO2 groups were normalized to 100%. The relative percentages of surviving pathogens in the C200 groups are shown. The illumination intensity was 3 × 104 lux (90 mW/cm2), and the reaction time was 30 min. * P < 0.05 and ** P < 0.01 compared with respective UV100 TiO2 groups. n = 6, three experiments with two replicates. Figure 3. Antibacterial properties of C200 NPs against vegetative bacteria and spores of Bacillus species. Bacteria B. subtilis, B. thuringiensis, B. cereus, and B. anthracis were photocatalyzed using UV100 TiO2 and C200 NPs, respectively. All vegetative bacteria (A) or spores (B) in the UV100 TiO2 groups were normalized to 100%. The relative percentages of surviving pathogens in the C200 groups Figure 3. Antibacterial properties of C200 NPs against vegetative bacteria and spores of Bacillus Figure 3. Antibacterial properties of C200 NPs against vegetative bacteria and spores of Bacillus UV100 TiO2 and C200 NPs, respectively. All vegetative bacteria (A) or spores (B) in the UV100 TiO2 groups were normalized to 100%. The relative percentages of surviving pathogens in the C200 groups are shown. The illumination intensity was 3 × 104 lux (90 mW/cm2), and the reaction time was 30 min. * P < 0.05 and ** P < 0.01 compared with respective UV100 TiO2 groups. n = 6, three experiments with two replicates. h l I f A h b p , g , , p y g TiO2 and C200 NPs, respectively. All vegetative bacteria (A) or spores (B) in the UV100 TiO2 groups were normalized to 100%. The relative percentages of surviving pathogens in the C200 groups are shown. The illumination intensity was 3 × 104 lux (90 mW/cm2), and the reaction time was 30 min. * P < 0.05 and ** P < 0.01 compared with respective UV100 TiO2 groups. n = 6, three experiments with two replicates. Figure 3. Antibacterial properties of C200 NPs against vegetative bacteria and spores of Bacillus species. Bacteria B. subtilis, B. thuringiensis, B. cereus, and B. anthracis were photocatalyzed using UV100 TiO2 and C200 NPs, respectively. All vegetative bacteria (A) or spores (B) in the UV100 TiO2 groups were normalized to 100%. The relative percentages of surviving pathogens in the C200 groups y f y Anthrax lethal toxin (LT), which is composed of two 2.4. Photocatalytic Inactivation of Anthrax LT by C200 NPs are shown. The illumination intensity was 3 × 104 lux (90 m * P < 0.05 and P < 0.01 compared with respective UV100 T To investigate whether photocatalysis can inactivate the protein components and detoxicate the anthrax toxin, LT was subjected to visible-light-activated photocatalysis on UV100 TiO2 and C200 NPs. As expected, C200 NP-mediated photocatalysis markedly reduced the potency of LT to induce cell death of macrophage J774A.1 cells, when compared with UV100 TiO2 groups (Figure 4). These results suggested that treatment with visible-light-responsive C200-mediated photocatalysis of particular Figure 4. Visible-light-responsive C200 NP-mediated inactivation of lethal toxin (LT). Macrophage J774A.1 cells were treated with LT with or without UV100 TiO2 and C200 photocatalysis for 3 h, and surviving cells of untreated groups were adjusted to 100%. Columns designated UV TiO2 or C200 represent that LT was pretreated with photocatalysis by using UV100 TiO2 or C200 NPs, respectively, objects not only eliminates the bacteria but also reduces the toxicity of LT. Figure 4. Visible-light-responsive C200 NP-mediated inactivation of lethal toxin (LT). Macrophage J774A.1 cells were treated with LT with or without UV100 TiO2 and C200 photocatalysis for 3 h, and surviving cells of untreated groups were adjusted to 100%. Columns designated UV TiO2 or C200 represent that LT was pretreated with photocatalysis by using UV100 TiO2 or C200 NPs, respectively, before being treated with J774A.1 cells. P < 0.01, compared with all other groups treated with LT (with or without additional treatments). n = 6, three experiments with two replicates. Figure 4. Visible-light-responsive C200 NP-mediated inactivation of lethal toxin (LT). Macrophage J774A.1 cells were treated with LT with or without UV100 TiO2 and C200 photocatalysis for 3 h, and surviving cells of untreated groups were adjusted to 100%. Columns designated UV TiO2 or C200 represent that LT was pretreated with photocatalysis by using UV100 TiO2 or C200 NPs, respectively, before being treated with J774A.1 cells. ** P < 0.01, compared with all other groups treated with LT (with or without additional treatments). n = 6, three experiments with two replicates. talysis by using p p p y y g p y Figure 4. Visible-light-responsive C200 NP-mediated inactivation of lethal toxin (LT). Macrophage J774A.1 cells were treated with LT with or without UV100 TiO2 and C200 photocatalysis for 3 h, and surviving cells of untreated groups were adjusted to 100%. Columns designated UV TiO2 or C200 represent that LT was pretreated with photocatalysis by using UV100 TiO2 or C200 NPs, respectively, before being treated with J774A.1 cells. we found that C200 NPs exhibited higher visible-light-responsive anti 2.3. Antibacterial and Antispore Activities of C200 against Bacillus Species Photocatalyst-mediated killing was performed to determine the an 2.3. Antibacterial and Antispore Activities of C200 against Bacillus Species The relative percentages of surviving pathogens in the C200 groups are shown. The illumination intensity was 3 × 104 lux (90 mW/cm2), and the reaction time was 30 min. * P < 0.05 and ** P < 0.01 compared with respective UV100 TiO2 groups. n = 6, three experiments with two replicates. cereus, B. thuringiensis, and B. anthracis vegetative bacteria (Figure 3A, * P < 0.05, ** P < 0.01). Consistent with the antibacterial experiment, in the spore analysis, C200 also exhibited a superior visible-light- responsive antispore activity compared with the UV100 TiO2 NPs, although this activity was less efficient (20%–30% killing/inactivation) (Figure 3B, * P < 0.05) compared with the results obtained in vegetative bacteria experiments (Figure 3A). Figure 3. Antibacterial properties of C200 NPs against vegetative bacteria and spores of Bacillus species. Bacteria B. subtilis, B. thuringiensis, B. cereus, and B. anthracis were photocatalyzed using UV100 TiO2 and C200 NPs, respectively. All vegetative bacteria (A) or spores (B) in the UV100 TiO2 groups were normalized to 100%. The relative percentages of surviving pathogens in the C200 groups h Th ill i ti i t it 3 104 l (90 W/ 2) d th ti ti 30 i giensis, and B. anthracis vegetative bacteria (Figure 3A, * P < 0.05, ** P < acterial experiment, in the spore analysis, C200 also exhibited a supe ispore activity compared with the UV100 TiO2 NPs, although this 30% killing/inactivation) (Figure 3B, * P < 0.05) compared with the re eria experiments (Figure 3A). Figure 3. Antibacterial properties of C200 NPs against vegetative bacteria and spores of Bacillus species. Bacteria B. subtilis, B. thuringiensis, B. cereus, and B. anthracis were photocatalyzed using UV100 TiO2 and C200 NPs, respectively. All vegetative bacteria (A) or spores (B) in the UV100 TiO2 groups were normalized to 100%. The relative percentages of surviving pathogens in the C200 groups are shown. The illumination intensity was 3 × 104 lux (90 mW/cm2), and the reaction time was 30 min. * P < 0.05 and ** P < 0.01 compared with respective UV100 TiO2 groups. n = 6, three experiments with two replicates. 2 4 Ph t t l ti I ti ti f A th LT b C200 NP Figure 3. Antibacterial properties of C200 NPs against vegetative bacteria and spores of Bacillus species. Bacteria B. subtilis, B. thuringiensis, B. cereus, and B. 2.5. In Vitro Phagocytic Clearance Analysis 2 5 I Vit Ph ti Cl A l i Our previous reports suggest that VLRP-mediated photocatalysis can injure bacteria [17–19,24,25] and lead to faster clearance of bacteria by phagocytes [24]. To investigate whether photocatalysis might injure the bacterium and make it more vulnerable to phagocyte-mediated killing, B. subtilis was used as a surrogate for B. anthracis to treat macrophage J774A.1 cells. To avoid a biased condition, we applied a subbacterial killing condition (3 × 104 for 5 min), under which bacterial survival could not be considerably suppressed (Figure 2B). The analysis results revealed that B. subtilis in groups without antibacterial photocatalytic properties (i.e., UV100 TiO2 groups and C200 dark groups; Figure 2) tended to have a low reduction rate (killing by macrophages) after being engulfed by the phagocytes (Figure 5). By contrast, the levels of surviving bacteria of macrophage-engulfed B. subtilis were markedly suppressed 8 h after ingestion, only in C200 groups with visible-light illumination (Figure 5, C200 light group). These results suggested that the C200-mediated photocatalysis also induced damage in bacterial cells that could be repaired after plating (Figure 2B, 5 min group, not signiﬁcant reduction), but resulted in a relatively vulnerable phenotype when encountering phagocytes (Figure 5, C200 light 8 h group). This suggested that, although the bacteria may not efﬁciently be killed when a subantibacterial dose of photocatalysis is applied, the bacteria that survive the photocatalysis are still more easily secured and eliminated by phagocytes in the immune system. In other words, the C200-mediated photocatalysis exhibits a protective effect against contaminated bacteria even under subbacterial killing conditions. 2.5. In Vitro Phagocytic Clearance Analysis Our previous reports suggest that VLRP-mediated photocatalysis can injure bacteria [17– 19,24,25] and lead to faster clearance of bacteria by phagocytes [24]. To investigate whether photocatalysis might injure the bacterium and make it more vulnerable to phagocyte-mediated killing, B. subtilis was used as a surrogate for B. anthracis to treat macrophage J774A.1 cells. To avoid a biased condition, we applied a subbacterial killing condition (3 × 104 for 5 min), under which bacterial survival could not be considerably suppressed (Figure 2B). The analysis results revealed that B. subtilis in groups without antibacterial photocatalytic properties (i.e., UV100 TiO2 groups and C200 dark groups; Figure 2) tended to have a low reduction rate (killing by macrophages) after being engulfed by the phagocytes (Figure 5). By contrast, the levels of surviving bacteria of macrophage- engulfed B. y f y Anthrax lethal toxin (LT), which is composed of two 2.4. Photocatalytic Inactivation of Anthrax LT by C200 NPs are shown. The illumination intensity was 3 × 104 lux (90 m * P < 0.05 and P < 0.01 compared with respective UV100 T P < 0.01, compared with all other groups treated with LT (with or without additional treatments). n = 6, three experiments with two replicates. Figure 4. Visible-light-responsive C200 NP-mediated inactivation of lethal toxin (LT). Macrophage J774A.1 cells were treated with LT with or without UV100 TiO2 and C200 photocatalysis for 3 h, and surviving cells of untreated groups were adjusted to 100%. Columns designated UV TiO2 or C200 represent that LT was pretreated with photocatalysis by using UV100 TiO2 or C200 NPs, respectively, before being treated with J774A.1 cells. P < 0.01, compared with all other groups treated with LT (with or without additional treatments). n = 6, three experiments with two replicates. Figure 4. Visible-light-responsive C200 NP-mediated inactivation of lethal toxin (LT). Macrophage J774A.1 cells were treated with LT with or without UV100 TiO2 and C200 photocatalysis for 3 h, and surviving cells of untreated groups were adjusted to 100%. Columns designated UV TiO2 or C200 represent that LT was pretreated with photocatalysis by using UV100 TiO2 or C200 NPs, respectively, before being treated with J774A.1 cells. P < 0.01, compared with all other groups treated with LT (with or without additional treatments). n = 6, three experiments with two replicates. Figure 4. Visible-light-responsive C200 NP-mediated inactivation of lethal toxin (LT). Macrophage J774A.1 cells were treated with LT with or without UV100 TiO2 and C200 photocatalysis for 3 h, and surviving cells of untreated groups were adjusted to 100%. Columns designated UV TiO2 or C200 represent that LT was pretreated with photocatalysis by using UV100 TiO2 or C200 NPs, respectively, before being treated with J774A.1 cells. ** P < 0.01, compared with all other groups treated with LT (with or without additional treatments). n = 6, three experiments with two replicates. 5 of 12 Nanomaterials 2016, 6, 237 2.5. In Vitro Phagocytic Clearance Analysis 2 5 I Vit Ph ti Cl A l i subtilis was treated with J774A.1 macrophage cells (multiplicity of infection (MOI): 0.1 bacteria/cell). Levels of surviving bacteria (colony-forming unit; CFU) harvested from macrophage cell lysate are shown. Columns designated UV TiO2 and C200 represent that anthrax spores were pretreated with photocatalysis by using UV100 TiO2 and C200 NPs, respectively. * P < 0.05, compared with all other groups under the 8 h treatment condition. n = 6, three experiments with two replicates. 2.5. In Vitro Phagocytic Clearance Analysis 2 5 I Vit Ph ti Cl A l i subtilis were markedly suppressed 8 h after ingestion, only in C200 groups with visible- light illumination (Figure 5, C200 light group). These results suggested that the C200-mediated photocatalysis also induced damage in bacterial cells that could be repaired after plating (Figure 2B, 5 min group, not significant reduction), but resulted in a relatively vulnerable phenotype when encountering phagocytes (Figure 5, C200 light 8 h group). This suggested that, although the bacteria may not efficiently be killed when a subantibacterial dose of photocatalysis is applied, the bacteria that survive the photocatalysis are still more easily secured and eliminated by phagocytes in the immune system. In other words, the C200-mediated photocatalysis exhibits a protective effect against t i t d b t i d bb t i l killi diti ntaminated bacteria even under subbacterial killing conditions. Figure 5. Surviving B. subtilis after clearance by macrophages. B. subtilis was treated with J774A.1 macrophage cells (multiplicity of infection (MOI): 0.1 bacteria/cell). Levels of surviving bacteria (colony-forming unit; CFU) harvested from macrophage cell lysate are shown. Columns designated UV TiO2 and C200 represent that anthrax spores were pretreated with photocatalysis by using UV100 TiO2 and C200 NPs, respectively. * P < 0.05, compared with all other groups under the 8 h treatment condition. n = 6, three experiments with two replicates. Figure 5. Surviving B. subtilis after clearance by macrophages. B. subtilis was treated with J774A.1 macrophage cells (multiplicity of infection (MOI): 0.1 bacteria/cell). Levels of surviving bacteria (colony-forming unit; CFU) harvested from macrophage cell lysate are shown. Columns designated UV TiO2 and C200 represent that anthrax spores were pretreated with photocatalysis by using UV100 TiO2 and C200 NPs, respectively. * P < 0.05, compared with all other groups under the 8 h treatment condition. n = 6, three experiments with two replicates. Figure 5. Surviving B. subtilis after clearance by macrophages. B. subtilis was treated with J774A.1 macrophage cells (multiplicity of infection (MOI): 0.1 bacteria/cell). Levels of surviving bacteria (colony-forming unit; CFU) harvested from macrophage cell lysate are shown. Columns designated UV TiO2 and C200 represent that anthrax spores were pretreated with photocatalysis by using UV100 TiO2 and C200 NPs, respectively. * P < 0.05, compared with all other groups under the 8 h treatment condition. n = 6, three experiments with two replicates. Figure 5. Surviving B. subtilis after clearance by macrophages. B. 3. Discussion 3. Discussion Disinfection is a vital method for controlling numerous pathogens in the sterilization of critical instruments, water treatment, food production, and hospital environments. Traditional chemical- based disinfectants, such as alcohols, aldehydes, iodine, phenols, and chlorine, have been used for centuries in environmental cleaning [18]. Among these, agents and methods such as formaldehyde, hypochlorite solutions, chlorine dioxide, and radiation have been used to inactivate anthrax spores [8]. Although these methods may be effective, they have drawbacks. Many of these disinfectants are volatile, and their byproducts can be toxic and carcinogenic to humans. The establishment and development of novel anti-anthrax strategies are necessary Disinfection is a vital method for controlling numerous pathogens in the sterilization of critical instruments, water treatment, food production, and hospital environments. Traditional chemical-based disinfectants, such as alcohols, aldehydes, iodine, phenols, and chlorine, have been used for centuries in environmental cleaning [18]. Among these, agents and methods such as formaldehyde, hypochlorite solutions, chlorine dioxide, and radiation have been used to inactivate anthrax spores [8]. Although these methods may be effective, they have drawbacks. Many of these disinfectants are volatile, and their byproducts can be toxic and carcinogenic to humans. The establishment and development of novel anti-anthrax strategies are necessary. development of novel anti-anthrax strategies are necessary. Compared with chemical disinfectants, the TiO2 photocatalyst is safe, nontoxic, and produces no hazardous byproducts [36,37]. Utilization of the excellent photocatalytic antibacterial effect of TiO2 appears to be a conceptually feasible technology for solving the aforementioned problem of chemical Compared with chemical disinfectants, the TiO2 photocatalyst is safe, nontoxic, and produces no hazardous byproducts [36,37]. Utilization of the excellent photocatalytic antibacterial effect of TiO2 appears to be a conceptually feasible technology for solving the aforementioned problem of 6 of 12 Nanomaterials 2016, 6, 237 chemical disinfectants. However, traditional antibacterial TiO2 photocatalysts are activated by UV irradiation, which is hazardous to humans [12,13,18]. The UV-responsive TiO2 photocatalysts are therefore unsuitable for application in indoor environments. By contrast, visible-light-activated TiO2 photocatalytic NPs exhibit at least four advantages. First, compared with UV-responsive materials, visible-light-activated TiO2 photocatalysts offer potential for use as a disinfectant in human living environments such as indoor spaces and public areas. Second, because TiO2 is a chemically stable and inert material, it can continuously exert antimicrobial action when illuminated by light. Third, the bactericidal activity can be switched on and off or modulated by controlling the light intensity. 3. Discussion 3. Discussion Fourth, the transportability of NPs offers greater adjustability. Because of these advantages, visible-light-activated photocatalytic NPs might be used complementarily with existing disinfection technologies against anthrax. g g Emerging nanomaterials and particular NPs have been implicated as having tremendous potential applications in environmental and disinfection use [38]. We previously demonstrated that photocatalytic ﬁlms can eliminate vegetative bacteria and spores of anthrax [24]. However, compared with nanostructured ﬁlms, which have to be precoated at the surfaces of particular objects, NPs frequently show superior portability [39]. For example, VLRP NPs can theoretically be easier to spread into a dead corner of spore-contamination-suspected objects, houses, territorial waters, and even aerosol spaces, when compared with VLRP ﬁlms. Therefore, in the present study, visible-light-responsive C200 NPs were used. For toxicity consideration, previous studies have shown that TiO2 NPs are generally considered safe for skin contact or even ingestion by healthy animals [40–42]. However, inhalation of TiO2 NPs can be toxic to the lungs [42]. As a result, in a real setting, aerosol TiO2 NPs should be avoided unless the nearby persons are provided with appropriate certiﬁed masks (e.g., N95) and/or air cleaning and ﬁltering equipment. A pioneering work demonstrated that supplements with various types of NPs, including TiO2 NPs, may reduce the growth of Escherichia coli and B. anthracis in bacterial culture [43]; however, it showed that the growth retardation effect was due to the presence of excessive NPs in the culture medium rather than bacterial killing. In addition, the photocatalytic effect of TiO2 NPs on B. anthracis has not yet been reported. Therefore, to determine a more efﬁcient way to eliminate the contaminated pathogenic bacteria, relevant veriﬁcations that involve using photocatalytic NPs are necessary. Carbon-containing titanium dioxide C200 NPs exhibit considerably superior photocatalytic properties under visible-light illumination compared with UV100 TiO2 NPs [17,19,24,26–28]. However, whether C200 NPs can eliminate spore-forming bacteria such as Bacillus species has remained elusive. Therefore, in the present study, we demonstrated that visible-light-responsive C200 NP-mediated photocatalysis can eliminate considerable levels of both vegetative bacteria and spores of Bacillus species, including B. subtilis, B. cereus, B. thuringiensis, and B. anthracis. The analysis data suggested that C200 NPs are useful for eliminating contamination of these spore-forming bacteria. Notably, in agreement with our previous ﬁndings [24], our data suggest that C200-mediated photocatalysis somehow injures the bacteria even under suboptimal antibacterial conditions, thus leading to faster clearance of the photocatalyzed bacteria by phagocytes (Figure 5). 3. Discussion 3. Discussion This suggests that C200 can still exert anti-anthrax properties even at a subantibacterial level. 4.2. Bacterial Strains and Culture Bacterial culture and maintenance were conducted according to previously described methods [24]. B. anthracis (ATCC 14186) containing both pXO1 and pXO2 plasmids to express functional LT and edema toxin (ET) was grown on blood agar plates (BAPs) and maintained in brain–heart infusion broth (BHIB) (Sigma-Aldrich, St. Louis, MO, USA) using previously described methods [17,24,30–34,45–47]. B. cereus (ATCC 13061) and B. thuringiensis (ATCC 35646) were maintained and cultured in BAPs or BHIB at 30 ◦C, and B. subtilis (ATCC 39090) was maintained and cultured in trypticase soy agar or broth (Sigma-Aldrich, St. Louis, MO, USA) at 37 ◦C [24,48]. Bacterial strains were stored in a 50% culture medium and 50% glycerol solution at −80 ◦C before use. To reactivate bacteria from frozen stock, 25 µL of bacterial stock solution was transferred to a test tube containing 5 mL of a freshly prepared culture medium and then incubated at 30 ◦C or 37 ◦C under agitation overnight (16–18 h). Spores of B. anthracis were prepared as previously described [24,49,50]. Overnight BHIB cultures of B. anthracis were diluted to approximately 107 colony-forming units (CFU)/mL in phosphate-buffered saline, and 0.1-mL aliquots were inoculated onto blood agar plates. The agar plates were incubated at 25–37 ◦C until 90%–99% phase-bright spores were observed using phase-contrast light microscopy. Spores were harvested and washed with cold sterile DI water as previously described [49] and stored in the freezer at −20 ◦C for up to 1 mol. The quality of spores was determined by two complementary criteria previously established for validating the presence of dormant spores [50]. The criteria consisted of evaluating (i) the absence of vegetative cells (rods) determined through microscopic examination as described, and (ii) the survival of spores in hydrochloric acid (2.5 N). Spore preparations of B. subtilis, B. cereus, and B. thuringiensis were conducted following the same protocol. 4.1. Preparation of Photocatalysts Visible-light-responsive carbon-containing mixed phase TiO2 nanoparticles [TiO2(C) NPs; C200 NPs] were prepared using a modiﬁed sol-gel method as previously described [17,26,27]. A measured quantity of tetrabutyl orthotitanate (50 mmol) was slowly introduced into 90 mL of anhydrate ethanol and 20 mL of deionized (DI) water in a 250 mL ﬂask. After complete dissolution, 4 mL of nitric acid was added to catalyze the hydrolysis and condensation reactions. The mixed solution was uniformly agitated at 500 rpm for 3 h, producing a precipitate of titanium hydroxide. After drying 7 of 12 7 of 12 Nanomaterials 2016, 6, 237 at 110 ◦C, the dried powder was calcined in air at 200 ◦C for 5 h and designated as C200 [17,24,26,27]. Details of preparing the production of C200, including structural properties and the sizes of primary particles, were reported in our earlier work [27]. The surface of C200 was found to contain unique anatase/rutile mixed crystalline phases that exhibit strong visible-light absorption and photocatalytic effects [22,23]. The carbonaceous species on TiO2(C) NPs exist in an amorphous form, as observed in Raman spectra [26–28]. One commercially available TiO2 NP (UV100, Sachtleben, Germany) that can exert photocatalytic properties only when illuminated by UV light was used for comparison. Because C200 samples often aggregate into larger clusters because of surface charges, in Van der Waals interactions, we dispersed the aggregates by using sonication (Transsonic Digital TP680DH, Elma Schmidbauer, Singen, Germany) before the bacteria-killing or bacteria-photocatalyst interaction experiments. The UV-Vis absorption spectra were recorded on a Hitachi 3300H spectrophotometer (Hitachi Taiwan, Taipei, Taiwan) [24,44]. Particle size and morphology were determined using a transmission electron microscope (Philips Tecnai F20 G2 FEI-TEM, Philips Taiwan, Taipei, Taiwan) and a scanning electron microscope (JEM-3010, JEOL, Tokyo, Japan) [17,21]. Material compositions were determined using X-ray photoelectron spectroscopy (Perkin Elmer SSI-M probe XPS system and S4800, Waltham, MA, USA). 4.3. Photocatalytic Reaction and Detection of Viable Bacteria In this study, bacterial concentrations were either determined using the standard plating method or inferred from optical density readings at 600 nm (OD600). For each Bacillus species, a factor for converting the OD600 values of the bacterial culture to concentration (CFU/mL) was calculated as follows. A fresh bacterial culture was diluted by factors of 10−1 to 10−7, and the OD600 of these dilutions was measured. The bacterial concentrations of these dilutions were determined using the standard plating method. To determine the bactericidal effects of the TiO2-related substrates, 200 µL of bacterial overnight culture was transferred into 5 mL of a culture medium and incubated at 37 ◦C until an OD600 of 0.3–0.6 (log phase) was reached. The bacterial concentrations were calculated using the previously determined conversion factor for the bacteria, and the cultures were diluted to 1 × 105 CFU/mL with the culture medium. The bacterial cultures (2.5 × 104 CFU) were mixed 8 of 12 Nanomaterials 2016, 6, 237 with the TiO2 or C200 NPs (200 µg/mL in 150 µL of normal saline) using a 200 µL pipetman and placed onto a 24-well cell culture dish. Photocatalytic reactions were carried out using an incandescent lamp (Classictone incandescent lamp, 60 W, Philips Taiwan, Taipei, Taiwan). A light meter (model LX-102, Lutron Electronic Enterprises, Taipei, Taiwan) was used to record the illumination density. In the dose-dependent experiments, illumination was carried out for 5 min at distances of 5, 10 and 15 cm from the lamp, corresponding to illumination densities of 3 × 104, 1.2 × 103 and 3 × 102 lux (lumen/m2) (equivalent to 90, 30 and 10 mW/cm2), respectively. In the kinetic analysis experiments, illumination was carried out for 1, 5, 10, 20 and 30 min at a distance of 5 cm, corresponding to an illumination density of 3 × 104 lux (90 mW/cm2). Unless speciﬁed, illumination was carried out in a 4 ◦C cold room to prevent overheating of the photocatalyst-containing solution. After illumination, the levels of surviving bacteria were determined using a standard plating method immediately after bacterial collection. In the spore experiments, 1 × 104 CFU (1 × 105 CFU/mL in 100 µL) was used, and the procedures followed the same protocols as those in the live bacteria experiments. 4.4. Cytotoxicity Analysis Cytotoxicity of anthrax LT was measured following previously described methods [30,45]. Anthrax LT is composed of two protein components, a PA (cell receptor-binding) and an LF (metalloprotease). The PA and LF were puriﬁed from B. anthracis (ATCC 14186) culture supernatants, as previously described [24,30–34,45,47]. The culture supernatants were ﬁlter-sterilized by being passed through a 0.22-mm ﬁlter (Millipore, Bedford, MA, USA) and concentrated using the Minitan Ultraﬁltration System (Millipore, Bedford, MA, USA). Protease inhibitor phenylmethylsulfonyl ﬂuoride 0.1 mM (Sigma-Aldrich, St. Louis, MO, USA) [51,52] was added to prevent toxin degradation. Ammonium sulfate was added to 75% to precipitate the protein; and the protein was collected and suspended in 20 mM tris(hydroxymethyl)aminomethane HCl (Tris-HCl) pH 8.0, and extensively dialyzed against the same buffer. The puriﬁcation was further performed using fast protein liquid chromatography Mono Q anion exchange (Pharmacia, Piscataway, NJ, USA) with a 20 mM Tris-HCl pH 8.0 buffer and linear 0–400 mM NaCl gradient elution over 40 min. The PA was eluted at 130–140 mM NaCl, and the LF was eluted at 250–270 mM NaCl. Lipopolysaccharide (LPS) contamination was monitored using a Limulus Amoebocyte Lysate QCL-1000 kit (Lonza, Walkersville, MD, USA) [31,35,53]. Batches of puriﬁed LT with an LPS contamination level of less than 1 EU/mg of LT were used [31]. The LT solution was mixed with the TiO2 or C200 nanoparticles (200 µg/mL in 150 µL of normal saline) by using a 200 µL pipetman and placed onto a 24-well cell culture dish. After inactivation in visible-light illumination for 30 min (3 × 104 lux; 90 mW/cm2), the LT (LF:PA = 1:5) was ready for use. Cytotoxic doses of LT (10 mg/L) [30], with or without pretreatment of photocatalytic inactivation using pure TiO2 and C200 NPs, was used to treat mouse macrophage J774A.1 cells. Three hours after the LT treatments, the cell viability of J774A.1 cells was measured using a WST-1 kit (Roche, Mannheim, Germany) [30,53,54], following manufacturer instructions. 4.5. Phagocytosis Analysis B. subtilis was dissolved in normal saline (100 µL, 1 × 105 CFU/mL) and then incubated with TiO2 and C200 NPs. The bacterial cultures (2.5 × 104 CFU) were mixed with TiO2 or C200 NPs (200 µg/mL in 150 µL of normal saline) using a 200 µL pipetman and placed onto a 24-well cell culture dish. The bacterium–photocatalyst mixtures were then illuminated with visible light (Classictone incandescent lamp, 60 W, Philips; 90 mW/cm2; lamp target distance 10 cm) for 30 min in 24-well plates. After illumination, the bacteria-containing solutions (85 µL) were recovered from the 24-well plates. The bacterial solution was then added into one well of a six-well cell culture dish containing conﬂuent murine macrophage J774A.1 cells (1 × 106 cells/well; MOI: 0.1 bacteria/cell). After phagocytosis was carried out for 1 h, the culture medium was removed, and 200 µL of a cell lysis buffer (100 mM Tris-HCl [pH 7.4], 10 mM MgCl2, 100 mM NaCl, 0.2% sucrose, 0.5% Triton X-100) modiﬁed from that described in previous literature [55,56] was then added to release the cell-engulfed or cell-bound bacteria. 9 of 12 Nanomaterials 2016, 6, 237 A serum-free and antibiotic-free cell culture medium (Dulbecco’s modiﬁed Eagle’s medium) was used in this analysis. 4.6. Statistical Analyses The means, standard deviations, and statistics for the quantiﬁable data were calculated using Microsoft Ofﬁce Excel 2003 (Microsoft, Redmond, WA, USA), SigmaPlot 10 (Systat Software, San Jose, CA, USA), and SPSS Statistics for Windows, Version 19.0 (IBM, Armonk, NY, USA). Signiﬁcance of the data was examined using one-way ANOVA followed by the post hoc Bonferroni-corrected test. The probability of type 1 error α = 0.05 was recognized to be the threshold of statistical signiﬁcance. 5. Conclusions In the present study, we demonstrated that C200 NPs are conceptually feasible nanomaterials for eliminating the vegetative cells and spores of B. anthracis. In particular, C200-mediated photocatalysis exhibits a protective effect against the bacteria, even under suboptimal conditions. Our results collectively suggest that C200 NPs and the extended concepts and technologies described in this report are useful for the development of new strategies against anthrax. Acknowledgments: This work was supported by Ministry of Science and Technology of Taiwan R.O.C. under Grant No. 95-2314-B-320-009-MY3 and 102-2221-E-259-005-MY3, and Ministry of Economic Affairs of Taiwan R.O.C. under Grant No. 98-EC-17-A-19-S2-0111. Author Contributions: Hsin-Hou Chang and Der-Shan Sun conceived and designed the experiments; Der-Shan Sun, Jyh-Hwa Kau, Wen-Shiang Wu and Hsin-Hsien Huang performed the experiments; Hsin-Hou Chang and Jyh-Hwa Kau analyzed the data; Yao-Hsuan Tseng and Hsin-Hsien Huang contributed materials; Hsin-Hou Chang wrote the paper. Conﬂicts of Interest: The authors declare no conﬂict of interest. ts of Interest: The authors declare no conﬂict of intere Abbreviations The following abbreviations are used in this manuscript: TiO2 titanium dioxide TiO2(C) carbon-containing TiO2 VLRP visible-light-responsive photoc NPs nanoparticles UV ultraviolet ROS reactive oxygen species H2O2 hydrogen peroxide •OH hydroxyl radicals O2− superoxide anions LT anthrax lethal toxin PA anthrax protective antigen LF anthrax lethal factor ET anthrax edema toxin MOI multiplicity of infection CFU colony-forming units LPS lipopolysaccharide TiO2 TiO2(C VLRP NPs UV ROS H2O2 •OH O2− LT PA LF ET MOI CFU LPS References Photobiol. B 2001, 64, 136–143. [CrossRef] 15. Wu, M.S.; Sun, D.S.; Lin, Y.C.; Cheng, C.L.; Hung, S.C.; Chen, P.K.; Yang, J.H.; Chang, H.H. Nanodiamonds protect skin from ultraviolet b-induced damage in mice. J. Nanobiotech. 2015, 13, 35. [CrossRef] [PubMed] 16. 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https://openalex.org/W2597065118	https://edoc.hu-berlin.de/bitstream/18452/20611/3/The_Landscape_of_Research_Data_Repositories_final.pdf	English	null	The Landscape of Research Data Repositories in 2015: A re3data Analysis	D-Lib magazine	2,017	cc-by	15,326	The Landscape of Research Data Repositories in 2015 A re3data Analysis Maxi Kindling maxi.kindling@hu-berlin.de 1, Heinz Pampel  heinz.pampel@gfz-potsdam.de 2, Stephanie van de Sandt  stephanie.van.de.sandt@cms.hu-berlin.de 1, Jessika Rücknagel ruecknagel@sub.uni-goettingen.de 1, Paul Vierkant  paul.vierkant@gfz-potsdam.de 2, Gabriele Kloska  gabriele.kloska@kit.edu 3, Michael Witt mwitt@purdue.edu 4, Peter Schirmbacher schirmbacher@hu-berlin.de 1, Roland Bertelmann roland.bertelmann@gfz-potsdam.de 2, and Frank Scholze frank.scholze@kit.edu 3 1Humboldt-Universität zu Berlin, Berlin School of Library and Information Science (BSLIS), Germany 2GFZ German Research Centre for Geosciences, Section 7.4 Library and Information Services (LIS) 3Karlsruhe Institute of Technology (KIT), KIT Library, Germany 4Purdue University Libraries, West Lafayette, Indiana, USA This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ Abstract This article provides a comprehensive descriptive and statistical analysis of metadata information on 1,381 research data repositories worldwide and across all research disciplines. The analyzed metadata is derived from the re3data database, enabling search and browse functionalities for the global registry of research data repositories. The analysis focuses mainly on institutions that 1 1 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 operate research data repositories, types and subjects of research data repos- itories (RDR), access conditions as well as services provided by the research data repositories. RDR differ in terms of the service levels they offer, lan- guages they support or standards they comply with. These statements are commonly acknowledged by saying the RDR landscape is heterogeneous. As expected, we found a heterogeneous RDR landscape that is mostly influenced by the repositories’ disciplinary background for which they offer services. Keywords: Research Data Repositories, RDR, Statistical Analysis, Metadata, re3data, Open Science, Open Access, Research Data, Persistent Identifier, Dig- ital Object Identifier, Licenses This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 1.1 Research data repositories for open research data The idea of Open Science is becoming increasingly important (Nielsen 2011; Bartling and Friesike 2014; OECD 2015). An essential part of Open Science is Open Access to research data (Pampel and Dallmeier-Tiessen 2014). By sharing research data and other research materials, third parties can assess scholarly knowledge based on these data. The availability of research data and research materials fosters transparency and trustworthiness in research processes as well as enabling the reuse of research data. Accurately generated and curated datasets can be reanalyzed to validate re- search findings or reused and repurposed to answer different research questions. If datasets are easily accessible, new discoveries are facilitated and duplicate work can be reduced (Simons and Richardson 2013). This may lead to the high economic benefit that open research data may have for research and public societies. Sev- eral studies expose this economic impact, which the curation and sharing of valuable research data produces (Beagrie and Houghton 2014; Houghton and Gruen 2014). Considering these benefits, research data have to be regarded as a core element of the scholarly record. It is for this reason that funding organizations established data policies to influence the data management practices of researchers receiving pub- lic funding (European Commission. Directorate-General for Research & Innovation 2016). This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 2 2 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 The term “research data” can mean many things. As Borgman stated correctly, “data is a difficult concept to define, as data takes many forms, both physical and digital” (Borgman et al. 2012). In Pampel, Vierkant, et al. (2013) we define digital research data as “a (descriptive) part or the result of a research process”, covering all stages of research. This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 1.1 Research data repositories for open research data “Digital research data occur in different data types, levels of aggregation and data formats, informed by the research disciplines and their methods.” (Pampel, Vierkant, et al. 2013) To share or reuse research data, researchers and other interested parties need to be able to find information about datasets and the respective source making research datasets available. In this respect research data repositories (RDR) greatly contribute to realize the sharing and reuse of research data. Policies and recommendations for proper research data management stipulate that research data should be made available in an appropriate RDR to comply with the principles of research integrity (Pampel, Vierkant, et al. 2013; The Royal Society 2012). A RDR is a technical and organizational information system that helps researchers to manage, store and provide their own datasets, and to easily find and access datasets from other sources. Different approaches exist for operating a RDR. It can be described as “a subtype of a sustainable information infrastructure which provides long-term storage and access to research data” (Rücknagel et al. 2015). RDRs are an essential part of the research infrastructure of different “facilities, resources and related services used by the scientific community to conduct top-level research in their respective fields.” (European Commission 2016) At the same time, it is not easy to find an appropriate RDR for storing or reusing datasets. Marcial and Hemminger (2010) conducted a websample study of 100 RDR, analyzing information which is presented on the respective webpages. As they stated “there are many differences in the size, types, and organizations” of RDR (Marcial and Hemminger 2010), in part with differing scopes of services. It is ambiguous how many RDR are operated, but a tendency of growth can be assumed. Thus, a heterogeneous (and constantly changing) landscape of RDR can be described (Pampel, Vierkant, et al. 2013). 3 3 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 1.2 re3data — Registry of Research Data Repositories The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 ◦Technical aspects such as information concerning supported persistent identifier systems, application programming interfaces or software in use if determinable. ◦Technical aspects such as information concerning supported persistent identifier systems, application programming interfaces or software in use if determinable. All research data repositories listed in re3data are indexed and reviewed by our re3data editorial team. Up to the end of the funding period in December 2015, the editorial team consisted of project members from the partner institutions. Dur- ing the funding period, we primarily checked other registries listing research data and other repositories to include them in re3data. The editorial team analyzes the website of a research data repository thoroughly using an internal handbook that pro- vides practical information on how to obtain the metadata properties of the re3data schema. All of the information gathered was then reviewed by a second editor to improve the quality of the metadata entries provided on the website re3data.org. We are currently planning to enlarge the editorial team led by DataCite, to enable the indexing of repositories which do not use English as the interface language. Every re3data record is persistently accessible and citable via a Digital Object Identi- fier (DOI). The registry offers a suggestion form to add RDR to re3data. By using the form, users and repository managers can provide detailed information about reposi- tories that have not yet been indexed. Furthermore, re3data enables machine access to the registry via an Application Programming Interface (API). Funding agencies such as the European Commission (Tarazona Rua et al. 2015) and the National Science Foundation (National Science Foundation. Directorate for Bi- ological Sciences 2015) include references to re3data in their guidelines and policies related to data management and sharing. Additionally, several publishers and jour- nals such as Copernicus Publications, PeerJ, PLOS ONE and Nature’s Scientific Data recommend re3data in their editorial policies as a tool for authors to deposit data that support research findings published in their journals. This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 1.2 re3data — Registry of Research Data Repositories re3data was a research project funded by the German Research Foundation (DFG) from 2012 until 2015 to create the Registry of Research Data Repositories called re3data. Project partners were the Library and Information Services (LIS) of the GFZ German Research Centre for Geosciences, the Library of the Karlsruhe Insti- tute of Technology (KIT) and the Berlin School of Library and Information Science (BSLIS) at Humboldt-Universität zu Berlin. The project team developed the re3data metadata schema and provided a web interface to facilitate RDR search and browse functionalities. In 2014, the two major international registries for RDR — DataBib and re3data — joined forces and merged to one service, making the Library of the Purdue University a project partner. Since January 2016, re3data has been a service of DataCite (Brase et al. 2015) to ensure the registry’s sustainable development. The objective of our service re3data is to index and describe RDR so as to present detailed information about existing services. Being a starting point for researchers, funders, publishers, and other stakeholders to find and evaluate suitable services that support the management, storage, access and usage of research data such as research data repositories, portals and other service providers, re3data also helps different target groups to decide which RDR is appropriate for different purposes. The registry currently indexes over 1,821 RDR (as of 26 February 2017) with an extensive metadata description. Our approach for re3data is to provide extensive and quality-approved descriptions of RDR. For this reason, the “Metadata Schema for the Description of Research Data Repositories” is a comprehensive set of 42 properties (Rücknagel et al. 2015). The re3data description of a RDR, also referred to as a re3data metadata entry, provides the following information: ◦General information about the RDR, such as the repository name, URL, disci- plinary scope and a descriptive paragraph. ◦General information about the RDR, such as the repository name, URL, disci- plinary scope and a descriptive paragraph. ◦Information concerning the responsible institutions of a RDR, such as the in- stitution’s name, type, location, and the type of responsibility. ◦Information concerning the responsible institutions of a RDR, such as the in- stitution’s name, type, location, and the type of responsibility. ◦Legal issues including access and upload regulations as well as the availability of policies. 4 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). 2 Methods By analyzing the re3data metadata entries, we want to reveal the state of the art within the field of RDRs by the end of 2015. Furthermore, the findings presented in this analysis are meant to provide initial information on the RDR landscape to help 5 5 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 identify areas requiring improvement. We explore the landscape of RDR focusing on the following research questions: identify areas requiring improvement. We explore the landscape of RDR focusing on the following research questions: ◦Which types of RDR can be identified? ◦Which types of RDR can be identified? ◦What is the institutional background of the RDR covered? ◦What are the disciplines and content types for which RDRs offer services? ◦What are the disciplines and content types for which RDRs offer services? ◦How do RDRs differ in terms of technical standards and access regulations? ◦How do RDRs differ in terms of technical standards and access regulations? To answer these research questions, we conducted a quantitative data analysis of a total of 1,381 RDR metadata entries in the re3data database at the end of the project funding phase, as of 3 December 2015. All metadata entries included in this database dump were indexed according to the “re3data.org Schema for the Description of Research Data Repositories” Version 2.2 (Vierkant et al. 2014) or below. re3data does not necessarily contain a representative sample of all RDR worldwide at that time. It is important to note that the schema was adjusted during the indexing process. Further the majority of the RDRs are indexed by our editorial team by analyzing RDRs’ websites. As a consequence of the re3data indexing process, we cannot assess whether a respective metadata entry contains all information applicable to the RDR. This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 2 Methods The analysis reflects the RDR landscape in the indexing period in re3data up to the end of 2015. The re3data metadata entries support drawing conclusions at the repository level. For instance, we can make statements on the technical capabilities of RDR to provide research data of a special content type such as text documents, audio and video material as often stated on the RDRs’ website. Accordingly this paper does not provide an investigation on the distribution of individual content types in research data repositories on the level of data sets and collections. The re3data database snapshot was provided as an SQL (Structured Query Language) dump and imported into a PostgreSQL database. A number of SQL queries based on the research questions were used to extract the required information from the database into a csv format (comma separated values). We used the unique internal database identifier of repositories (“re3data repository id”) that served as foreign key within the relation We used the unique internal database identifier of repositories re3data.repository_id”) that served as foreign key within the relational database 6 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 for the statistical analysis of the metadata entries. Most properties of a repository are modeled as 1:n relation in the re3data metadata schema, allowing the selection of more than one value (such as more than one “content type”). Thus multiple tuples of each identifier and the appropriate property could occur. This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 3.1 Institutional background re3data provides information on the institutions that are responsible for developing, maintaining, funding, etc. RDR listed in re3data. The metadata schema differenti- ates between subcategories further describing the type of responsibility, whether an institution is profit or non-profit and the institution’s country of origin. Institutions can have more than one type of responsibility for a respective RDR, whereas only the head quarter’s country is indexed. The categories are explained within the metadata schema on page 20 (Vierkant et al. 2014). 2 Methods The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 lower than 0.3 is considered to be a low effect size. Values equal to 0.3 and lower than 0.5 are considered to be medium effect (Cohen 1988). The research data set consisting of five data tables, a matrix of all correlations and a research data documentation is publicly available (Sandt et al. 2017). The correlation matrix can be used as an overview on all results of our analysis, whereas this article only presents results that are of main importance. The research data documentation provides detailed information on the methods and data transformation and aims to help reproduce the data analysis. This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 2 Methods In order to obtain a useful dichotomous system for the statistical analysis with SPSS (mainly used SPSS Version 22.0.0 Mac OS X on Mac OS X 10.7.5) we modeled the existence of repository characteristics with a binary representation, transposing the property values into new variables for each research data repository identifier (ID) with the help of a Python script. This form allowed us to perform analytical tests for correlations with SPSS Statistics. The script matched rows that are dependent on the internal database identifier. If a value exists for an ID, it will receive a 1 as new value for this variable. For example if a RDR has “plain text” and “raw data” as values in the variable “content type”, it receives a 1 for the variable “contenttype:plain text” and “contenttype:raw data”, but a 0 as a value for all other “content type” variables. Moreover, data cleansing included to consolidate multiple equal values for one RDR to a single value (e.g. some IDs have several “other” certificates etc.). Hence, the parent population of data differs in some cases from the original database population. In the following text, we always refer to this cleansed dataset. We needed the raw quantities only in one case, which is explained at that point. As the given data are dichotomous nominal values, our range of statistical methods was limited to nonparametric procedures. Apart from the presentation of numbers and relative occurrences of variables, we also identified correlations between nomi- nal variables in the data, e.g. dependencies between “content type” and “subject”. We chose contingency tables and chi-square distribution (χ2) tests combined with Cramer’s V value for this. By using contingency tables we compare expected values in case of statistical inde- pendence calculated by SPSS with the observed values to reject the null hypothesis in order to find significant correlations. Mostly those correlations substantiate pre- liminary assumptions on the disciplinary influence on RDR. A number of correlation effects that passed the test on at least a 95% significance level are presented in this article. Unless otherwise stated, the presented correlations are significant on the 95 % level. The strength of dependency was tested by Cramer’s V, whereby a value 7 7 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). 3.1.1 Responsibility types of institutions We list a total of 4,311 institutions that are responsible for the 1,381 registered RDR. One institution may occur multiple times and with differing names or organizational units. Each RDR may also have multiple responsible institutions (1:n relation). The re3data metadata schema distinguishes between “general”, “funding”, “technical” and “sponsoring” responsibility of institutions. Nearly half of the listed institutions have a general responsibility for the associated RDR (43.0 %). That means the particular institution is responsible for the content as well as for the management of the RDR. One third are funding institutions (33.4 %). Additionally, 21.7 % of the institutions are technical hosts while only 1.9 % of all institutions are RDR sponsoring 8 8 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 institutions, meaning that funds are granted to a research data repository in exchange for advertising. About one third of all repositories (29.3 %) are related to institutions with more than one area of responsibility. institutions, meaning that funds are granted to a research data repository in exchange for advertising. About one third of all repositories (29.3 %) are related to institutions with more than one area of responsibility. The distribution of responsibility types of institutions according to the total amount of 1,381 RDR indexed in re3data is shown in a Venn diagram (cf. Figure 1). It is important to note that occurrences of multiple responsibility types are reduced to one for each type. Most RDR (49.4 %) are operated by institutions that are responsible in general, technical and funding terms; 20.5 % are operated by technically and generally responsible institutions. Only 2.3 % of all RDR are operated by institutions of all types of responsibility. This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 9 3.1.2 Profit or non-profit institutions Nearly all (96.6 %) of the above mentioned institution entries in re3data (n = 4,311) are non-profit organizations. Most of the repositories are funded solely by non-profit organizations, but hybrid forms also exist (4.9 % of all RDR have more than one institution type) as well as those that are profit institutions (2.1 %). 3.1.1 Responsibility types of institutions Figure 1: Responsibility types of institutions operating research data repositories in- dexed in re3data (n = 1,381) Figure 1: Responsibility types of institutions operating research data repositories in- dexed in re3data (n = 1,381) 9 9 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 3.1.3 Breakdown by country For the analysis of the institutions’ countries, we focused on the absolute number of institutions for each RDR regardless of multiple values (a RDR might be related to several institutions from identical countries. For example a RDR can be supported by five institutions, all of them from the USA). We therefore refer to the uncleansed dataset. Most institutions (n = 1,936) are from the USA. Germany (n = 521), Great Britain (n = 378) and Canada (n = 216) are also prevalent origins of institutions. Fifty-seven countries are represented below a 5 % level. International cooperation seems to be widely spread depending on the repository context, whereby the number of different countries per RDR varies widely between 1 and 22 (cf. Figure 2). Important to consider is that these findings depict only RDR indexed in re3data requiring an English graphical user interface (GUI) according to the re3data registration policy until the end of 2015. Figure 2: Countries of the responsible institutions operating research data reposito- ries indexed in re3data (n = 1,381, multiple values possible) Figure 2: Countries of the responsible institutions operating research data reposito- ries indexed in re3data (n = 1,381, multiple values possible) This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 10 10 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 3.2 Repository types To describe the registered RDR in more detail, re3data collects some general in- formation about the repository. This includes a description of the repository type. The re3data metadata schema distinguishes between “disciplinary”, “institutional” and “other” repository types, defined in the metadata schema on pages 18 and 19 (Rücknagel et al. 2015). Figure 3: Types of research data repositories indexed in re3data (n = 1,379, 2 RDR with missing values, multiple values possible) Figure 3: Types of research data repositories indexed in re3data (n = 1,379, 2 RDR with missing values, multiple values possible) As shown in the Venn diagram above the majority (86.2 %) of the registered RDR are “disciplinary”. Also, 29.5 % of the repositories are “institutional” and 12.2 % fulfill the “other” criteria. This “other” category includes, among others, portals or commercial data storage services. Further, 0.8 % of the repositories fulfill all categories, like the “Australian Ocean Data Network Portal” (re3data.org: Australian Ocean Data Network Portal 2014) (cf. Figure 3). This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 11 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 With 19.5 % there is a high number of RDR that are described as being institutional as well as disciplinary. Of all RDR, 72.9% can be clearly categorized into one type: 109 institutional RDR, 832 disciplinary RDR, and 62 other RDR. With 19.5 % there is a high number of RDR that are described as being institutional as well as disciplinary. Of all RDR, 72.9% can be clearly categorized into one type: 109 institutional RDR, 832 disciplinary RDR, and 62 other RDR. This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 12 3.3.1 Subjects re3data implemented the classification of subject areas of the German Research Foun- dation (Rücknagel et al. 2015). The following Venn diagram (cf. Figure 4) shows the distribution of registered RDR according to the four main categories “Humanities and Social Sciences” (SSH), “Life Sciences”, “Natural Sciences” and “Engineering Sciences”. A RDR can offer research data and services for more than one subject. Figure 4: The four main subject categories of research data repositories indexed in re3data (n = 1,381, multiple values possible) Figure 4: The four main subject categories of research data repositories indexed in re3data (n = 1,381, multiple values possible) 12 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 Only 5.8 % of 1,381 RDR cover all four subject areas, Humanities and Social Sci- ences, Natural Sciences, Life Sciences and Engineering Sciences, and can be described as being multidisciplinary RDR. Most RDR have a disciplinary focus on one subject area (mean = 1.4, median = 1.0, standard deviation = 0.8). RDR covering Natural Sciences (51.5 %) and Life Sciences (49.8 %) are the most frequent, while RDR cov- ering Humanities and Social Sciences are represented with 27.1 % and RDR covering Engineering Sciences with 12.0 % (cf. Figure 4). This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 3.3.2 Content types re3data differentiates between 15 content types according to the PARSE.insight sur- vey (PARSE.insight team 2016) that are represented in the following table (cf. Ta- ble 1). All 15 content types are covered by “Research Data Australia” (re3data.org: Research Data Australia 2015), “Europeana” (re3data.org: Europeana 2015) covers 14 categories. 13 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 re3data content type Number (n = 6,340) Number (n = 1,381) Count (n) Percentage (%) Scientific and statistical data for- mats 881 63.8 Standard office documents 786 56.9 Plain text 690 50.0 Images 686 49.7 Raw data 586 42.4 Structured graphics 541 39.2 Structured text 490 35.5 Other 446 2.3 Archived data 339 25.5 Software applications 258 18.7 Audiovisual data 253 18.3 Databases 204 14.8 Networkbased data 104 7.5 Source code 49 3.5 Configuration data 27 2.0 Table 1: Content types of research data repositories indexed in re3data (n = 1,381, multiple values possible) This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 Table 1: Content types of research data repositories indexed in re3data (n = 1,381, multiple values possible) This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 3.3.3 Content types according to the main subject categories In the following we differentiate RDR content types according to the four main subject categories. The mean value of the content type variety shown according to the four main subject categories is 4.59 different types of content per RDR (median = 4.00, standard deviation = 2.13). Research data in Humanities and Social Sciences’ RDR contain more “Standard office documents”, “Plain text” or “Images” than expected. The number of occurrences is only significantly higher (n = 218) than the expected value (n = 186.9) for “Plain text”. The test for independence showed a significant 14 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 dependency between the variables “contentType” and “subject” with a low effect size (Cramer’s V = 0.101). The existence of “Images” and the subject Humanities and Social Sciences correlate with a low effect size (Cramer’s V = 0.156). The observed value (n = 185.8) is much higher than was expected (n = 138) on a 99.9 % level. “Standard office documents” are clearly overrepresented in this discipline (observed n = 276; expected n = 212.9), but also with a low effect size (Cramer’s V = 0.208). For all cases mentioned in this paragraph, the test indicated a dependency on 99.9% level. Life Sciences’ repositories cover more “other” types of content than other disciplines (observed n = 252 instead of expected n = 221.9). The dependency is significant around 99 %, but with a very low effect size (Cramer’s V = 0.093). 3.3.3 Content types according to the main subject categories We did not find any statistically significant dependency on other content types, though we observed more “Structured text”, “Software”, “Scientific and statistical data formats” and “Structured graphics” than was statistically to be expected. “Images” as a content type largely depends on the discipline. There are significantly more “Images” in Natural Sciences’ RDR than expected (observed n = 429; expected n = 353.2). The test is significant on 99.9 % level, though the effect size is low (Cramer’s V = 0.220). The dependency between Natural Sciences and “Raw data” (observed n = 357, expected n = 301.7) reflects the same probability, even though the effect size is just as low (Cramer’s V = 0.162). The value of 479 “Scientific and statistical data formats” was higher than expected (n = 453.6). The dependency is on a 96 % level with a minor effect (Cramer’s V = 0.077). In Engineering Sciences more research data repositories cover “Audiovisual data” (observed n = 67) than expected (n = 30.2; Cramer’s V = 0.212). There is also significantly more “other” content than expected (observed n = 74 instead of expected n = 53.39 on 99.9 % level and Cramer’s V = 0.099). This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 3.4 Policies Of the 1,381 RDR, 85.4 % provide at least one policy document of some kind. A policy document is a document that expresses a guiding framework for a RDR regulating different aspects of the implementation or operation of the repository (Martín and 15 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 Ballard 2010). Thus, different thematic issues and priorities can be depicted. The property “policyType” did not exist in version 2.2 of the re3data metadata schema, on which this metadata analysis is based. It was integrated in the metadata schema version 3.0. Before this, only the names of the respective policies were collected. Since numerous names are used for policy documents, we cannot provide a quanti- tative analysis revealing the most popular policy types. Based on the observations of the policies’ content, and with regard to the team’s knowledge of the repository landscape, we gathered the following policy types “Access policy”, “Collection pol- icy”, “Data policy”, “Metadata policy”, “Preservation policy”, “Submission policy”, “Terms of use”, “Usage policy” and “Quality policy”. In order to provide information concerning conditions of use we strongly encourage RDR to have at least one policy in place. It should cover general issues including the aspects in table 2 (cf. Table 2, following Australian National Data Service 2010). The policy types mentioned above are defined in the re3data metadata schema 3.0 on page 21 (Rücknagel et al. 2015). Policy aspect Details Name of the policy E.g. “Policy of the RDR” Purpose of the document E.g. commitment to long term & key principles preservation Application of the policy E.g. This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ Table 2: Policy aspects of research data repositories This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 16 3.4 Policies user of the website or person uploading research data; responsibilities Licensing and copyright matters E.g. obligation or recommendation of standard licences Access & usage regulations Embargo period, restrictions, privacy issues Retention period Removal of datasets Formal Dates (commencement, review, versioning), contact information, links to other relevant documents, glossary Table 2: Policy aspects of research data repositories 16 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 3.5 Access and Licenses re3data distinguishes between “open”, “restricted” and “closed” as access categories with respect to the following levels: to the RDR (“database”), to the research data itself (“data”) and to the data submission services (“upload”) (Rücknagel et al. 2015). Each level can be “open”, “restricted”, or “closed”. If the repository, research data and submission services can be accessed without financial and technical barriers, it means that the value for the respective property is “open”. Access barriers to the RDR and its services that a user can overcome are “restricted”, e.g. a user account needs to be created or an agreement has to be signed by the user to obtain access to the RDR, the data sets or to submit research data. “Closed” access means that an external user cannot overcome access barriers, e.g. if a service is solely for a respective community that he/she cannot become a member of. The access type “embargoed” is also used for research data. “Embargoed” research data cannot be accessed by third persons until the data sets have been released for “open” or “restricted” access (Rücknagel et al. 2015). Each RDR can have several access values for each level since, e.g., parts of data collections can be accessed openly and other parts may be restricted. 17 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 3.5.1 Access to the RDR and database licenses The vast majority of all RDR (n = 1,381) are openly accessible (95.5 %). A few RDR have access restrictions to the database (4.3 %) while only 2 RDR (0.1 %) are closed (cf. Figure 5). Figure 5: Access to the database of research data repositories indexed in re3data (n = 1,381) Figure 5: Access to the database of research data repositories indexed in re3data (n = 1,381) Three hundred forty-two database licenses are mentioned that belong to 336 RDR. Six RDR make use of more than one license. Despite the fact, that we are thus unable to make any statements on the use of license information for most RDR, we do know that copyright information is the most common, followed by “other” license information and the use of one license type from the Creative Commons license family. All other license information does not seem to be widespread at all. The distribution of all license information is shown in the table below (cf. Table 3). 18 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 Database license Number (n = 342) Number (n = 1,381) Count (n) Percentage (%) Copyrights 139 10.1 Other (e.g. a policy to clarify legal as- pects) 114 8.3 CC (Creative Commons license family) 51 3.7 Apache License 2.0 19 1.4 ODC (Open Data Commons) 8 0.6 BSD (Berkeley Software Distribution) 6 0.4 Public Domain 4 0.3 CC0 (Creative Commons Public Do- main Dedication) 1 0.1 Table 3: Database licenses found in research data repositories indexed in re3data (n = 342, multiple values possible) Table 3: Database licenses found in research data repositories indexed in re3data (n = 342, multiple values possible) This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 3.5.2 Access to the research data As shown in the Venn diagram below, most RDR offer “open” access to the research data at least partly (86.2 %). However, 45.8 % of the RDR provide “restricted” or at least partly “restricted” access, 7.5 % “closed” access and 0.8 % “embargoed” access. RDR provide research data in all four access categories (cf. Figure 6). Overlapping categories in the Venn diagram such as RDR that are “open” and “restricted” at the same time mean that a RDR can be accessed openly in parts (e.g. “Australian Data Archive”) (re3data.org: Australian Data Archive 2015). 19 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 Figure 6: Access to the research data of repositories indexed in re3data (n = 1,381, multiple values possible) Figure 6: Access to the research data of repositories indexed in re3data (n = 1,381, multiple values possible) A correlation of access to the research data and the aforementioned four subject categories shows a low effect size on “closed” access (Cramer’s V = 0.156) for all RDR covering Humanities and Social Sciences. More RDR (n = 53) provide research data in “closed” access than was expected (n = 27.9). This also becomes obvious when comparing the relative numbers of “Humanities and Social Sciences” with all other disciplines. One effect of this is that there are significantly fewer “open” data access repositories (observed n = 302) than statistically expected (n = 322.5). This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 3.5.2 Access to the research data The effect size is 0.097 (Cramer’s V). In addition, more RDR offer “restricted” data access (observed n = 242 instead of expected n = 171.49). The effect size is a little stronger here (Cramer’s V = 0.231). This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 20 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 3.5.3 Access restriction to the research data For 839 RDR, re3data provides metadata information concerning access restriction to the research data. “Restricted” data access includes restrictions such as “registration” (33.9 % of 1381), “other” (18.3 %, e.g. request data sets via email) , “institutional membership” (1.2 %), and “feeRequired” (7.4 %) (cf. Figure 7). Figure 7: Access restrictions to the research data of repositories indexed in re3data (n = 839) Figure 7: Access restrictions to the research data of repositories indexed in re3data (n = 839) For RDR covering Life Sciences, a significance was proven for “embargoed” data access with an effect size of Cramer’s V = 0.105. More repositories offering “embar- goed” data access (n = 65) than expected (n = 46.8) may indicate that scientists in these fields require more time to analyze their data before allowing third parties to access the research data. Another reason may be the need to protect research data for patent submissions. Slightly more “open” data access repositories (observed n = 616, expected n = 592.5) are indicated in Life Sciences’ fields that already have an established open access and data sharing culture to facilitate scientific progress. The effect is low though (Cramer’s V = 0.099). There are fewer “restricted” RDR in Life Sciences (n = 284, expected n = 314.9) on nearly the same effect size (Cramer’s V = 0.090). Engineering Sciences has an effect (Cramer’s V = 0.113) on “embargoed” data access RDR (n = 24, expected n = 11.2). No dependencies above the 95 % significance level were identified for Natural Sciences. This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 21 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 22 Table 4: Research data licenses found in research data repositories indexed in re3data (n = 1,895, multiple values possible) This work is licensed under a Creative Commons Attribution 4.0 International License: This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ 3.5.4 Research data licenses There are a total of 2,122 metadata entries on research data licenses for all unique 1,381 RDR. As some RDR have more than one “other” license, the cleansed data only counts 1895 licenses. The table below shows the distribution of different kinds of legal use and license information RDR provide to access and use research data. The largest group of data licenses within re3data relate to individual license information prepared by the RDR on regulations for use. These licenses are recorded within the “Other” category (57.2 %). Copyright information is provided by 38.6 % of the 1,381 RDR. The Creative Commons license family is the most frequently used (21.8 %). Research data is declared to be “public domain” less often (14.2 %), while “ODC” (2.0 %), CC0 (1.9 %), OGL (0.9 %) were almost negligible (cf. Table 4). Research data license Number (n = 1895) Number (n = 1381) Count (n) Percentage (%) Other 790 57.2 Copyrights 553 38.6 CC (Creative Commons license fam- ily) 301 21.8 Public Domain 196 14.2 ODC (Open Data Commons) 27 2.0 CC0 (Creative Commons Public Do- main Dedication) 26 1.9 OGL (Open Government License) 12 0.9 BSD (Berkeley Software Distribution) 6 0.4 Apache License 2.0 2 0.1 OGL C (Open Government License) 1 0.1 RL (Restrictive License) 1 0.1 Table 4: Research data licenses found in research data repositories indexed in re3data (n = 1,895, multiple values possible) 22 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 The effect size on using a “Creative Commons” license in RDR covering Humanities and Social Sciences is high (Cramer’s V = 0.132). We observed 115 RDR using “Creative Commons” licenses (expected n = 81.5). Sixteen RDR (instead of the 7 expected) use “CC0” for their Humanities and Social Sciences research data, though there is only a low correlation (Cramer’s V = 0.107). 3.5.4 Research data licenses There is also a low effect size on the “BSD” data license (Cramer’s V = 0.084) for the Humanities and Social Sciences because more RDR (n = 5) use these than statistically expected (n = 1.6). In Life Sciences, there is again a tendency towards an established culture of data sharing. This is depicted by a significantly (Cramer’s V = 0.152) higher usage of “Creative Commons” licenses (observed n = 193, expected n = 149.7) and slightly more research data repositories make use of “Public Domain” (observed n = 121) than expected (n = 97.5), with a low effect size (Cramer’s V = 0.098). Significantly more “Creative Commons” licenses (observed n = 67, expected n = 36, Cramer’s V = 0.168), “Apache” licenses (observed n = 2, expected n = 0.2, Cramer’s V = 0.103) and “BSD” licenses (observed n = 5, expected n = 0.7, Cramer’s V = 0.145) were found for research data repositories in Engineering Sciences. This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 3.5.5 Access and restriction to research data upload The majority of RDR restricts data upload (57 %). Slightly fewer RDR are “closed” in terms of data upload (39.5 %). This means that no external data submissions are accepted for inclusion in the data collection of the RDR. “Closed” data uploads are found for RDR that collect project-specific research data or institutional output in particular. Only 3.9 % offer an open data upload, meaning that there is no reg- istration or contact information required to submit research data to the RDR (cf. Figure 8). There are rare overlapping values indicating, e.g., that access to upload is partly open or that there are several options with varying restrictions for submitting data to a repository (e.g. “NCBI Genome repository”) (re3data.org: NCBI Genome repository 2015). 23 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 Figure 8: Research data upload access to the repositories indexed in re3data (n = 1,379, 2 RDR with missing values, multiple values possible) Th d t l d i lik l t h b “ i t ti ” (37 6 %) “ th ” h i This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 25 3.5.5 Access and restriction to research data upload The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 Figure 8: Research data upload access to the repositories indexed in re3data (n = 1,379, 2 RDR with missing values, multiple values possible) Figure 8: Research data upload access to the repositories indexed in re3data (n = 1 379 2 RDR with missing values multiple values possible) Figure 8: Research data upload access to the repositories indexed in re3data (n = 1,379, 2 RDR with missing values, multiple values possible) The data upload is likely to happen by “registration” (37.6 %) or “other” mechanisms, such as sending data media by postal mailing (16.0 %). Only 4.1 % of the 1,381 RDR provide the upload services solely to “institutional members”, and only 0.7 % require “submission fees” (cf. Table 5). The number of RDR in re3data missing information on research data upload restriction in general or on the specific type of upload restriction is 576. Specifying the type of upload restriction was not required in the indexing process. The data upload is likely to happen by “registration” (37.6 %) or “other” mechanisms, such as sending data media by postal mailing (16.0 %). Only 4.1 % of the 1,381 RDR provide the upload services solely to “institutional members”, and only 0.7 % require “submission fees” (cf. Table 5). The number of RDR in re3data missing information on research data upload restriction in general or on the specific type of upload restriction is 576. Specifying the type of upload restriction was not required in the indexing process. 24 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). This work is licensed under a Creative Commons Attribution 4.0 International License: t // ti /li /b /4 0/ 25 Table 6: Persistent Identifier systems used by research data repositories indexed in re3data (n = 1,421, multiple values possible) tps://creativecommons.org/licenses/by/4.0/ 25 3.5.5 Access and restriction to research data upload The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 Research data upload restriction Number (n = 805) Number (n = 1,381) Count (n) Percentage (%) Registration 519 37.6 Other 221 16.0 Institutional Membership 56 4.1 Fee required 9 0.7 Table 5: Research data upload restrictions found in research data repositories indexed in re3data (n = 805, multiple values possible) 3.6 Services 3.6.1 Persistent Identifier systems PID system Number (n = 1,421) Number (n = 1,381) Count (n) Percentage (%) None 924 66.9 DOI (Digital Object Identifier) 275 19.9 Handle 102 7.4 Other 77 5.6 PURL (Persistent Uniform Resource Locator) 16 1.6 URN (Uniform Resource Name) 16 1.6 ARK (Archival Resource Key) 11 0.8 Table 6: Persistent Identifier systems used by research data repositories indexed in re3data (n = 1,421, multiple values possible) This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 Research data upload restriction Number (n = 805) Number (n = 1,381) Count (n) Percentage (%) Registration 519 37.6 Other 221 16.0 Institutional Membership 56 4.1 Fee required 9 0.7 Table 5: Research data upload restrictions found in research data repositories indexed in re3data (n = 805, multiple values possible) Table 5: Research data upload restrictions found in research data repositories indexed in re3data (n = 805, multiple values possible) PID system Number (n = 1,421) Number (n = 1,381) Count (n) Percentage (%) None 924 66.9 DOI (Digital Object Identifier) 275 19.9 Handle 102 7.4 Other 77 5.6 PURL (Persistent Uniform Resource Locator) 16 1.6 URN (Uniform Resource Name) 16 1.6 ARK (Archival Resource Key) 11 0.8 Table 6: Persistent Identifier systems used by research data repositories indexed in re3data (n = 1,421, multiple values possible) Number (n = 1,421) Number (n = 1,381) Table 6: Persistent Identifier systems used by research data repositories indexed in re3data (n = 1,421, multiple values possible) 25 25 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 3.5.5 Access and restriction to research data upload The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 Figure 9: Persistent Identifier systems used by research data repositories indexed in re3data (n = 1421, multiple values possible) Figure 9: Persistent Identifier systems used by research data repositories indexed in re3data (n = 1421, multiple values possible) Figure 9: Persistent Identifier systems used by research data repositories indexed in re3data (n = 1421, multiple values possible) Only 36 % of 1381 RDR use a Persistent Identifier (PID) system to ensure the per- sistent citability of the provided research data. For 46 RDR we found more than one PID system applied. The most common system is the “Digital Object Identifier (DOI)” system, with 19.9 % RDR in re3data using it. DOI is used widely in all four subject categories (as shown in the correlation matrix) (Sandt et al. 2017). Less than half as many RDR offer the “Handle” system (7.4 %). Least popular are the “URN” (1.6 %) and “ARK” systems (0.8 %) (cf. Table 6, Figure 9). This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 26 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 3.6.2 Repository software Repository software Number (n = 1,166) Number (n = 1,381) Count (n) Percentage (%) Unknown 765 55.4 Other 271 19.6 DSpace 36 2.6 DataVerse 30 2.2 CKAN (Comprehensive Knowledge Archive Network) 16 1.2 Fedora 14 1.0 EPrints 11 0.8 MySQL 10 0.7 Nesstar 10 0.7 dLibra 2 0.1 eSciDoc 1 0.1 Table 7: Repository software used by research data repositories indexed in re3data (n = 1,166, multiple values possible) 3.6.2 Repository software Table 7: Repository software used by research data repositories indexed in re3data (n = 1,166, multiple values possible) We can make statements on the software used to run the RDR for 396 repositories of 1,381. The small number of registered software types within re3data is most certainly due to our manual indexing process. This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 3.5.5 Access and restriction to research data upload In most cases the repository software is not explicitly stated on the website and as a consequence not indexed. Furthermore, it is important to know that the “unknown” value was not part of the Metadata Scheme in the first version 1.0. Thus re3data includes RDR with unknown software that have no metadata information as well as RDR with the value “unknown” from the later indexing process. What we can say is that from the captured repository software, DSpace and DataVerse are the most prevalent followed by CKAN, Fedora and Eprints, MySQL and Nesstar. Combinations of different software solutions were rarely observed (0.4 %). The majority of RDR use another type of software (19.6 %) that is not explicitly listed in the controlled vocabulary of the metadata schema version 2.2 (cf. Table 7). This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 27 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 3.6.3 Application Programming Interfaces (API) API Number (n = 830) Number (n = 1,381) Count (n) Percentage (%) FTP (File Transfer Protocol) 284 20.6 Other 178 12.9 REST (Representational State Trans- fer) 164 11.9 OAI-PMH (Open Archives Initiative Protocol for Metadata Harvesting) 85 6.2 SOAP (Simple Object Access Proto- col) 52 3.8 SWORD (Simple Web-service Offering Repository Deposit) 21 1.5 NetCDF (Network Common Data For- mat) 20 1.4 OPeNDAP (Open Source Project for a Network Data Access Protocol) 16 1.2 SPARQL (SPARQL Protocol and RDF query language) 10 0.7 Table 8: APIs used by research data repositories indexed in re3data (n = 830, multiple values possible) 3.6.3 Application Programming Interfaces (API) Table 8: APIs used by research data repositories indexed in re3data (n = 830, multiple values possible) We found 608 (44 %) RDR that provide information concerning APIs for data and metadata exchange. The “Alaska Ocean Observing System” (re3data.org: Alaska Ocean Observing System 2015) repository offers a total of seven “APIs” as an extreme. The average number of APIs per RDR is 1.49 (median = 1.00, standard deviation = 0.84). This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 3.5.5 Access and restriction to research data upload Most RDR (20.6 %) offer data and metadata exchange via “FTP”, 12.9 % via “other” APIs, followed by “REST”, “OAI-PMH”, and “SOAP” (cf. Table 8). Similar to the “software type” property, our editorial team experienced difficulties collecting information concerning API systems in use by RDR. Therefore we assume that the depicted information is biased by the fact that some interfaces are easier to identify 28 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 by our editors, such as the FTP API. by our editors, such as the FTP API. Apart from this fact, we found subject dependencies for the distribution of the API types. We observed far fewer “FTP” APIs for RDR in Humanities and Social Sciences (n = 22) than expected (n = 76.9) on a medium low correlation level (Cramer’s V = 0.221), but a lot more “OAI-PMH” APIs (observed n = 62 instead of expected n = 23.0). The correlation strength is slightly stronger here (Cramer’s V = 0.264). “SOAP” is rarely used in this subject area (observed n = 3 instead of expected n = 14.1; Cramer’s V = 0.095). A “REST” API seems to be significantly popular among Life Sciences (observed n = 104 instead of expected n = 81.6; Cramer’s V = 0.100). We observed the same popularity for a “NetCDF” API (observed n = 20 instead of expected n = 10.3; Cramer’s V = 0.118) and “OpenDAP” (observed n = 16 instead of expected n = 8.2; Cramer’s V = 0.105) for Natural Sciences RDR. A “REST” API seems to be significantly popular among Life Sciences (observed n = 104 instead of expected n = 81.6; Cramer’s V = 0.100). We observed the same popularity for a “NetCDF” API (observed n = 20 instead of expected n = 10.3; Cramer’s V = 0.118) and “OpenDAP” (observed n = 16 instead of expected n = 8.2; Cramer’s V = 0.105) for Natural Sciences RDR. Just as we had fewer “FTP” APIs in Humanities and Social Sciences, the same phenomena could be observed in Engineering Sciences RDR (observed n = 10 instead of expected n = 33.9; Cramer’s V = 0.132). Both subject classes also show the same behavior for “OAI-PMH”. Engineering Sciences offer 27 “OAI-PMH” APIs instead of the statistically expected 20.2 if both variables were independent (Cramer’s V = 0.156). 29 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 3 by our editors, such as the FTP API. The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 3.6.4 Certificates Certificates Number (n = 261) Number (n = 1,381) Count (n) Percentage (%) Other 109 7.9 WDS (World Data System) 72 5.2 DSA (Data Seal of Approval) 52 3.8 CLARIN certificate B (Common Lan- guage Resources and Technology In- frastructure) 14 1.0 RatSWD (German Data Forum) 6 0.4 TRAC (Trustworthy Repositories Au- dit & Certification) 3 0.2 DINI Certificate (Deutsche Initiative für Netzwerkinformation e.V. — DINI- Zertifikat) 2 0.2 DIN 31644 (Deutsches Institut für Normung German Institute for Stan- dards) 1 0.1 ISO 16363 (International Standard Or- ganisation) 1 0.1 Trusted Digital Repository 1 0.1 Table 9: Certificates used by research data repositories indexed in re3data (n = 261, multiple values possible) 3.6.4 Certificates Table 9: Certificates used by research data repositories indexed in re3data (n = 261, multiple values possible) able 9: Certificates used by research data repositories indexed in re3data (n = 261, multiple values possible) Two hundred sixty-one of 1,381 RDR (18.9 %) have been awarded a certificate ac- cording to the re3data metadata. The metadata schema offers 9 options for known certificates or standards and the “other” category. Only one RDR has a maximum of three certificates (re3data.org: UK.Data Archive 2015). Most RDR only have one certificate, the mean value is 1.14 (median = 1.00, standard deviation = 0.36). The World Data System (“WDS”) certificate is awarded most often (5.2 %, n = 72), followed by 52 RDR (3.8 %) that are compliant with the “Data Seal of Approval”. 30 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 Only 109 RDR (7.9 %) offer information on any “other” kinds of certification and standards compliance (cf. Table 9). Only 109 RDR (7.9 %) offer information on any “other” kinds of certification and standards compliance (cf. Table 9). With respect to the RDR’s subjects, we can prove a low effect for Humanities and Social Sciences (Cramer’s V = 0.166) with 14 observed “CLARIN certificate B” (ex- pected n = 3.8). Since the “CLARIN certificate B” clearly focuses on Humanities and Social Sciences’ disciplines, we consequently have no further RDR from another subject that is compliant. by our editors, such as the FTP API. The effect on the Data Seal of Approval (“DSA”) for Humanities and Social Sciences is average (0.307). Here we identified 50 certificates. In contrast to Humanities and Social Sciences, Life Sciences have a certificate signif- icantly less often (effect size is 0.167). 4.1 General observations As expected, we found a heterogeneous landscape of RDR that is mostly influenced by the repositories’ disciplinary background for which they offer services. Most repos- itories are disciplinary services (cf. Figure 3). We identified statistically significant dependencies in connection with the four main subject categories Humanities and So- cial Sciences, Natural Sciences, Life Sciences, and Engineering Sciences for a number of properties. This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 4.2 Access Openness seems to be a common factor because the vast majority of the RDR grant Open Access to their databases and at least partly to dataset collections (cf. Figures 6 and 7). Research data repositories in the Life Sciences group appear to be most willing to share data sets openly. The re3data analysis provides a first overview of access with respect to information at the repository level. Before any assumptions can be made on the distribution of open research data datasets and collections itself, it’s metadata needs to be investigated on the data level. The most frequent data license types This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 31 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 according to re3data observed on the repository level are “other” and “copyright” (cf. Table 4). This means that self-written policy documents or copyright notices are mainly used to regulate access and usage conditions for the database and the content it provides. The dissemination of standard licenses, especially for research data and metadata, should be accorded even greater support. Although the Creative Commons License family is used by 21.8 % of the repositories (cf. Table 4), this number could be improved and needs to be investigated further e.g. in terms of license types in use. Creative Commons is an approved standard to clarify how content can be used, commonly in a digital form. Research data repository operators should consider adding appropriate standard licenses to their range of licenses a data submitter can choose from. More than half of the RDR offer restricted access to upload research data through registration or other means, e.g. data upload via API or postal mailing (cf. Figure 8 and Table 5). 4.2 Access The authentication of a data submitter seems to be a major concern for research data repository to restrict the upload processes. Hardly any repository listed in re3data is requiring a submission fee. There are two possible reasons for this: either very few RDR demand such a fee, or the information was not found during the indexing process. To clarify access and usage regulations and further determine the terms on which users can submit data sets to a repository’s collection, it is of utmost importance to provide this information on the research data repository’s website. This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 4.3 Services Well-established standards and services in the field of text repositories are far from common within the RDR environment. Aspects such as the persistent citability of research data sets and collections are most important to realize an adequate reuse of research data sets. Consequently, the provision of PID systems needs to be expanded from this aspect, as do other issues. With respect to those repositories that use a PID system, the DOI and Handle system are the most widespread solutions to enable a persistent reference to research data. DOI and Handle together account for 27.3 % (cf. Table 6 and Figure 9). Over 50 % of all captured PID values are DOIs (55.3 %). This number indicates that DOI is a well-established standard. The foundation of This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 32 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 DataCite can be named as one possible reason for this development, as the well-known non-profit organization has been providing PIDs for data sets since 2009. DataCite can be named as one possible reason for this development, as the well-known non-profit organization has been providing PIDs for data sets since 2009. 4.4 Repository software and API As for the software solutions in use to run a research data repository, we realize that the software types used by the majority of repositories are hard to record since the information is not readily provided by all RDR. What we can indicate is that a large number of “other” software types were registered during the indexing pro- cess (cf. Table 7). This shows that several repositories have developed their own software solution. Presumably, some repositories have special needs that require a self-developed software tool or the existing (standard) software packages do not match the institutional research infrastructure development strategy. Far more RDR than were recorded need to have (standardized) APIs to provide metadata for service providers and enable metadata search to improve findability of research data sets and collections. Enabling metadata aggregation is an important feature of appropriate repository software to improve discovery of research data. This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 33 4.5 Certificates Around 19 % of all RDR have a certificate or comply with a standard. This number seems rather small despite the fact that re3data does not only list repositories with a long-term commitment to preserve data sets. Complex standards such as DIN and ISO in particular, which require a high implementation effort, can only be demon- strated for a few exceptions. The Data Seal of Approval is an approach that seems to be well on it’s way to becoming a common “soft” standard for data curation (cf. Table 9). WDS is also widespread and ensures the sustainability of an information system, even though the certification process requires the membership of responsible institutions. Apart from these fundamental, quality-proven standards and audits that focus on the repository level, quality standards for the research data and metadata itself are crucial for the research community to guarantee that RDR are perceived as reliable information systems suitable for the indefinite storage of valuable research data sets and materials. This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 33 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 34 6 Recommendations The discussion on research data management has made big steps forward in the last years. The “FAIR Guiding Principles for scientific data management and steward- ship” (findable, accessible, interoperable, reusable research data) from 2016 describe the current state of the art in a short and precise manner (Wilkinson et al. 2016). Quite a large number of the re3data indexed repositories began to operate long before that. We therefore recommend RDR managers to take a closer look at the following issues: 5 Conclusions The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 community by managing research data in a sustainable way. Less information on sustainability and long-term preservation is provided with respect to the metadata schema of re3data. Nevertheless, this is of utmost importance for a sustainable landscape of Open Science services and infrastructure. The metadata entries re3data provides do not claim to be complete or accurate. Our editorial board indexes and updates the RDR manually. Information might change over time or could not be found during the indexing process. The active cooperation of the community is needed to keep the re3data metadata entries as up- to-date as possible. These findings should also help the RDR community to improve their services or build new services if research institutions deem this necessary. For this reason, we have prepared the following recommendations based on our indexing experience as well as the analysis of the metadata collected. This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 35 5 Conclusions RDR differ in terms of the service levels they offer, languages they support or stan- dards which they comply with. These statements are commonly acknowledged by say- ing that the RDR landscape is heterogeneous. By conducting an analysis of re3data metadata entries, we were able to further present differences between the 1,381 listed RDR on a statistical basis. Although this analysis is limited to the re3data metadata entries indexed by the end of 2015, we identified tendencies concerning the global landscape of RDR. One outstanding fact is that compliance with important standards of the Information Infrastructure as well as the Library and Information Science (LIS) community are underused. This data analysis supports this conclusion especially regarding the pro- vision of Persistent Identifiers for research data sets or the use of common APIs (cf. Tables 6 and 8). The API entries recorded within our sample do not allow extensive remarks on the possibilities of cross-institutional metadata exchange on the data level. The compatibility with community-approved standards nevertheless is essential for the implementation of any service that serves one or several research communities. This factor should be considered when planning, operating and improving a RDR. The use, trust and reputation of repository services are of vital importance if they are to be widely recognized as a reliable information system. We also demonstrated that there are several RDR that are already recognized services since they are rec- ommended in funder and publisher policies (Pampel, Vierkant, et al. 2013). Based on the findings of this initial analysis further research on the landscape of RDR can differentiate and sharpen our exemplary picture of RDR. Within the re3data metadata schema, and consequently in the course of the analysis, we used a broad subject classification on the first level of main subject categories. A more in-depth analysis of the sub-categories may reveal significant differences between subjects that are summarized, for example, under the category “Humanities and Social Sciences”. Similar applies for the content type category. More granular information on the RDR’s content, such as data formats, might be a starting point for follow up research. An active community of stakeholders (researchers, research organization, funding possibilities) is needed to either build or evaluate RDR that support the research 34 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). 6.1 Visibility of a research data repository 1. Register RDR in re3data or similar registries to improve the repository’s visi- bility. Services offered by the research data repository to the community are presented in an adequate and transparent way. 2. Review the metadata entry for the listed research data repository within re3data 2. Review the metadata entry for the listed research data repository within re3data 35 35 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This work is licensed under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/ 6.2 Functionalities of a research data repository 36 36 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 This is a self-archiving version of an article published in D-Lib Magazine (D-Lib volume 23, number 3/4). The final publisher’s version is available online at: https://doi.org/10.1045/march2017-kindling Please use this identifier to cite this postprint: https://doi.org/10.18452/19814 Acknowledgements We would like to thank all colleagues for their helpful input and comments on this article. The project re3data and writing of this paper was enabled by a grant from the German Research Foundation (DFG). We would like to thank the DFG for supporting our work. We would also like to thank the global repository community for its help and support in the establishment of re3data. re3data is operated as a service of DataCite. 6.2 Functionalities of a research data repository 3. Support PID systems to provide each dataset that is stored within the research data repository with a persistent identifier, thus allowing the persistent citation of the relevant dataset. 3. Support PID systems to provide each dataset that is stored within the research data repository with a persistent identifier, thus allowing the persistent citation of the relevant dataset. 3. Support PID systems to provide each dataset that is stored within the research data repository with a persistent identifier, thus allowing the persistent citation of the relevant dataset. 4. Use a data license to clarify access and usage conditions for the data sets pro- vided. Existing machine-readable standard licenses can be supported to ensure that the license is legally effective and globally understandable. 5. Make metadata and related research data sets available to other services and research organizations through an API. This networking approach improves interoperability of the services, the visibility and findability of data sets within the research community and facilitates new services. 6. Ensure compliance with certificates and standards to ensure the reliability and trustworthiness of the RDR. It should be clearly communicated which certifi- cates and standards are met, so that users can evaluate the service quality. 7. The institutional responsibility has to be clarified and communicated to the user, e.g. included in a data policy or a mission statement. 8. Create policies to describe the services offered, the terms under which the repos- itory may be used and to clarify the principles that the RDR deems important. 8. Create policies to describe the services offered, the terms under which the repos- itory may be used and to clarify the principles that the RDR deems important. 9. When choosing or developing a repository software, ensure that the software supports technical standards that for example are described in the certificates mentioned above. 9. When choosing or developing a repository software, ensure that the software supports technical standards that for example are described in the certificates mentioned above. Taking into account the number of existing RDR as well as their different focuses in terms of subjects, content types and services, we strongly recommend to consider whether one or more of the listed RDR might be suitable for certain requirements. Before a single institution begins to build another service, probably with a similar orientation, re3data could be consulted to assess the appropriateness of available RDR. 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https://openalex.org/W2514636650	https://europepmc.org/articles/pmc4982142?pdf=render	English	null	Contrasting origin of B chromosomes in two cervids (Siberian roe deer and grey brocket deer) unravelled by chromosome-specific DNA sequencing	BMC genomics	2,016	cc-by	12,230	* Correspondence: alex.makunin@gmail.com 1Institute of Molecular and Cell Biology, Novosibirsk, Russia 2Theodosius Dobzhansky Center for Genome Bioinformatics, Saint-Petersburg State University, Saint-Petersburg, Russia Full list of author information is available at the end of the article Makunin et al. BMC Genomics (2016) 17:618 DOI 10.1186/s12864-016-2933-6 Makunin et al. BMC Genomics (2016) 17:618 DOI 10.1186/s12864-016-2933-6 Contrasting origin of B chromosomes in two cervids (Siberian roe deer and grey brocket deer) unravelled by chromosome- specific DNA sequencing Alexey I. Makunin1,2, Ilya G. Kichigin1, Denis M. Larkin3, Patricia C. M. O’Brien4, Malcolm A. Ferguson-Smith4, Fengtang Yang5, Anastasiya A. Proskuryakova1, Nadezhda V. Vorobieva1, Ekaterina N. Chernyaeva2, Stephen J. O’Brien2, Alexander S. Graphodatsky1,6 and Vladimir A. Trifonov1,6 © 2016 The Author(s). 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: alex.makunin@gmail.com 1Institute of Molecular and Cell Biology, Novosibirsk, Russia 2Theodosius Dobzhansky Center for Genome Bioinformatics, Saint-Petersburg State University, Saint-Petersburg, Russia Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Background genome shotgun sequencing (WGS) with short reads (e.g. Illumina) does not allow the assembly of short DNA fragments into chromosomes directly – this pro- cedure requires long-insert libraries or longer reads (e.g. PacBio) and subsequent anchoring to chromosomes using various genome maps. In B chromosome research, comparative WGS of individuals with and without Bs can be used to identify B-specific blocks of non-repetitive sequence by demonstrating increased read depth in B- carriers [16]. B chromosomes are dispensable karyotype elements found in some species of animals, plants and fungi. They seem to originate from duplications and translocations of host genomic regions or result from inter-species hybridization. The number of B chromosomes can vary between individuals and even tissues or cells. They are present in a significant proportion of individuals within populations and are effectively transferred through gen- erations. These features distinguish Bs from other types of supernumerary chromosomes, such as small supernumer- ary marker [1] or double minute chromosomes [2]. Until re- cently, little was known about their genetic content, only repetitive elements and high copy number genes such as ribosomal RNA and histone genes were reported [3]. In mammals, duplicated unique protein-coding genes were found on the B chromosomes of canids (red fox and rac- coon dog [4–6]) and Siberian roe deer. In the latter case, the copies of genes on B chromosomes were also transcription- ally active [7]. Similar findings were reported in fish, insects and plants (reviewed in [8, 9]). Alternatively, NGS can be also applied to individual chromosomes isolated by flow-sorting or microdissection. These methods usually produce low amounts of DNA (although high-performance sorting is possible [17]) and require subsequent whole-genome amplification (WGA). In our study, we focused on degenerate-oligonucleotide- primed PCR (DOP-PCR) [18] with semi-random primers, a method routinely used for chromosome paint probe construction. Here, we performed Illumina MiSeq sequencing of flow-sorted B chromosomes of Siberian roe deer [12] and grey brocket deer. Our aim was to analyse the genetic content of mammalian Bs by NGS for the first time and to compare the evolutionary pathways of B chromosomes in two cervid species. In Cetartiodactyla, B chromosomes have been de- scribed in the Siberian roe deer (Capreolus pygargus) [10], grey brocket deer (Mazama gouazoubira) [10, 11], red brocket deer (M. americana) [10, 11], Brazilian dwarf brocket deer (M. nana) [11, 12], small red brocket deer (M. bororo) [11, 13], brown brocket deer (M. Abstract Background: B chromosomes are dispensable and variable karyotypic elements found in some species of animals, plants and fungi. They often originate from duplications and translocations of host genomic regions or result from hybridization. In most species, little is known about their DNA content. Here we perform high-throughput sequencing and analysis of B chromosomes of roe deer and brocket deer, the only representatives of Cetartiodactyla known to have B chromosomes. Results: In this study we developed an approach to identify genomic regions present on chromosomes by high-throughput sequencing of DNA generated from flow-sorted chromosomes using degenerate-oligonucleotide- primed PCR. Application of this method on small cattle autosomes revealed a previously described KIT gene region translocation associated with colour sidedness. Implementing this approach to B chromosomes from two cervid species, Siberian roe deer (Capreolus pygargus) and grey brocket deer (Mazama gouazoubira), revealed dramatically different genetic content: roe deer B chromosomes consisted of two duplicated genomic regions (a total of 1. 42-1.98 Mbp) involving three genes, while grey brocket deer B chromosomes contained 26 duplicated regions (a total of 8.28-9.31 Mbp) with 34 complete and 21 partial genes, including KIT and RET protooncogenes, previously found on supernumerary chromosomes in canids. Sequence variation analysis of roe deer B chromosomes revealed a high frequency of mutations and increased heterozygosity due to either amplification within B chromosomes or divergence between different Bs. In contrast, grey brocket deer B chromosomes were found to be more homogeneous and resembled autosomes in patterns of sequence variation. Similar tendencies were observed in repetitive DNA composition. (Continued on next page) (Continued on next page) © 2016 The Author(s). 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 14 Makunin et al. BMC Genomics (2016) 17:618 (Continued from previous page) Conclusions: Our data demonstrate independent origins of B chromosomes in the grey brocket and roe deer. Abstract We hypothesize that the B chromosomes of these two cervid species represent different stages of B chromosome sequences evolution: probably nascent and similar to autosomal copies in brocket deer, highly derived in roe deer. Based on the presence of the same orthologous protooncogenes in canids and brocket deer Bs we argue that genomic regions involved in B chromosome formation are not random. In addition, our approach is also applicable to the characterization of other evolutionary and clinical rearrangements. Keywords: B chromosome Evolution, High-throughput Sequencing, DOP-PCR, Siberian Roe Deer, Grey Brocket Deer, Protooncogenes Results In this study we analysed B-chromosome-specific DNA derived from flow-sorted chromosomes of Siberian roe deer and grey brocket deer. As controls, we took two samples of chromosome-specific DNA obtained by flow sorting and DOP-PCR amplification from the well- characterized mammalian genomes – dog (Canis lupus familiaris) chromosome 12 (CFA12) and a bovine (Bos taurus) mixed peak containing chromosomes 23, 26, 28, and 29 (BTA23, BTA26, BTA28, BTA29). Statistics on sequencing and analysis are presented in Tables 1 and 2. Background nemorivaga) [14], and Siberian musk deer (Moschus sibiricus) [15]. With the exception of an unconfirmed case in the musk deer, these species belong to the subfamily Capreolinae of Cervidae. Our study aims to characterize the B chromo- some genetic content in two cervid species: Siberian roe deer and grey brocket deer. In both of these species B chromosomes are among the smallest karyotype elements (only grey brocket deer Y chromosome is tinier). A previ- ous study revealed a 2 Mbp region with three genes (TNNI3K, LRRIQ3 and FPGT) on roe deer B chromo- somes [7], while B chromosomes of Mazama have not yet been studied by molecular methods. Automation of target region detection Isolated chromosome sequencing data are known to be contaminated with whole-genome DNA to some extent [21, 22]. We aimed to develop a method that discrimi- nates target regions corresponding to sampled chromo- somes from this noise. First, we took samples from species with well-assembled reference genomes – dog chromosome 12 (CFA12) and a mixed peak (BTAMix) containing bovine chromosomes 23, 26, 28, and 29 (BTA23, BTA26, BTA28, and BTA29). Upon visual inspection of read alignments in the UCSC genome browser, we derived two statistics suitable for detection of target chromosomes: pairwise distances between consecutive DOP-positions and read coverage inside DOP-positions. We collected and summarized these statistics for every chromosome in the reference genomes (Fig. 1 for cattle mixed peak). Pairwise distances were significantly lower in the target chromosomes, while read coverage exhibited more outlier high values without significant changes of median values. We also identified additional enrichment peaks corresponding to chromo- somes similar in size and CG content (i.e. located closely in the flow karyotype): for CFA12 we observed minor In the further search for target regions (i.e. regions corresponding to the sampled chromosomes), overlapping reads were referred as positions corresponding to DOP- PCR amplicons (or DOP-positions). It should be noted that this estimation could be inflated to some extent by non-overlapping reads from ends of longer amplicons. Two peaks of DOP-position sizes were observed for all samples: about 100 bp and 180-200 bp (for example, Additional file 1 Figure S1). Given that we used the Nextera protocol which includes enzymatic DNA frag- mentation, it is tempting to attribute the shorter peak to randomly fragmented amplicons and the longer peak to virtually intact ones. Properties of chromosome-specific DNA amplified with DOP-PCR In our study we adopted an approach used in an- cient DNA analyses [19], where reads are aligned to tar- get (dog for CFA12 or cattle for BTAMix, CPYB, and MGOB) and contamination (human) genomes and reads with better alignment to human genome than to target genome are discarded. We used mapping quality as an alignment metric in contrast to initially proposed edit distance, which we found to be prone to alignment length alterations. With this approach we eliminated 1.1–4.3 % of human contamination reads that could affect downstream analysis (Table 1). those involving whole-genome amplification from small amounts of starting material, such as microdissected chromosomes [16], ancient DNA [19] or single cells [20]. In our study we adopted an approach used in an- cient DNA analyses [19], where reads are aligned to tar- get (dog for CFA12 or cattle for BTAMix, CPYB, and MGOB) and contamination (human) genomes and reads with better alignment to human genome than to target genome are discarded. We used mapping quality as an alignment metric in contrast to initially proposed edit distance, which we found to be prone to alignment length alterations. With this approach we eliminated 1.1–4.3 % of human contamination reads that could affect downstream analysis (Table 1). Properties of chromosome-specific DNA amplified with DOP-PCR The next generation sequencing (NGS) technologies are actively developing, but their use for the characterization of isolated chromosomes is still quite limited. Whole- Human DNA is an inevitable source of contamination in high-throughput sequencing experiments, especially in Makunin et al. BMC Genomics (2016) 17:618 Page 3 of 14 Table 1 Statistics of sequencing and mapping of sorted chromosome-specific DNA Sample Reference # reads Reads, bp % contam % target CFA12 canFam3 746,338 198,383,243 1.3 40.2 BTAMix bosTau7 708,228 184,977,360 1.1 49.3 CPYB1 bosTau7 1,033,322 304,920,244 1.7 13.8 CPYB2 bosTau7 886,978 268,895,542 1.1 12.7 MGOB bosTau7 716,158 239,022,974 4.2 31.9 Samples: CFA12 – dog chromosome 12; BTAMix – cattle sorting peak with chromosomes 23, 26, 28, and 29; CPYB1, CPYB2 – Siberian roe deer B chromosomes (technical replicates); MGOB – grey brocket deer B chromosomes. # reads, reads bp – initial number and volume of sequence reads. # contam – percent of reads mapped to human genome (see text for details). # target – percent of reads mapped to target genome with MAPQ > 20 after contamination removal Table 1 Statistics of sequencing and mapping of sorted chromosome-specific DNA amplified samples of flow-sorted chromosomes prepared with the TruSeq protocol (based on adapter end-ligation to DNA fragments) and observed only one size peak at about 200 bp (data not included in this study). It is inter- esting that some amplicon lengths were overrepresented, potentially due to PCR duplicates or DOP-sites located within repeats. Depending on the sample, 9.7 to 50 thou- sand DOP-positions were recovered throughout the refer- ence genome (Table 2). The fraction of DOP-positions falling in target regions depends on sorting quality, target region size and sequence divergence, e.g. 82 % of positions belong to 202.4 Mbp of target chromosomes in the mixed cattle chromosome sample while only 8–9 % belong to the 2.0 Mbp B chromosome-specific region in the Siberian roe deer (Table 2). DOP-positions cover 3–4 % of target region sequences for autosomes and 13–15 % for B chro- mosomes (1 position per 3.6–4.9 kbp and per 1.7–2.2 kbp, respectively). those involving whole-genome amplification from small amounts of starting material, such as microdissected chromosomes [16], ancient DNA [19] or single cells [20]. Sample names as in previous; Positions (bp) in genome – number of non-repetitive DOP-positions throughout the reference genome and their cumulative size; Target region size, bp – size of regions present on chromosome determined with our method; % positions (occupancy/coverage) in target regions: % positions – percent of DOP-positions in target region relative to the whole genome (column 2), occupancy – percent of target region size (column 3) occupied by DOP-positions Automation of target region detection We sequenced the DOP-PCR- Table 2 Positions occupied by DOP-clones (DOP-positions) and target regions Sample Positions (bp) in genome Target region size, bp % positions (occupancy) in target regions CFA12 36,819 (5,285,070) 72,498,081 53.4 (4.4) BTAMix 50,035 (6,997,158) 202,419,725 82.1 (3.0) CPYB1 12,158 (1,530,385) 1,979,679 8.3 (14.6) CPYB2 9,665 (1,182,269) 1,979,679 9.4 (13.2) MGOB 12,530 (1,934,874) 9,311,710 43.5 (14.3) Sample names as in previous; Positions (bp) in genome – number of non-repetitive DOP-positions throughout the reference genome and their cumulative size; Target region size, bp – size of regions present on chromosome determined with our method; % positions (occupancy/coverage) in target regions: % positions – percent of DOP-positions in target region relative to the whole genome (column 2), occupancy – percent of target region size (column 3) occupied by DOP-positions Sample names as in previous; Positions (bp) in genome – number of non-repetitive DOP-positions throughout the reference genome and their cumulative size; Target region size, bp – size of regions present on chromosome determined with our method; % positions (occupancy/coverage) in target regions: % positions – percent of DOP-positions in target region relative to the whole genome (column 2), occupancy – percent of target region size (column 3) occupied by DOP-positions Page 4 of 14 Makunin et al. BMC Genomics (2016) 17:618 Fig. 1 Mean pairwise distances between consecutive DOP-positions (a) and position coverage with reads (b) in the flow sorting peak containing bovine chromosomes 23, 26, 28, and 29. Note the signal on chromosome 6 corresponding to KIT region translocation to BTA29 Fig. 1 Mean pairwise distances between consecutive DOP-positions (a) and position coverage with reads (b) in the flow sorting peak containing bovine chromosomes 23, 26, 28, and 29. Note the signal on chromosome 6 corresponding to KIT region translocation to BTA29 Target regions detection in both samples yielded two regions at BTA3:74.55–76.49 Mbp and BTA28:11.36– 11.40 Mbp (Table 3, Additional file 2) as well as a small 6–8 kbp artefact region (12 and 16 positions in CPYB1 and CPYB2, respectively) at the end of BTA10 resulting from spurious mapping of telomeric DNA reads. Mar- gins of the larger region on BTA3 were previously esti- mated with B-specific cDNA sequencing and bovine BAC clone localizations [7]. Our sequencing results provided higher resolution and also indicated that the region had two putative deletions, i.e. regions not cov- ered by reads (74.88–75.30 Mbp and 75.68–75.82 Mbp) (Fig. 2). B chromosomes of the grey brocket deer Similarly to the roe deer Bs, the grey brocket Bs were re- solved by flow cytometry into a distinct peak [26]. The specificity of the flow-sorted B chromosome DNA was evaluated by FISH (Additional file 1: Figure S3). The se- quence analysis of the sample revealed 26 regions of sig- nificant homology in the cattle genome, ranging from 23 to 1827 kbp in size (Table 3). Of these regions, three could not be detected by our script due to their small Automation of target region detection The first deletion was previously found to be located on the Siberian roe deer Bs by bovine BAC clone mapping, thus it may be a result of DOP-amplification bias or a mapping problem. The second deletion coincides with a cattle genome assembly gap. As previously shown, the BTA3:74.55–76.49 Mbp region includes RefSeq genes LRRIQ3, FPGT and the 5’ part of TNNI3K. The 42-kb re- gion on BTA28 is located between genes CHRM3 and ZNF33B. peaks at CFA14 and CFA22, which were expected from sorting [23], for BTAMix – BTA18, BTA19. The cattle mixed peak sample was evaluated with fluorescence in situ hybridization (FISH), which yielded signals on the chromosomes identified by sequencing (Additional file 1: Figure S2). peaks at CFA14 and CFA22, which were expected from sorting [23], for BTAMix – BTA18, BTA19. The cattle mixed peak sample was evaluated with fluorescence in situ hybridization (FISH), which yielded signals on the chromosomes identified by sequencing (Additional file 1: Figure S2). g We then wrote an R script dividing the entire genome into regions with differing mean values of pairwise dis- tances between consecutive DOP-positions (PD) (see Methods for the procedure), where low values of mean PD are characteristic for target regions and higher values – for whole-genome contamination. To test the performance of this method we first analysed control libraries CFA12 and BTAMix (Additional file 2). The script tended to identify multiple regions within target chromosomes, but all of these regions had the lowest mean PD as expected. In the case of the mixed bovine chromosome sample, a relatively small region at BTA6:72,525,912–73,007,603 was detected as target. This region includes the entire KIT gene, and this rearrangement was previously described as associated with colour sidedness [24]. Localization of a canine BAC clone with the KIT gene on cattle chromosomes confirmed the translocation of this region to one of these small auto- somes (Fig. 2a). B chromosomes of the Siberian roe deer Siberian roe deer B chromosomes were readily separated from other chromosomes during sorting due to their relatively small size and high GC content [25]. We se- quenced and analysed two samples (CPYB1 and CPYB2) from independent collections in one sorting experiment. Page 5 of 14 Makunin et al. BMC Genomics (2016) 17:618 Fig. 2 Localization of canine BAC clone with KIT gene on chromosomes of (a) cattle and (b) grey brocket deer. Note that in brocket deer, autosomal copies of KIT are located on MGO24 corresponding to a region of BTA6 [27]. Translocation of a 480-kbp regions from BTA6 to BTA29 is a previously characterized rearrangement associated with colour sidedness in cattle [24] Fig. 2 Localization of canine BAC clone with KIT gene on chromosomes of (a) cattle and (b) grey brocket deer. Note that in brocket deer, autosomal copies of KIT are located on MGO24 corresponding to a region of BTA6 [27]. Translocation of a 480-kbp regions from BTA6 to BTA29 is a previously characterized rearrangement associated with colour sidedness in cattle [24] size (23, 55, 90 kbp), but were obvious upon visual in- spection of the pairwise distance plot (Additional file 2). This pattern differs dramatically from the one observed in the roe deer, where only two homology regions (neither of which was detected in the grey brocket deer) were dis- covered. The total size of the regions present on brocket deer Bs was 9.31 Mbp or less due to 1.03 Mbp in 10 puta- tive deletions inferred from long fragments lacking posi- tions covered by reads. According to flow sorting and cytological data, Bs are among the smallest chromosomes in the grey brocket deer karyotype (only Y chromosome is smaller), approximately 10 Mbp in size. Taken together, these findings indicate a lack of extensive amplification of unique genomic regions in grey brocket deer Bs. containing the KIT gene gave signals on B chromo- somes and on a pair of autosomes (most likely MGO24 according to the roe deer painting results on bovine chromosomes and the presumed conservation of 70- chromosomal karyotype in cervids [27]) (Fig. 2b). We also performed total cDNA enrichment using the selection of hybrids by affinity capture with sorted chromosome-specific DNA of B chromosomes [7]. B chromosomes of the Siberian roe deer Sanger sequencing of 17 clones yielded 5 repetitive se- quences, 3 clones originated from BTA29:49.34–50.25 Mbp region, 2 clones – from BTA7:21.38–21.65 Mbp re- gion and BTA10:28.36–29.13 Mbp each, 1 clone from BTA23:8.99–9.08 Mbp. One clone originated from a non-specific region and two clones could not be localized. The remaining single clone was located on the non- chromosomal 65-kbp scaffold Un_JH125058. Upon visual inspection in the genome browser this scaffold showed a position density characteristic of the target region. Ortho- logous alignment to the human genome (chain/net track from UCSC browser) indicated scaffold homology to Hsa4:46,58–46,66 Mbp. Coordinates of neighbouring sites in the human genome were transferred to the cattle genome by LiftOver which allowed us to attribute the scaffold (reverse complement) to the assembly gap at BTA6:67,529,404–67,612,958. Thus, we were able to include this scaffold in the previously detected BTA6:67.48–69.30 Mbp region on the grey brocket deer Bs. To test the target regions observed from sequencing data we localized 9 bovine BAC-clones (Additional file 1: Table S1). For the region BTA10:28.36–29.13 Mbp two BAC clones were hybridized: CH240–472E2 covered the left margin of the region, and showed hybridization sig- nals on B chromosomes (Additional file 1: Figure S4); and CH240–449 J24 (located 50 kbp away from the re- gion detected by sequencing) did not show any FISH sig- nal on B chromosomes. For BTA29:49.34–50.25 Mbp three BACs (CH240–374 L11, CH240–244 F2, and CH240–235P20) located within the region and the BAC CH240–39 K2 overlapping the left margin of the region all showed strong hybridization signals on Bs. For BTA7:21.38–21.65 Mbp two BACs not overlapping with the target region were used and no hybridization signals were observed as expected. The canine BAC clone A total of 34 complete and 21 partial RefSeq genes were found on grey brocket deer B chromosomes Page 6 of 14 Makunin et al. Sequence variation on B chromosomes Using sequencing data it became possible to analyse the variants specific to B chromosomes. To get a robust estimate of B chromosome variation patterns we had to ensure that the DOP-PCR protocol does not intro- duce significant biases into the amplified DNA nucleo- tide composition. Also, it was necessary to discriminate B-specific variants from sequence divergence between cattle and deer genomes. The mixed peak of cattle chromosomes 23, 26, 28, and 29 was used to establish variant calling methodology and to ensure that it worked properly. The statistics of the called variants (variant density, heterozygosity, and pro- portions of different variant types – see Table 4) were similar to the values obtained in SNP discovery by gen- ome resequencing in cattle [28, 29]. We then compared our callset to known SNPs from bovine dbSNP 138 [30]. 12,367 of 19,188 (64.4 %) variants detected from sequen- cing were novel compared to that dataset. We hypothe- sized that novel variants with a low read coverage could result from various artefacts, such as sequencing errors, presence of contaminant DNA etc. We applied read depth filters and observed 6,662 novel variants of 10,052 total (66.3 %) at minimum depth 4 and 1,939 of 3,247 (59,7 %) at minimum depth 6. Without significant de- crease in number of novel variants (potential errors), we decided to use the entire callsets, but to treat individual variants with caution. Sequence variation in interspecies alignment of B chromosomes is hard to interpret, as it can be attributed to the divergence of either orthologous sequences be- tween lineages or paralogous sequence duplicates during B chromosome evolution. In the case of the Siberian roe deer we took advantage of the recently sequenced gen- ome of the closely related European roe deer (Capreolus capreolus) [31], which is also known to have incomplete reproductive isolation from the Siberian species [32–34]. The genome was assembled only to contigs with N50 = 10,813 kbp, thus we aligned these contigs to the bo- vine genome and called all variants in uniquely mapped contigs, obtaining a set of derived positions of European Coordinates in cattle genome assembly (bosTau7) are given. Only RefSeq cattle genes are listed, (f) – partial gene fragments located at region margins, (f?) – genes overlapping putative deletions not covered by reads Coordinates in cattle genome assembly (bosTau7) are given. B chromosomes of the Siberian roe deer BMC Genomics (2016) 17:618 Table 3 Regions detected on B chromosomes of Siberian roe deer and grey brocket deer with flow-sorted chromosome sequencing Region Genes Siberian roe deer (Capreolus pygargus) chr3:74,548,027-76,486,586 LRRIQ3, FPGT, TNNI3K (f) chr28:11,359,721-11,400,841 - Grey brocket deer (Mazama gouazoubira) chr1:80,557,678-80,815,945 LPP (f) chr1:132,195,520-132,248,825 PIK3CB (f) chr3:53,939,956-54,035,661 GFI1 chr3:97,346,492-97,530,439 ACOT11 (f), SSBP3 (f) chr5:112,038,039-112,092,762 CCND2 chr6:67,475,289-69,302,666 COX7B2, GABRA4, GABRB1 (f?), LOC536190 (f?), ATP10D, NFXL1, CNGA1, NIPAL1, TXK chr6:72,725,708-72,802,304 KIT (f) chr6:101,762,919-101,983,089 COQ2, HPSE, MIR2446, MRPS18C, FAM175A (f) chr6:115,148,215-115,688,073 MIR2448, FBXL5, CD38 (f?), FGFBP1 chr7:11,112,904-11,187,619 MAN2B1 (f) chr7:21,377,048-21,645,864 - chr8:78,145-782,808 ANXA10, MIR2466 chr10:28,363,651-29,133,083 TMCO5B chr14:5,823,596-6,377,396 KHDRBS3 (f) chr17:46,546,364-46,775,437 MBD3L1, CHFR, ZNF268 (f) chr19:59,129,525-59,366,644 CDC42EP4, FAM104A, COG1 (f) chr22:1,765,826-1,920,639 EOMES (f) chr22:11,626,319-11,984,586 ACAA1, MYD88, OXSR1, XYLB, ACVR2B, DLEC1 (f) chr23:8,994,141-9,084,277 C23H6orf106 (f), SNRPC (f) chr23:50,678,412-50,701,238 SERPINB9 chr25:37,085,021-37,276,232 - chr25:41,480,442-41,787,784 - chr28:628,155-915,003 TRIM67, FAM89A, ARV1, TTC13 (f) chr28:12,110,619-12,174,951 RET chr29:34,728,621-35,503,130 OPCML (f) chr29:49,338,779-50,252,639 DHCR7 (f?), NADSYN1, CARS (f) Coordinates in cattle genome assembly (bosTau7) are given. Only RefSeq cattle genes are listed, (f) – partial gene fragments located at region margins, (f?) – genes overlapping putative deletions not covered by reads We performed functional enrichment clustering ana- lysis for both complete and complete + partial gene lists. Enrichment signals were not very strong, both gene lists yielded functional clusters associated with ATP-binding/ kinase (score 1.33 and 1.30), transit to mitochondria (0.81 and 0.45), cell cycle (0.68 and 0.35), Zn-ion binding/ Zn-finger (0.5 and 0.66), and membrane (0.39 and 0.26). Complete + partial genes were also enriched with the cell proliferation/differentiation (1.32) and positive regulation of protein kinase activity (1.00), but such an associ- ation disappeared in the list containing only complete genes. Sequence variation on B chromosomes Only RefSeq cattle genes are listed, (f) – partial gene fragments located at region margins, (f?) – genes overlapping putative deletions not covered by reads (Table 3). Among these, the KIT gene was previously found on the B chromosomes of three canid species: red fox and Chinese and Japanese raccoon dogs [4–6]. The RET gene was found only in some B chromosomes in the Chinese raccoon dog [6]. The margins of the KIT re- gion present on the brocket deer Bs were not identical to those in the cattle chromosome 29 and in the B chro- mosomes of canids (the latter also included the neigh- bouring gene KDR). Makunin et al. BMC Genomics (2016) 17:618 Page 7 of 14 Page 7 of 14 Table 4 Sequence variation of DOP-PCR amplified chromo- some-specific DNA relative to bosTau7 genome classified relative to bovine RefSeq genes BTAMix CPYB CPYB-CCA MGOB A Total 6,068,678 344,775 297,042 1,332,441 Intergenic 4,518,518 262,448 226,421 991,640 Intron 1,501,565 80,987 69,282 327,012 5` UTR 1,337 0 0 27 3` UTR 15,213 0 0 2,780 Coding 32,045 1,340 1,339 10,982 B Total 19188 (6374) 15267 (2915) 3086 (1929) 46592 (1735) Intergenic 14829 (4970) 12187 (2349) 2476 (1530) 35292 (1334) Intron 4556 (1474) 3134 (558) 605 (392) 10102 (320) 5` UTR 4 (2) - - 2 (0) 3` UTR 49 (18) - - 101 (8) Coding syn. 46 (8) 17 (3) 1 (1) 113 (2) Coding non-syn. 44 (15) 26 (10) 12 (9) 103 (12) C Total 316 (952) 23 (118) 96 (154) 29 (768) Intergenic 305 (909) 22 (112) 91 (148) 28 (743) Intron 330 (1,019) 26 (145) 115 (177) 32 (1,022) 5` UTR 334 (669) - - 14 (-) 3` UTR 310 (845) - - 28 (348) Coding 356 (1,393) 31 (103) 103 (134) 51 (732) A. Lengths of cattle genome regions covered by reads. Samples: BTAMix – mixed peak of cattle chromosomes 23, 26, 28 and 29; CPYB – Siberian roe deer B chromosomes (combination of samples CPYB1 and CPYB2); CPYB-CCA – same, but regions not covered by European roe deer contigs are excluded; MGOB – grey brocket deer B chromosomes B. Number of total and heterozygous (in brackets) variants called and their annotation. Numbers of variants do not add up due to overlapping annotations and excluded NMD_target_transcript annotation. Intergenic variants also include up/downstream variants; coding non-synonymous variants include missense, stop codon gain/loss, frameshift, inframe indels. Sequence variation on B chromosomes Lengths of cattle genome regions covered by reads. Samples: BTAMix – mixed peak of cattle chromosomes 23, 26, 28 and 29; CPYB – Siberian roe deer B chromosomes (combination of samples CPYB1 and CPYB2); CPYB-CCA – same, but regions not covered by European roe deer contigs are excluded; MGOB – grey brocket deer B chromosomes Sequence variation on B chromosomes Sample descriptions – see 3A, except for CPYB-CCA – CPYB variants excluding the variants observed in Capreolus capreolus genomic contigs C. Variant density (bp per 1 variant) calculated as length of sequence covered by reads divided by number of called variants. Numbers are given for all and heterozygous (in brackets) variants. Sample descriptions – see 3B Table 4 Sequence variation of DOP-PCR amplified chromo- some-specific DNA relative to bosTau7 genome classified relative to bovine RefSeq genes ratio was equal 1.48 – much higher than 0.50 in bovine autosomes), most of CPYB-CCA variants in protein- coding regions were non-synonymous and heterozygous. The high level of sequence divergence and the extent of protein-disrupting variants were interpreted as indicators of the pseudogenisation process, while the high level of heterozygosity could have resulted either from the pre- viously discovered amplification of protein-coding genes on roe deer B chromosomes or from differences between the 8 Bs present in the karyotype of the cell culture sample [7]. p To our knowledge, no brocket genomes have been se- quenced yet, thus we could not apply the variant filtra- tion strategy implemented for roe deer. We could only extrapolate some observations from the Siberian roe deer Bs. First, heterozygosity of grey brocket deer B chromosomes was lower than that for the Siberian roe deer Bs: 1 variant per 768 bp compared to 1 per 118 for all roe deer B variants and 1 per 154 for CPYB-CCA variants. In fact, it was closer to the heterozygosity level detected in bovine autosomes (1 variant per 952 bp). This observation together with the larger size of non- repetitive genomic regions compared to roe deer Bs supports the hypothesis for the lack of gene amplifica- tion in grey brocket deer Bs, proposed on the basis of their estimated size. Second, no bias towards non- synonymous variants in protein-coding regions was ob- served, in contrast to roe deer Bs (Table 4B), which we interpret as weak evidence for the lack of pseudogenisa- tion. Similar conclusions were made from the slightly decreased density of derived positions relative to the cattle genome (1 per 29 bp in grey brocket deer Bs ver- sus 1 per 23 bp in Siberian roe deer Bs). Thus, in con- trast to the Siberian roe deer, B chromosomes of the grey brocket deer appear to be composed of non- amplified and presumably more conserved duplicated regions. A. Repetitive DNA analysis We adopted a strategy for repeat characterization by read clustering previously widely used in isolated plant chromosome studies [21]. For the mixed cattle chromo- some sample, the content of repetitive DNA identified in read analysis with RepeatExplorer [35, 36] differed sig- nificantly from the one obtained from RepeatMasker annotation of cattle chromosomes (Additional file 1: Table S2). RepeatExplorer reported a higher proportion of unannotated repeats and centromeric satellite DNA, which was as expected due to unassembled centromeres in the cattle genome. Other repeat families were mostly under-represented in RepeatExplorer output, sometimes drastically, e.g. 0.08 % versus 2.29 % for LINE L2. A notable exception was a SINE Alu repeat detected only in sorted chromosome samples, evidently resulting roe deer relative to cattle genome. We subtracted the resulting variants from the variants conventionally called from Siberian roe deer B chromosomal reads (CPYB) uni- fied from two samples CPYB1 and CPYB2. The resulting variants (denoted 'CPYB-CCA' in Table 4) were more B-chromosome-specific, although they still included population-level differences. Several peculiarities of this reduced callset were noted: the variation density remained high (1 variant per 96 bp compared to 1 variant per 316 bp in bovine autosomes), many variants were hetero- zygous (about 2/3 of heterozygous variants remained in the reduced callset, heterozygous to homozygous variation Page 8 of 14 Page 8 of 14 Makunin et al. BMC Genomics (2016) 17:618 from contamination with human DNA. The total per- centage of repeats was similar for both methods. These observations imply that the results of RepeatExplorer analysis of DOP-PCR amplified samples sequencing are not directly comparable to the RepeatMasker annota- tion of the assembled chromosomes. breaking up millions of cells. Chromosome microdis- section in principle produces cleaner results, but most successful experiments yield only several chromosome copies. Our results for flow-sorted chromosomes indi- cated that while chromosomes within the peak were not readily separated, discrimination from whole- genome background was feasible. Contamination with human DNA is considerable, but can be removed dur- ing read mapping, and thus affects mostly repeat ana- lysis – Alu repeats, the major component of the human genome, formed a cluster of up to 2 % of all reads. Thus, we chose to compare RepeatExplorer results be- tween sequences of cattle autosomes and B chromo- somes and came to several conclusions (Additional file 1: Table S2 and S3). Repetitive DNA analysis First, in both roe deer and brocket deer, B chromosomes bear a higher proportion of repeats com- pared to bovine autosomes. Second, annotation results were reproducible between two independent amplifica- tions of roe deer B chromosomes. Repeat family compos- ition of grey brocket deer B chromosomes was similar to cattle autosomes, while the largest repeat clusters from Si- berian roe deer Bs were often unannotated or comprised of low complexity and satellite repeats (Additional file 1: Table S3). In brocket deer Bs, of interest is the cluster 5 that contained virtually intact copies of LTR retrotran- spons from the ERVK family, indicating a recent re- peat expansion probably due to retroviral infection (Additional file 1: Table S3C). Only high-throughput sorting produces amounts of DNA suitable as input for NGS. Several studies used over 106 copies of sorted chromosomes (~0.1 pg for 100Mbp chromosome): 1.3 million human chromosome X [17] and 300 ng of mouse chromosome 17 [42] for SNP and CNV discovery based on Illumina GA; 80– 620 ng of every sorting peak in a Chinese Hamster Ovary cell line for separated chromosome assembly based on Illu- mina HiSeq [43]. The DNA yields from a few hundreds of flow-sorted chromosomes and a few copies of microdis- sected chromosomes are far too low and require amplifica- tion prior to NGS. p In our study we utilized DOP-PCR, a method for Whole-Genome Amplification (WGA) based on PCR with semi-random primers [18] previously applied in high throughput sequencing of the microdissected whole human chromosome 1 and the short arm of chromo- some 6 with only summary statistics of mapping calcu- lated [22]. In our study we found that DOP-PCR derived sequencing data is suitable for characterization of both non-repetitive and repetitive DNA in sampled chromo- somes, although biases in DNA composition resulted in uneven DOP-position distribution, e.g. false deletion observed in CPYB (Fig. 3) and distorted repeat content. On the other hand, due to the non-random 3`-end of DOP-primer, the same amplicons can be recovered from different samples. For example, in technical repli- cates CPYB1 and CPYB2 2,133 DOP-positions were re- peatedly recovered from both libraries across all cattle chromosomes. 782 (totalling in 205,979 bp) of those were located in the detected target regions. This is over three quarters of DOP-positions in target regions for both samples, indicating saturation of sequencing results with the recoverable DOP-amplicons. Repetitive DNA analysis In this respect, sequencing of DOP-PCR amplified DNA is similar to re- duced representation sequencing approaches, such as RAD-seq [44], with a benefit of coping with individual chromosomes obtained by flow sorting or microdissection. NGS of isolated chromosomes Several aspects are important in the design of the chromo- some studies with NGS: DNA source (whole genome or isolated chromosome), isolation method (flow sorting or microdissection), amplification (none or DOP-PCR or MDA). All of these affect the bioinformatic analysis in the ways discussed below. The supernumerary nature of B chromosomes allows for whole-genome shotgun sequencing (WGS) of indi- viduals with and without B chromosomes, and B-specific blocks can be identified by the increased read depth upon mapping to the reference genome [16]. This method provides high resolution, but sequence variation and repeat composition are hard to interpret because of the main genome background. The usage of isolated chromosomes for sequencing overcomes most of the WGS problems, but it also has several drawbacks. Two methods for chromosome isola- tion have been extensively used: flow sorting and micro- dissection. Flow sorted chromosome-specific DNA proves to be highly efficient for the detection of chromo- somal rearrangements by molecular cytogenetics techniques [37, 38] and array-comparative genome hybridization [39, 40]. However, the sorting method has several draw- backs: an inability to separate chromosomes of similar size and GC-content, e.g. human chromosomes 9–12 [41] or cattle chromosomes 23, 26, 28, 29 in our study; contamination with whole-genome and organelle DNA during sample preparation which inevitably involves Detection of chromosomal rearrangement margins based on NGS of isolated chromosomes was one of the main objectives of our study. Previously, a similar problem was resolved for balanced translocations in human [45, 46]. In these studies, Illumina reads of Makunin et al. BMC Genomics (2016) 17:618 Page 9 of 14 Fig. 3 Region of BTA3 present on B chromosomes of Siberian roe deer. BAC clone localization (green – present, red – absent) and cDNA data taken from [7]. Two samples from one flow-sorting experiment were used to produce reads 1 and 2. Read coverage data omitted. Note two regions lacking reads – one is covered by BACs, the other corresponds to an assembly gap receptor tyrosine kinase crucial for differentiation of haematopoietic, melanoblast and primordial germ cells. Mutations and amplification of this gene activate cancer- ogenesis in humans [47, 48] and lead to various pigmen- tation phenotypes in mammals (reviewed in [9, 49]). It is interesting that the control sample of mixed cattle chro- mosomes 23, 26, 28, and 29 indicates a translocation of a 480-kbp genomic region including KIT from BTA6 to one of these small autosomes. NGS of isolated chromosomes This translocation was previously described as linked with colour sidedness phenotype and the target chromosome was identified as BTA29 [24]. Sizes and exact coordinates of rearrange- ments involving KIT vary: 480 kbp region including the entire KIT gene in cattle translocation, 76 kbp region encompassing exons 1–20 of KIT in grey brocket Bs, 202 kbp including exons 2–21 of KIT in fox Bs. MDA-amplified flow-sorted chromosomes with trans- locations were mapped to the reference human gen- ome. Breakpoints were detected as changes in coverage at 1-bp resolution with a maximum likelihood-based algorithm. This approach was not applicable in our case, due to incomplete genome coverage by DOP-positions. Here we used the distances between positions covered by reads to locate the margins of the target regions: lower distances are characteristic for target regions present on chromosomes. Our classification script currently works only as a provisional tool to indicate putative target re- gions: visual inspection revealed that several positions can be erroneously included in or excluded from the target re- gion. Finally, the exact margins of the rearrangements have to be then verified by means of PCR. MDA-amplified flow-sorted chromosomes with trans- locations were mapped to the reference human gen- ome. Breakpoints were detected as changes in coverage at 1-bp resolution with a maximum likelihood-based algorithm. This approach was not applicable in our case, due to incomplete genome coverage by DOP-positions. Here we used the distances between positions covered by reads to locate the margins of the target regions: lower distances are characteristic for target regions present on chromosomes. Our classification script currently works only as a provisional tool to indicate putative target re- gions: visual inspection revealed that several positions can be erroneously included in or excluded from the target re- gion. Finally, the exact margins of the rearrangements have to be then verified by means of PCR. y In general, our approach utilizing NGS of isolated chromosomes amplified with DOP-PCR is relatively low cost method, allowing for over 10 samples to be analysed in a single Illumina MiSeq run. Resolution of margins of the rearrangements by the read mapping method was about 3-5 kbp (mean distance between neighbouring DOP positions), the minimum size of detected regions was 20 kbp, and the only errors discovered thus far were false-negative deletions (marginal shrinking of the regions is also expected). Spectra of repeats were biased but com- parable between samples. NGS of isolated chromosomes Recovery of multiple sequence variants allowed the analysis of their functional dis- tributions (coding/non-coding, synonymous/non- nonsynonymous) and heterozygosity. Reproducibility of the genomic positions of DOP-amplicons enables further phylogenetic analysis. p g RET represents another protooncogene found on B chromosomes of both grey brocket and a canid (Chinese raccoon dog). It encodes tyrosine-kinase and is frequently translocated in various cancers (reviewed in [50]). Among other cancer-related genes present on grey brocket Bs are: PIK3CB (phosphoinositide-3-kinase, catalytic, beta poly- peptide) – an isoform of outer membrane kinase con- nected with DNA double-strand break repair and various types of cancer [51, 52]; CCND2 (cyclin D2), controlling G1/S transition in cell cycle known to be translocated in mantle-cell lymphoma [53]; OXSR1 (oxidative-stress re- sponsive 1) – a serine/threonine kinase participating in stress response and cytoskeleton regulation, also was once identified as a weak candidate tumour suppressor [54]. According to the functional enrichment analysis, the most significant cluster (enrichment score 1.27) included 4 genes controlling embryonic development and cell differentiation: ACVR2B, EOMES, GFI1, and KIT. The second cluster (score 1.22) overlaps with the first one and includes kinases of proteins and amino acids DNA content of B chromosomes in cervids The KIT gene has been found on the B chromosomes of the grey brocket (this study) and two distinct species of canids [4–6]. KIT is a protooncogene encoding the Makunin et al. BMC Genomics (2016) 17:618 Page 10 of 14 Page 10 of 14 Mazama species) awaits to be addressed by future studies. (tyrosine – KIT, RET, TXK; serine/threonine – ACVR2B, OXSR1), nucleotides (PIK3CB) and carbohydrates (XYLB – xylulokinase homolog). A closely connected cluster of kinase positive regulation genes (score 0.91) includes three kinases (ACVR2B, PIK3CB, KIT), as well as CCND2. Most of these genes are located in separate re- gions, only OXSR1, XYLB, ACVR2B are found in a sin- gle 360-kbp region from cattle chromosome 22. If a connection between gene function and acquisition to B chromosome exists, it does not operate in the majority of cases: 5 out of 26 B-chromosomal regions bear cancer-related genes, 5 – kinases and their regulatory genes, other functional categories score even less regions. Conclusions We have developed a method for the analysis of DOP- PCR amplified chromosome-specific DNA using Next- Generation Sequencing. It includes breakpoint mapping at a resolution of several thousand nucleotides, as well as information on sequence variation and repeat con- tent. This approach is cost-effective: about 10 chromo- somes can be analysed in a single Illumina MiSeq run. We propose a range of applications besides B chromo- some research including chromosome attribution for scaffolds in genome assembly and characterization of evolutionary and clinical rearrangements. As an example, we a identified a 480-kbp translocation in cattle, which is associated with colour sidedness. Non-random content of B chromosomes fits well with the current knowledge of other types of genome instabil- ity on various time scales. For example, some genomic regions are more frequently amplified and translocated in human cancer than others [55]. Human small super- numerary marker chromosomes (sSMC) observed with an estimated population frequency of 1/2000 in most cases originate from a restricted number of genomic sites [1]. Reuse of some breakpoints in mammalian karyotype evolution and their enrichment with various features have been reported [56]. Using this method we have found that B chromosomes of the Siberian roe deer are more derived than the ones of the grey brocket deer, and they have undergone amp- lification and intensive pseudogenisation processes, while the B chromosomes of the grey brocket deer have retained a low copy number of autosomal genes on B chromo- somes without obvious signs of the degeneration of both unique and repetitive sequences. These patterns may re- flect differences in either the age or evolutionary fate of Bs in these two lineages. The B chromosomes of the Siberian roe deer incorpor- ate only two regions less than 2-Mbp in size character- ized by a high pseudogenisation and amplification of unique regions. A high degree of repeated sequences degradation further proves a degenerate nature of these genomic segments. Grey brocket deer Bs are completely different – there are 26 segments derived from different autosomal regions continuously ranging from a dozen kpb to over a Mbp in size. Pseudogenisation, internal amplification and repeat degradation were not signifi- cant. In contrast, a relatively new dispersal of LTR retro- transposons was detected. Next Generation Sequencing Sequencing of DOP-PCR chromosome-specific DNA was prepared using the Nextera DNA Library Preparation kit, which includes random fragmentation ofBEDTools: a flexible suite of utilities for comparing genomic features template DNA. 8 barcoded samples were sequenced in a single Illumina MiSeq run with read length equal to 150 bp and thus obtained 0.71–1.03 million reads per sample (Table 1). Sequencing data are freely available from NCBI read archive [SRA:PRJNA285957]. cDNA library construction and sequencing All procedures for grey brocket deer cDNA library con- struction, enrichment cloning and sequencing were per- formed as described [7]. Briefly, total RNA was extracted from grey brocket deer fibroblast tissue cell culture. A total cDNA library was constructed followed by enrichment for transcripts homologous to B chromosomes by selection of hybrids with affinity capture. Individual cDNA fragments were cloned and Sanger sequenced. Within the target regions, pairwise distances between positions were highly unevenly distributed with numer- ous long gaps (e.g., Fig. 2). We propose several explana- tions for this: 1) deletions or rapid sequence divergence occurred after duplication 2) reads cannot be mapped significantly to duplicated and repetitive genome regions, for example, human contamination reads are more or less evenly distributed throughout the genome, but no reads are mapped to short arms of chromosomes 21 and 22, where clusters of ribosomal genes are located; 3) reads cannot be mapped to gaps, including conventional 3 Mbp gaps at centromeres; 4) genomic sequence biases resulting in lack of ATGTGG motif and 5) PCR amplifi- cation biases. We accounted for factor 3) as gap posi- tions throughout the genome are known. Combination of factors 2) and 4) can theoretically be assessed by map- ping of putative DOP-positions flanked by the ATGTGG hexamer, but this method is prone to nucleotide substi- tutions, especially for cross-species alignment. We accounted for stochastic DOP-fragment retention by in- dependent generation of two samples from one sorting ex- periment of Siberian roe deer B chromosomes. The observed level of agreement was high enough to make this factor negligible. BAC clone localization in both Siberian roe deer [7] and grey brocket deer (Additional file 1: Table S1) were in agreement with sequencing-derived regions, even partial overlap of the BAC and the region was sufficient to generate a hybridization signal. The only exception occurred in Siberian roe deer, where the BAC clone signals were positive for a region not Cell cultures and chromosome sorting Fibroblast tissue cultures of the Siberian roe deer [38], grey brocket deer [61], cattle, and dog [62] were taken from the collections of the Department of Genome Di- versity and Evolution, Institute for Molecular and Cell Biology, Novosibirsk and the Cambridge Resource Centre for Comparative Genomics, Department of Veterinary Medicine, Cambridge University, UK. The numbers of B chromosomes in cell cultures of the cervids used to chromosome flow sorting were: eight for the Siberian roe deer and three for the grey brocket deer. p Brocket deers and roe deers belong to different tribes of subfamily Capreolinae (Odocoileini and Capreolini, respectively) that diverged ca 7.4–7.8 Mya, early in Cer- vidae evolution [57]. An independent origin of Bs in these species coincides with the absence of Bs in other representatives of the subfamily. As an interesting excep- tion, B chromosomes were found in 4 of ca. 10 Mazama species [10, 11]. Studies of the B chromosome genetic content in other Mazama lineages would test if they are of common or independent origins and thus specify their age as up to 5 million years (an estimated date of genus radiation [58]). As for the Siberian roe deer, the origin of B chromosomes could have occurred after its divergence from the European species [59], which has been dated by the mitochondrial control region phyl- ogeny as approximately 2 Mya [60]. The resulting poten- tial controversy (i.e., young but differentiated Bs in Siberian roe deer versus older but virtually intact Bs in Cell culturing, metaphase preparation and chromosome flow sorting were performed as previously described [7, 26]. In flow sorting experiments we collected about 300 copies of B chromosomes, which were well re- solved and distinct from non-B chromosomal peaks. Generation of chromosome-specific DNA with DOP-PCR Degenerate oligonucleotide-primed polymerase chain re- action (DOP-PCR) [18] with 6-MW primer (5'-CCGA CTCGAGNNNNNNATGTGG-3') was used for the generation of chromosome-specific DNA from sorted chromosomes as previously described [63]. Page 11 of 14 Page 11 of 14 Makunin et al. BMC Genomics (2016) 17:618 Detection of target regions g g BAM files with mapped reads were converted to BED files with positions and coverage information using BED- tools [66] “bamtobed -i” and “merge -n -i” commands. The genome was divided into regions with differing mean distance between positions with a custom R script region_dnacopy.R. First, for each chromosome distances are calculated between every DOP-position and the pos- ition on its left (PD) (for the first position in the chromosome, distance to 0-coordinate is taken). Then, changes of mean PD along the chromosomes are lo- cated. This step relies on the DNAcopy R package which uses circular binary segmentation and was initially de- signed for the detection of copy number variations from microarray genotyping data [67]. For this analysis, we found it useful to remove outlier values: big PD values corresponding to genomic regions without any positions, which are most likely unmappable (gaps or repeats); small values representing non-overlapping reads from one amplicon. Lastly, the script plots the results (Additional file 2) and outputs a table with chromosomes divided into regions and several statistics for each region. Regions with lowest mean PD are candidates for being target (i.e. present on the sampled chromosome). Cattle BAC clones from the CHORI-240 library listed in Additional file 1: Table S1 were used. Coordinates in the cattle genome (Baylor Btau_4.6.1 or bosTau7 in UCSC Genome Browser notation) were obtained from the NCBI Clone database or estimated from alignment of in- sert draft sequence to the genome with BLAST. Canine BAC-clone from RPC181 canine 8.1-fold BAC library containing KIT gene was previously characterized [4]. Fluorescence in situ hybridization Probes for fluorescence in situ hybridization (FISH) were labelled directly by DOP-PCR (for chromosome-specific DNA) or by using a nick translation kit (Invitrogen, UK) for bovine BACs. FISH was performed using a standard protocol [23, 62]. QC, alignment to reference genomes and elimination of human contaminant reads Sequencing adapters and DOP-primer were trimmed using cutadapt [64]. Three reference genomes were used: cattle (Baylor Btau_4.6.1 or bosTau7 in UCSC Genome Browser notation), dog (Broad CanFam_3.1 or canFam3) and human (GRCh37 or hg19). Reads were aligned to human (contamination) and either cattle or dog (target) genomes (see Table 1) using bowtie2 [65] in paired-end mode with “–local” option enabled for optimization of divergent sequence alignments. Only highly significant and unique alignments to the target genome with MAPQ > 20 were left. After that we removed the reads that aligned better to contamination than to the target genome based on MAPQ scores. Page 12 of 14 Makunin et al. BMC Genomics (2016) 17:618 reads) were annotated with mammalian RepeatMasker database (http://www.repeatmasker.org). covered by reads in both libraries (Fig. 2, clone CH240- 515C3). As BAC-clone localization evidence is direct, we should recognize the extent of reproducible biases in DOP-PCR amplified DNA composition. Sequence variation analysis Additional file 2: Distance between positions plots. Plots are presented for all chromosomes of reference genomes (canFam3 for CFA12, bosTau7 for BTAMix, CPYB1, CPYB2, and MGOB). Axes: X – ln(distance between consecutive DOP-positions, bp), Y – cumulative chromosome length, bp. Black dots – individual distances, red lines – mean values. (PDF 958 kb) For chromosome-specific read alignments sequence vari- ants were called using GATK HaplotypeCaller [70] with default options. Variant manipulation, comparison and statistics calculation were made with appropriate GATK instruments for all and heterozygous only variants. Vari- ant density was calculated by dividing the total size of positions covered by reads in target regions by the num- ber of variants called. Variants were annotated using Variant Annotation Integrator at UCSC genome browser [71] based on cattle RefSeq genes. Size of gene features (intergenic, intron, 5` UTR, 3` UTR, coding sequence) covered by reads were calculated by intersection with the appropriate fraction of cattle RefSeq genes (track used for variant annotation). Funding This study was supported with RFBR grant 15-29-02384 and Budget Projects 0310-2014-0003, 0310-2014-0008, and 0310-2014-0009. Availability of data and material High-throughput sequencing data are freely available from NCBI read archive [SRA:PRJNA285957]. The source code for the data analysis pipeline DOPseq_analyzer is available on GitHub: https://github.com/ilyakichigin/ DOPseq_analyzer. Additional file 1 includes supplementary figures and tables. Additional file 2 includes position density data for all chromosomes of reference genomes (canFam3 for CFA12, bosTau7 for all other samples); axes: X – ln(distance between consecutive DOP-positions, bp), Y – cumulative chromosome length, bp. The bovine dbSNP 138 dataset [30] was downloaded from UCSC genome browser ftp. The dataset was con- verted to GATK-compatible format. About 40 thousand of the 22.3 million variants were filtered out due to malformations. Authors’ contributions PO, MFS, FY, and VT performed chromosome sorting. AP, NV, and VT performed molecular cytogenetics experiments. EC performed high-throughput sequencing. AM and IK developed the pipeline and performed sequencing data analysis. AG, VT, DL, SO, and AM designed the study. AM wrote the manuscript. All authors have revised and corrected the manuscript. All authors read and approved the final manuscript. To obtain sequence variants of the European roe deer, its genomic contigs [31] were aligned to bosTau7 genome using BWA-MEM [72]. All variants present in uniquely mapped contigs were called with GATK UnifiedGenotyper with options “-glm BOTH -minIndelCnt 1 -indelGOP 80 -stand_call_conf 0 -stand_emit_conf 0” in order to output all variants present in every contig. We called all variants for regions with >1 contig aligned simultaneously (i.e. pu- tative duplications in European roe deer versus cattle), thus the total number of variants might be inflated. Consent for publication Not applicable. Consent for publication Not applicable. Repetitive DNA content We implemented a de novo repeat characterization ap- proach utilizing clustering and annotation of reads with RepeatExplorer [35, 36]. Trimmed but not filtered reads of length >19 bp were submitted to RepeatExplorer clus- tering algorithm on Galaxy-based server (http:// www.repeatexplorer.org/). Read similarities over 55 % of the read length were interpreted as edges connect- ing the similar reads (nodes). Clusters of frequently connected reads (each including >0.01 % of initial Additional files Additional file 1:Table S1. Bovine BAC clone localization on grey brocket deer chromosomes. Table S2. Repeat family composition. Table S3. Major repeat clusters revealed in chromosome-specific DNA with RepeatExplorer. Figure S1. Insert length distribution for chromosome-specific DNA contain- ing bovine chromosomes 23, 26, 28 and 29) inferred from paired-end read mapping to cattle genome. Figure S2. Fluorescence in situ hybridization of chromosome-specific DNA samples containing mixed bovine chromosomes 23, 26, 28 and 29 (red - CY3) and chromosome 10 (green - FITC) to cattle metaphase and corresponding DAPI staining. Figure S4. Fluorescence in situ hybridization of bovine BAC clone CH240-472E2 to grey brocket deer metaphase and corresponding DAPI staining. (DOC 633 kb) Functional gene enrichment analysis Lists of complete and complete + partial genes present on B chromosomes of the grey brocket deer were ana- lysed for GO function enrichment with DAVID [68, 69]. As a background we used the cattle gene list from the database. Competing interests p g The authors declare that they have no competing interests. Ethics approval and consent to participate h l d b h The protocol was approved by the Committee on the Ethics of Animal Experiments of the Institute of Molecular and Cellular Biology SB RAS. Received: 9 March 2016 Accepted: 12 July 2016 References 25. Sokolov VE, Orlov VN, Chudinovskaya GA, Danilkin AA. Chromosomal differences between two roe subspecies (Capreolus capreolus L. and Capreolus capreolus pygargus Pall.). Zoologicheskii Zhurnal. 1978;57:1109– 1112. References 1. Liehr T, Mrasek K, Kosyakova N, Ogilvie CM, Vermeesch J, Trifonov V, et al. Small supernumerary marker chromosomes (sSMC) in humans; are there B chromosomes hidden among them. Mol Cytogenet. 2008;1:12. 2. Balaban-Malenbaum G, Gilbert F. Double minute chromosomes and the homogeneously staining regions in chromosomes of a human neuroblastoma cell line. Science. 1977;198:739–41. 3. Camacho JPM, Sharbel TF, Beukeboom LW. B-chromosome evolution. Philos Trans R Soc Lond B Biol Sci. 2000;355:163–78. 4. Graphodatsky AS, Kukekova AV, Yudkin DV, Trifonov VA, Vorobieva NV, Beklemisheva VR, et al. The proto-oncogene C-KIT maps to canid B-chromosomes. Chromosome Res. 2005;13:113–22. 5. Yudkin DV, Trifonov VA, Kukekova AV, Vorobieva NV, Rubtsova NV, Yang F, et al. 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https://openalex.org/W3085634769	https://iris.unica.it/bitstream/11584/296958/2/mSystems-2020-Vascellari-e00561-20.full.pdf	English	null	Gut Microbiota and Metabolome Alterations Associated with Parkinson’s Disease	MSystems	2,020	cc-by	9,951	Gut Microbiota and Metabolome Alterations Associated with Parkinson’s Disease on September http://msystems.asm.org/ Downloaded from on September 15, 2020 at Universita Cagliari http://msystems.asm.org/ Downloaded from on September 15, 2020 at Universita Cagliari http://msystems.asm.org/ Downloaded from Sarah Vascellari,a Vanessa Palmas,a Marta Melis,b Silvia Pisanu,a Roberto Cusano,c Paolo Uva,c Daniela Perra,a Veronica Madau,a Marianna Sarchioto,b,d* Valentina Oppo,b Nicola Simola,e Micaela Morelli,e Maria Laura Santoru,f Luigi Atzori,f Maurizio Melis,b Giovanni Cossu,b Aldo Manzina aDepartment of Biomedical Sciences, Section of Microbiology and Virology, University of Cagliari, Cagliari, Italy bNeurology Service and Stroke Unit, AO Brotzu Hospital, Cagliari, Italy cCRS4, Science and Technology Park Polaris, Piscina Manna, Pula, Cagliari, Italy dDepartment of Medical Sciences and Public Health, University of Cagliari, Cagliari, Italy eDepartment of Biomedical Sciences, Section of Neuropsychopharmacology, University of Cagliari, Cagliari, Italy fDepartment of Biomedical Sciences, Oncology and Molecular Pathology Unit, University of Cagliari, Cagliari, Italy ABSTRACT Parkinson’s disease is a neurodegenerative disorder characterized by the accumulation of intracellular aggregates of misfolded alpha-synuclein along the cerebral axis. Several studies report the association between intestinal dysbiosis and Parkinson’s disease, although a cause-effect relationship remains to be established. Herein, the gut microbiota composition of 64 Italian patients with Parkinson’s dis- ease and 51 controls was determined using a next-generation sequencing approach. A real metagenomics shape based on gas chromatography-mass spectrometry was also investigated. The most signiﬁcant changes within the Parkinson’s disease group highlighted a reduction in bacterial taxa, which are linked to anti-inﬂammatory/neu- roprotective effects, particularly in the Lachnospiraceae family and key members, such as Butyrivibrio, Pseudobutyrivibrio, Coprococcus, and Blautia. The direct evalua- tion of fecal metabolites revealed changes in several classes of metabolites. Changes were seen in lipids (linoleic acid, oleic acid, succinic acid, and sebacic acid), vitamins (pantothenic acid and nicotinic acid), amino acids (isoleucine, leucine, phenylalanine, glutamic acid, and pyroglutamic acid) and other organic compounds (cadaverine, ethanolamine, and hydroxy propionic acid). Most modiﬁed metabolites strongly cor- related with the abundance of members belonging to the Lachnospiraceae family, suggesting that these gut bacteria correlate with altered metabolism rates in Parkin- son’s disease. on September 15, 2020 at Universita Cagliari m.org/ IMPORTANCE To our knowledge, this is one of the few studies thus far that corre- lates the composition of the gut microbiota with the direct analysis of fecal metabo- lites in patients with Parkinson’s disease. Overall, our data highlight microbiota mod- iﬁcations correlated with numerous fecal metabolites. RESEARCH ARTICLE Host-Microbe Biology crossm Gut Microbiota and Metabolome Alterations Associated with Parkinson’s Disease This suggests that Parkinson’s disease is associated with gut dysregulation that involves a synergistic relationship between gut microbes and several bacterial metabolites favoring altered homeosta- sis. Interestingly, a reduction of short-chain fatty acid (SCFA)-producing bacteria in- ﬂuenced the shape of the metabolomics proﬁle, affecting several metabolites with potential protective effects in the Parkinson group. On the other hand, the extensive impact that intestinal dysbiosis has at the level of numerous metabolic pathways could encourage the identiﬁcation of speciﬁc biomarkers for the diagnosis and treat- ment of Parkinson’s disease, also in light of the effect that speciﬁc drugs have on the composition of the intestinal microbiota. Received 22 June 2020 Accepted 24 August 2020 Published 15 September 2020 KEYWORDS 16S RNA, gut microbiota, PD, metabolome September/October 2020 Volume 5 Issue 5 e00561-20 msystems.asm.org 1 Vascellari et al. P arkinson’s disease (PD) is a neurodegenerative disorder characterized by the intra- cellular accumulation of -synuclein (-syn) aggregates (Lewy bodies) at various levels of the cerebral axis, including the central nervous system (CNS) and the enteric nervous system (ENS) (1). Clinical and neuropathological evidence indicates that the neurodegenerative changes in PD are accompanied by gastrointestinal (GI) dysregula- tion; however, whether this precedes or follows motor impairment remains to be established (2). Constipation is the most common premotor symptom in PD; it can affect more than 70% of PD patients (3) and can promote pathogenesis more than 10 years before the onset of clinical symptoms (2). Nevertheless, accumulation and ag- gregation of misfolded -syn in the gut, coupled with neurodegeneration in the ENS (4), seems to start up to 20 years before the onset of neurodegeneration in the CNS (5), thus supporting the “gut-to-brain” hypothesis (6). Interactions between the intestine and brain are known to be modulated by the intestinal microbiota through immuno- logical, neuroendocrine, and direct neurochemical mechanisms (7). Gut bacteria and bacterial metabolites could play a role in the pathogenesis of PD by promoting a proinﬂammatory environment in the gut (8, 9). Furthermore, intestinal inﬂammation associated with dysbiosis may contribute to the misfolding of -syn (10, 11). P on September 15, 2020 at Universita Cagliari http://msystems.asm.org/ Downloaded from Recently, different studies have described gut microbial alterations associated with PD patients. The results are somewhat heterogeneous, and there is no explicit agree- ment about which bacteria community might be involved (12–20). Moreover, gut microbiota modiﬁcations could reﬂect changes in bacterial metabo- lites. Gut Microbiota and Metabolome Alterations Associated with Parkinson’s Disease To date, several studies have investigated the metabolomics proﬁle of PD patients in different biological samples, such as cerebrospinal ﬂuid, blood, and urine (21–26). However, only a few studies have explored the fecal metabolome that mirrors the status of colonic bacteria and also links the symbiotic microbiota and health. A better understanding of the microbiota and metabolomics proﬁle in fecal samples could elucidate the interactions between host/bacterial metabolisms, gut microbes, and disease. In turn, this knowledge may provide critical information for the implementa- tion of better diet in PD. Therefore, this study aimed to investigate the composition and structure of the fecal microbiota in a cohort of Italian PD patients (PDs) compared to healthy subjects (HCs) using 16S rRNA gene sequencing. Furthermore, intending to understand the functional contribution of the microbial community, we used direct analysis of the fecal metabo- lome to identify potential metabolic alterations associated with the microbiota in PD patients. September/October 2020 Volume 5 Issue 5 e00561-20 RESULTS Patients with Parkinson’s disease display altered microbiota composition com- pared to healthy subjects. Characteristics of the 64 patients with PD and 51 HCs are shown in Table 1. The bacterial communities in the PD patients and controls were analyzed at different taxonomic levels. Firmicutes, Bacteroidetes, Proteobacteria, Actino- bacteria, and Verrucomicrobia were the ﬁve most abundant phyla, and altogether, they comprised more than 96% of all sequences. We detected no signiﬁcant differences between cases and controls in alpha-diversity (species richness of a group) indexes (P 0.05) (data not shown). We also assessed potential community-level differences between samples using beta-diversity analysis, and the weighted and unweighted dissimilarity signiﬁcantly differed between cases and controls (R2 0.079, P 0.001, weighted; R2 0.028, P 0.001, unweighted) (Fig. 1b and c). The Mann-Whitney U test followed by the Benjamini and Hochberg false discovery rate (FDR) correction test for multiple comparisons was implemented to identify the different taxa within the groups. The FDR-corrected signiﬁcant differences were also plotted using linear discriminant analysis (LDA) effect size (LEfSe) method (Fig. 2). The relative abundance of each taxon in the two study groups is reported in Table 2. Actinobacteria, Proteobacteria, and Verrucomicrobia were signiﬁcantly enriched in diseased subjects, while Bacteroidetes and Cyanobacteria were signiﬁcantly decreased; also, members of the Firmicutes were msystems.asm.org 2 September/October 2020 Volume 5 Issue 5 e00561-20 Gut Microbiota, Metabolomics, and PD TABLE 1 Subject characteristicsa Variable PD patients (n 64) Control subjects (n 51) Age (yr), mean SD 71.39 10.99 51.67 12.42 BMI, mean SD 26.07 4.18 23.70 3.46 Sex, n (%) Male 44 (68.75) 31 (60.78) Female 20 (31.25) 20 (39.22) Constipation, n (%) 37 (57.81) 0 (0) Coffee consumption, n (%) Yes 40 (62.50) 44 (86.27) No 24 (37.50) 7 (13.73) Smoking status, n (%) Yes 7 (10.94) 18 (35.29) No 57 (89.06) 33 (64.71) Phenotype, n (%) Tremor-dominant 22 (34.37) Rigid-akinetic 26 (40.63) Dyskinetic 16 (25.00) Treatment, n (%) L-DOPA 64 (100) aPD, Parkinson’s disease; n, case number; SD, standard deviation; BMI, body mass index; L-DOPA, L-3,4-dihydroxyphenylalanine. on September 15, 2020 at Universita Cagliari http://msystems.asm.org/ Downloaded from decreased; however, there was no signiﬁcant difference in distribution between the PD and HC groups (P 0.05). A total of 225 families in PDs and 217 in HCs were detected. The abundance of 16 families was signiﬁcantly modiﬁed in PDs versus HCs. msystems.asm.org 3 September/October 2020 Volume 5 Issue 5 e00561-20 RESULTS for confounding factors was performed using the generalized linear at was adjusted for sex, age, body mass index (BMI), coffee consump- ng. No differences were observed in terms of the Mediterranean diet sis. The plot was generated using a Galaxy computational tool. (a) The bar plots represent the signiﬁcantly differential taxa between PD d controls (ctrl) (red), based on effect size (LDA score [log 10] 2). Enriched taxa in PD patients (positive LDA score) and enriched taxa in LDA score). Differences among classes were obtained by the Kruskal-Wallis test ( 0.05). (b) Cladogram showed the differences in enriched versus enriched taxa in controls (red). Differences among classes were obtained by the Kruskal-Wallis test ( 0.05). on September 15, 2020 at Universita Cagliari http://msystems.asm.org/ Downloaded from on September 15, 2020 at Universita Cagliari http://msystems.asm.org/ Downloaded from on September 15, 2020 at Universita Cagliari sm.org/ FIG 2 LEfSE analysis. The plot was generated using a Galaxy computational tool. (a) The bar plots represent the signiﬁcantly differential taxa between PD patients (green) and controls (ctrl) (red), based on effect size (LDA score [log 10] 2). Enriched taxa in PD patients (positive LDA score) and enriched taxa in controls (negative LDA score). Differences among classes were obtained by the Kruskal-Wallis test ( 0.05). (b) Cladogram showed the differences in enriched taxa in PD (green) versus enriched taxa in controls (red). Differences among classes were obtained by the Kruskal-Wallis test ( 0.05). FIG 2 LEfSE analysis. The plot was generated using a Galaxy computational tool. (a) The bar plots represent the signiﬁcantly differential taxa between PD patients (green) and controls (ctrl) (red), based on effect size (LDA score [log 10] 2). Enriched taxa in PD patients (positive LDA score) and enriched taxa in controls (negative LDA score). Differences among classes were obtained by the Kruskal-Wallis test ( 0.05). (b) Cladogram showed the differences in enriched taxa in PD (green) versus enriched taxa in controls (red). Differences among classes were obtained by the Kruskal-Wallis test ( 0.05). Escherichia, Biﬁdobacterium, Streptococcus, Clostridium, and Serratia. An increase in some genera, such as Veillonella, Prosthecobacter, Enterobacter, and Slackia, was also observed. In contrast, several genera were signiﬁcantly reduced: Bacteroides, Blautia, Lachnospira, Butyrivibrio, Roseburia, Pseudobutyrivibrio, Brevibacterium, Dolichospermum, Coprococcus, and Odoribacter. Interestingly, within Firmicutes, the major differences concerned Lachnospiraceae, whose different genera were signiﬁcantly reduced in the PD group. September/October 2020 Volume 5 Issue 5 e00561-20 msystems.asm.org 4 RESULTS For instance, among the most relevant in PDs, Verrucomicrobiaceae, Biﬁdobacteriaceae, Streptococcaceae, and Desulfo- halobiaceae were increased, while Bacteroidaceae, Lachnospiraceae, Brevibacteriaceae, and Sphingobacteriaceae families were reduced. Differences were also observed at the genus level (601 in PDs versus 563 in HCs). The microbiota of PD patients was characterized by signiﬁcantly higher levels of several genera, such as Akkermansia, FIG 1 Beta-diversity analysis was presented as a two-dimensional (2D) plot based on principal coordinate analysis (PCoA). The statistical signiﬁcance was assessed using permutational multivariate analysis of variance (PERMANOVA). (a) Bray Curtis (P 0.001; F 4.217; R2 0.036); (b) UniFrac weighted (P 0.001; F 9.628; R2 0.079); (c) UniFrac unweighted (P 0.001; F 3.255; R2 0.028). CTRL, control. FIG 1 Beta-diversity analysis was presented as a two-dimensional (2D) plot based on principal coordinate analysis (PCoA). The statistical signiﬁcance was assessed using permutational multivariate analysis of variance (PERMANOVA). (a) Bray Curtis (P 0.001; F 4.217; R2 0.036); (b) UniFrac weighted (P 0.001; F 9.628; R2 0.079); (c) UniFrac unweighted (P 0.001; F 3.255; R2 0.028). CTRL, control. September/October 2020 Volume 5 Issue 5 e00561-20 Vascellari et al. FIG 2 LEfSE analysis. The plot was generated using a Galaxy computational tool. (a) The bar plots represent the signiﬁcantly differential taxa between PD patients (green) and controls (ctrl) (red), based on effect size (LDA score [log 10] 2). Enriched taxa in PD patients (positive LDA score) and enriched taxa in controls (negative LDA score). Differences among classes were obtained by the Kruskal-Wallis test ( 0.05). (b) Cladogram showed the differences in enriched taxa in PD (green) versus enriched taxa in controls (red). Differences among classes were obtained by the Kruskal-Wallis test ( 0.05). dobacterium, Streptococcus, Clostridium, and Serratia. An increase in uch as Veillonella, Prosthecobacter, Enterobacter, and Slackia, was also ntrast, several genera were signiﬁcantly reduced: Bacteroides, Blautia, yrivibrio, Roseburia, Pseudobutyrivibrio, Brevibacterium, Dolichospermum, d Odoribacter. within Firmicutes, the major differences concerned Lachnospiraceae, genera were signiﬁcantly reduced in the PD group. oidetes, the major reductions concerned Bacteroidaceae and members view of abundant taxa of the gut microbiota composition from each PD bject is represented as a heat map using statistical analysis of metag- (STAMP) software (Fig. 3). RESULTS As for Bacteroidetes, the major reductions concerned Bacteroidaceae and members therein. An overview of abundant taxa of the gut microbiota composition from each PD and healthy subject is represented as a heat map using statistical analysis of metag- enomic proﬁles (STAMP) software (Fig. 3). The analysis for confounding factors was performed using the generalized linear model (GLM) that was adjusted for sex, age, body mass index (BMI), coffee consump- tion, and smoking. No differences were observed in terms of the Mediterranean diet among the different participants recruited for this study. GLM analysis conﬁrmed only, in part, the results obtained by the univariate analysis, indicating that some confound- ing factors inﬂuenced the microbiota community of our samples. The statistically signiﬁcant differences in the composition of the intestinal microbiota in the PD group compared to the HC group were, however, maintained at various taxonomic levels (Table 3). In particular, Lachnospiraceae was signiﬁcantly depleted in PD patients. RESULTS September/October 2020 Volume 5 Issue 5 e00561-20 msystems.asm.org 4 Gut Microbiota, Metabolomics, and PD ABLE 2 Statistically signiﬁcant differences in bacterial taxa between PD patients versus healthy controlsa TABLE 2 Statistically signiﬁcant differences in bacterial taxa between PD patients versus healthy controlsa Phylum Family Genus 2/1b MDc PD (%)d HC (%)d P value FDR- adjusted P valuee Firmicutes Thermoanaerobacterales Incertae sedis Caldicellulosiruptor 1 0.226 0.121 0.058 0.010 0.014 Veillonellaceae Veillonella 1 0.599 1.994 0.277 0.000 0.000 Clostridiaceae Clostridium 1 0.169 3.133 2.154 0.012 0.016 Streptococcaceae 1 0.526 1.737 0.155 0.000 0.000 Streptococcus 1 0.513 1.730 0.153 0.000 0.000 Eubacteriaceae 1 0.287 0.120 0.062 0.000 0.000 Acetobacterium 1 0.273 0.115 0.059 0.000 0.000 Lachnospiraceae 2 0.253 8.000 12.460 0.001 0.002 Anaerostipes 1 0.406 0.236 0.144 0.027 0.030 Blautia 2 0.243 3.778 5.928 0.001 0.002 Lachnospira 2 0.444 0.535 1.086 0.000 0.000 Butyrivibrio 2 0.655 0.021 0.113 0.000 0.000 Roseburia 2 0.668 1.066 1.777 0.000 0.000 Pseudobutyrivibrio 2 0.562 0.160 0.444 0.000 0.000 Coprococcus 2 0.409 0.522 0.954 0.003 0.005 Bacteroidetes 2 0.269 28.546 47.455 0.000 0.000 Bacteroidaceae 2 0.297 17.454 29.910 0.000 0.000 Bacteroides 2 0.297 17.454 30.383 0.000 0.000 Odoribacteriaceae Odoribacter 2 0.432 0.191 0.331 0.001 0.002 Porphyromonadaceae Parabacteroides 2 0.245 1.858 2.269 0.046 0.050 Rikenellaceae 1 1.460 0.112 0.000 0.000 0.000 Sphingobacteriaceae 2 0.177 1.045 1.471 0.044 0.049 Proteobacteria 1 0.177 10.548 6.542 0.020 0.023 Desulfovibrionaceae Desulfovibrio 1 0.278 0.293 0.245 0.020 0.023 Desulfohalobiaceae 1 0.355 0.185 0.123 0.002 0.003 Desulfonauticus 1 0.304 0.184 0.122 0.002 0.003 Sutterellaceae Sutterella 2 0.643 0.258 0.819 0.000 0.000 Alcaligenaceae 2 0.642 0.270 0.837 0.000 0.000 Comamonadaceae 2 0.440 0.045 0.127 0.001 0.002 Enterobacteriaceae Enterobacter 1 0.559 0.404 0.173 0.001 0.002 Escherichia 1 1.003 3.737 0.259 0.000 0.000 Serratia 1 1.011 0.672 0.050 0.000 0.000 Klebsiella 2 0.723 0.668 0.961 0.000 0.000 “Candidatus Blochmannia” 2 1.673 0.042 1.392 0.000 0.000 Actinobacteria 1 0.313 5.524 2.263 0.002 0.003 Biﬁdobacteriaceae 1 0.362 4.191 1.289 0.014 0.018 Biﬁdobacterium 1 0.356 4.173 1.288 0.016 0.020 Coriobacteriaceae 1 0.240 0.944 0.562 0.012 0.016 Slackia 1 0.309 0.309 0.158 0.001 0.002 Microbacteriaceae 1 0.441 0.677 0.333 0.000 0.000 Brevibacteriaceae 2 0.288 0.072 0.121 0.000 0.000 Brevibacterium 2 0.288 0.072 0.120 0.000 0.000 Verrucomicrobia 1 0.614 5.429 1.020 0.002 0.003 Verrucomicrobiaceae 1 0.629 5.396 1.009 0.006 0.008 Akkermansia 1 0.464 4.669 0.879 0.015 0.019 Prosthecobacter 1 0.511 0.133 0.022 0.000 0.000 Cyanobacteria 2 0.156 0.509 0.730 0.000 0.000 Aphanizomenonaceae Dolichospermum 2 1.152 0.009 0.391 0.000 0.000 aThe results were obtained by Mann-Whitney U test performed on Statistical Package for the Social Sciences (SPSS) version 25.0, followed by the Benjamini and Hochberg false discovery rate (FDR) correction test for multiple comparisons. September/October 2020 Volume 5 Issue 5 e00561-20 RESULTS b2, Signiﬁcantly reduced in PD patients; 1, signiﬁcantly increased in PD patients. cMD, mean difference between the logarithmic value of relative abundance in the PD and HC groups. dAverage relative abundance (as a percentage) of each taxon in the PD and HC groups. eFDR-corrected P values with FDR 0.05. on September 15, 2020 at Universita Cagliari http://msystems.asm.org/ Downloaded from on September 15, 2020 at Universita Cagliari http://msystems.asm.org/ Downloaded from msystems.asm.org 6 TABLE 3 Statistically signiﬁcant differences of gut microbiome in PD versus HC groupsa TABLE 3 Statistically signiﬁcant differences of gut microbiome in PD versus HC groupsa TABLE 3 Statistically signiﬁcant differences of gut microbiome in PD versus HC groupsa Phylum Family Genus 2/1b MDc P value Bonferroni- adjusted P valued Firmicutes Lachnospiraceae 2 0.567 0.009 0.002 Blautia 2 0.596 0.007 0.010 Butyrivibrio 2 0.951 0.004 0.003 Coprococcus 2 0.873 0.039 0.019 Veillonellaceae Veillonella 1 1.556 0.002 0.000 Proteobacteria Enterobacteriaceae “Candidatus Blochmannia” 2 1.62 0.000 0.000 Actinobacteria Brevibacteriaceae 2 0.640 0.001 0.000 Brevibacterium 2 0.640 0.001 0.000 Cyanobacteria Aphanizomenonaceae Dolichospermum 2 1.364 0.001 0.000 aAnalysis of covariance (ANCOVA) performed using generalized linear model (GLM) followed by Bonferroni correction for multiple comparisons in Statistical Package for the Social Sciences version (SPSS) 25.0 for Windows. The differences in microbiota composition between PD patients versus HCs were adjusted for sex, age, BMI, coffee consumption, and smoking status covariates. b2, Signiﬁcantly reduced in PD patients; 1, signiﬁcantly increased in PD patients. cMD, mean difference between logarithmic value of relative abundance in the PD and HC groups. dBonferroni-corrected P values with P 0.05. on September 15, 2020 at Universita Cagliari http://msystems.asm.org/ Downloaded from on Septembe http://msystems.asm.org/ Downloaded from on September 15, 2020 at Universita Cagliari http://msystems.asm.org/ Downloaded from on September 15, 2020 at Universita Cagliari ems.asm.org/ that included organic compounds, lipids, amino acids, and vitamins. The results of the orthogonal partial least-square discriminant analysis (OPLS-DA) model obtained from the comparisons of PD and HC groups using multivariate statistical analysis (MVA) are shown in Fig. 4a. OPLS-DA model quality parameters (R2Y, 0.6; Q2, 0.36) and the respective permutation test (R2 intercept, 0.0, 0.335; Q2 intercept, 0.0, 0.187) are shown in Fig. 4b and c, displaying the statistical validity of the analysis and indicating distinct metabolic proﬁles in the two different groups (Fig. 4). FIG 4 Metabolomics multivariate statistical analysis (MVA). (a) OPLS-DA score plots of PD patients versus control subjects. (b) Validation parameters. R2X and R2Y indicated the cumulative explained fraction of the variation of the X block and Y block for the extracted components. Q2 values indicated cumulative predicted fraction of the variation of the Y block for the extracted components. R2 and Q2 intercept values are indicative of a valid model. (c) The permutation test was evaluated on the corresponding partial least-square discriminant analysis (PLS-DA) model. FIG 4 Metabolomics multivariate statistical analysis (MVA). (a) OPLS-DA score plots of PD patients versus control subjects. (b) Validation parameters. on September 15, 2020 at Universita Cagliari http://msystems.asm.org/ Downloaded from September/October 2020 Volume 5 Issue 5 e00561-20 msystems.asm.org 5 Vascellari et al. Vascellari et al. FIG 3 Heat maps of microbiota composition in PD cases and controls performed using the statistical analysis of metagenomic proﬁles (STAMP) software. The results were tested for statistical signiﬁcance by the Mann-Whitney U test (Statistical Package for the Social Sciences Version [SPSS] 25.0) (60). on September 15, 2020 at Universita Ca http://msystems.asm.org/ Downloaded from on September 15, 2020 at Universita Cagliari http://msystems.asm.org/ Downloaded from on September 15, 2020 at Universita Cagliari stems.asm.org/ FIG 3 Heat maps of microbiota composition in PD cases and controls performed using the statistical analysis of metagenomic proﬁles (STAMP) software. The results were tested for statistical signiﬁcance by the Mann-Whitney U test (Statistical Package for the Social Sciences Version [SPSS] 25.0) (60). FIG 3 Heat maps of microbiota composition in PD cases and controls performed using the statistical analysis of metageno results were tested for statistical signiﬁcance by the Mann-Whitney U test (Statistical Package for the Social Sciences Vers Accordingly, Blautia, Butyrivibrio, and Coprococcus were signiﬁcantly reduced in the same group of subjects, while the only alteration in terms of richness was observed in the Veillonella genus. Concerning Proteobacteria, “Candidatus Blochmannia” was signif- icantly reduced in PD patients. Also, Brevibacteriaceae and Brevibacterium belonging to Actinobacteria, as well as Dolichospermum belonging to Cyanobacteria, were signiﬁ- cantly reduced. In our work, Bacteroidetes and Verrucomicrobia did not show any signiﬁcant difference as opposed to the results of the univariate analysis. Evaluation of fecal metabolites reveals alterations in the gut metabolome in PD patients. We performed a direct evaluation of fecal metabolites by gas chromatography-mass spectrometry (GC-MS). A total of 90 metabolites were identiﬁed msystems.asm.org 6 msystems.asm.org 6 September/October 2020 Volume 5 Issue 5 e00561-20 Gut Microbiota, Metabolomics, and PD msystems.asm.org 7 TABLE 3 Statistically signiﬁcant differences of gut microbiome in PD versus HC groupsa R2X and R2Y indicated the cumulative explained fraction of the variation of the X block and Y block for the extracted components. Q2 values indicated cumulative predicted fraction of the variation of the Y block for the extracted components. R2 and Q2 intercept values are indicative of a valid model. (c) The permutation test was evaluated on the corresponding partial least-square discriminant analysis (PLS-DA) model. msystems.asm.org 7 September/October 2020 Volume 5 Issue 5 e00561-20 Vascellari et al. FIG 5 Statistically signiﬁcant metabolites in fecal samples of PD patients versus control subjects comparison. Discriminant metabolites obtained with the MVA underwent a Mann-Whitney U test with Holm-Bonferroni correction to determine which metabolites were statistically signiﬁcantly different. The resulted metabolites obtained are shown and expressed on the y axes of the graphs as ranks (data transformation in which numerical or ordinal values are replaced by their rank when the data are sorted). The levels of signiﬁcance are indicated by asterisks as follows: , P 0.05; , P 0.01. Vascellari et al. Vascellari et al. on September 15, 2020 at Universita Cagliari http://msystems.asm.org/ Downloaded from on September 15, 2020 at Universita Cagliari http://msystems.asm.org/ Downloaded from FIG 5 Statistically signiﬁcant metabolites in fecal samples of PD patients versus control subjects comparison. Discriminant metabolites obtained with the MVA underwent a Mann-Whitney U test with Holm-Bonferroni correction to determine which metabolites were statistically signiﬁcantly different. The resulted metabolites obtained are shown and expressed on the y axes of the graphs as ranks (data transformation in which numerical or ordinal values are replaced by their rank when the data are sorted). The levels of signiﬁcance are indicated by asterisks as follows: , P 0.05; *, P 0.01. Both up- and downregulation of metabolites were observed in the PD group compared to the HC group. The PD fecal samples clearly showed higher levels of several metabolites, such as cadaverine, ethanolamine, hydroxypropionic acid, isoleu- cine and leucine, phenylalanine, and thymine. In contrast, linoleic acid, oleic acid, nicotinic acid, glutamic acid, pantothenic acid, pyroglutamic acid, succinic acid, and sebacic acid were signiﬁcantly decreased (Fig. 5). Gut microbiota and fecal metabolite alterations are signiﬁcantly related in PD patients. We correlated the different patterns of changes observed in the microbiota composition with microbiota metabolites in PD and HC groups. The Spearman correlation analysis showed several signiﬁcant associations of gut bacteria with metabolites in the two groups (Fig. 6). TABLE 3 Statistically signiﬁcant differences of gut microbiome in PD versus HC groupsa In PDs, Lachnospira, Pseudobutyr- ivibrio, and Roseburia genera showed strong positive correlations with nicotinic acid and pantothenic acid. Negative correlations were instead obtained between the Ser- ratia genus and nicotinic acid, while Biﬁdobacterium correlated with pantothenic acid. Streptococcaceae and Streptococcus showed positive associations with cadaverine and, at the same time, a negative correlation with the Sphingobacteriaceae family. Negative correlations were also observed between the Biﬁdobacteriaceae family, and the related genus Biﬁdobacterium, with pyroglutamic acid. A positive correlation was obtained between this amino acid and the Sphingobacteriaceae family in the HC group. Con- versely, negative correlations were observed with this amino acid and Enterobacter and Serratia genera. A similar trend was obtained between the Sphingobacteriaceae family and Enterobacter genus with glutamic acid. Prosthecobacter showed a positive correla- tion with leucine. Last, a positive correlation between Bacteroidaceae and linoleic acid was observed. September/October 2020 Volume 5 Issue 5 e00561-20 msystems.asm.org 8 DISCUSSION The present study conﬁrmed and extended previous studies by showing that the overall composition of gut bacterial microbiota in PD patients and HCs is signiﬁcantly September/October 2020 Volume 5 Issue 5 e00561-20 msystems.asm.org 8 Gut Microbiota, Metabolomics, and PD FIG 6 The heat maps represent Spearman correlation of the relative abundance of differential bacteria, selected by the linear discriminant analysis effect size (LEfSe) method followed by FDR correction test, and the concentrations of metabolites, selected by the MVA, underwent a Mann-Whitney U test with Holm-Bonferroni correction. (a) Heat map control (n 51); (b) heat map PD (n 64). The r values are represented by gradient colors, where red and green cells indicate positive and negative correlations, respectively. The asterisks indicate levels of signiﬁcance as follows: , P 0.05; , P 0.01; , P 0.001; **, P 0.0001. on September 15, 2020 at Universita Cagliari http://msystems.asm.org/ Downloaded from FIG 6 The heat maps represent Spearman correlation of the relative abundance of differential bacteria, selected by the linear discriminant analysis effect size (LEfSe) method followed by FDR correction test, and the concentrations of metabolites, selected by the MVA, underwent a Mann-Whitney U test with Holm-Bonferroni correction. (a) Heat map control (n 51); (b) heat map PD (n 64). The r values are represented by gradient colors, where red and green cells indicate positive and negative correlations, respectively. The asterisks indicate levels of signiﬁcance as follows: , P 0.05; , P 0.01; , P 0.001; ***, P 0.0001. different. Moreover, evaluation of gut microbiota composition in relation to some confounding factors showed that some of these factors inﬂuenced the microbiota community, although the differences in the composition of the intestinal microbiota were maintained between PDs and HCs. In recent years, the PD research community has given increasing attention to the alteration of gut microbiota because of its putative implication in the disease patho- genesis (27). Several reports on this topic have shown a different abundance of distinct bacterial taxa between PD patients and HCs. It has been postulated that this could be due to differences in the methodologies and patient enrollment criteria. This can also explain why data are only partially in agreement with each other (12–20). The results presented herein show a distinctive proﬁle of the gut microbial com- munity in patients with PD compared to HCs. September/October 2020 Volume 5 Issue 5 e00561-20 DISCUSSION However, our results are in partial agreement with those previously produced by others. A possible explanation is that the statistical signiﬁcance can be affected by the correction for confounders. As an exam- ple, in the case of Firmicutes, although univariate analysis showed a signiﬁcant differ- ence between patients and controls, after correction for different covariates (age, sex, msystems.asm.org 9 Vascellari et al. TABLE 4 PD-associated abundance of SCFA-producing bacteriaa TABLE 4 PD-associated abundance of SCFA-producing bacteriaa Phylum Family Genus 2/1b FDR- adjusted P valuec SCFA production Actinobacteria 1 0.003 Acetate Biﬁdobacteriaceae 1 0.018 Acetate Biﬁdobacterium 1 0.020 Acetate Bacteroidetes 2 0.000 Propionate Bacteroidaceae 2 0.000 Propionate Bacteroides 2 0.000 Propionate Odoribacteriaceae Odoribacter 2 0.002 Butyrate Firmicutes Clostridiaceae Clostridium 1 0.016 Butyrate Lachnospiraceaed 2 0.002 Butyrate Blautiad 2 0.002 Butyrate Lachnospira 2 0.000 Butyrate Veillonellaceae Veillonellad 1 0.000 Acetate/propionate Verrucomicrobia 1 0.003 Acetate/propionate Verrucomicrobiaceae 1 0.008 Acetate/propionate Akkermansia 1 0.019 Acetate/propionate aAbundance of SCFA-producing bacteria in PD patients versus healthy controls using Mann-Whitney U test, followed by the Benjamini and Hochberg false discovery rate (FDR) correction test for multiple comparisons (FDR 0.05). b2, Signiﬁcantly reduced in PD patients; 1, signiﬁcantly increased in PD patients. cBonferroni-corrected P values with P 0.05. dAbundance of SCFA-producing bacteria in PD patients versus controls adjusted for sex, age, and BMI covariates using analysis of covariance (ANCOVA) performed using a generalized linear model (GLM) followed by Bonferroni correction for multiple comparisons in Statistical Package for the Social Sciences Version (SPSS) 25.0 for Windows. on September 15, 2020 at Universita Cagliari http://msystems.asm.org/ Downloaded from aAbundance of SCFA-producing bacteria in PD patients versus healthy controls using Mann-Whitney U test, followed by the Benjamini and Hochberg false discovery rate (FDR) correction test for multiple comparisons (FDR 0.05). BMI, coffee, and smoking), it did not match even with our data. However, in PD patients, the results showed a signiﬁcant decrease in the abundance of taxa belonging to Firmicutes, particularly in Lachnospiraceae and key members therein, such as Blautia, Coprococcus, and Butyrivibrio. Our data are in agreement with other studies that showed a reduction of the Lachnospiraceae family and related genera in fecal samples from PD patients (12, 13, 18). Several members of the Lachnospiraceae family have progressively captured attention due to their ability to produce short-chain fatty acids (SCFAs) (28) (Table 4). September/October 2020 Volume 5 Issue 5 e00561-20 DISCUSSION Acetate, propionate, and butyrate are the primary SCFA mole- cules produced from gut bacteria fermentation and are endowed with anti- inﬂammatory properties. These metabolites appear to play an important role in or- chestrating the function of the enteric nervous system and in promoting gastrointestinal integrity and motility (29). A reduction of SCFAs may also contribute to the development of gastrointestinal motility dysfunctions, thus highlighting the po- tential role of SCFA-producing bacteria in the pathogenesis of PD. We have investigated whether a reduction of the aforementioned SCFA producers correlates with the deple- tion of these metabolites in fecal samples from PDs. Although the levels of butyric acid, propionic acid, and acetic acid, in line with the microbiota proﬁle relative to Lachno- spiraceae members, were decreased in PD patients, no statistically signiﬁcant variations were observed after FDR correction (P 0.05). This ﬁnding is in contrast with the results by another research group that found a reduction in the fecal SCFAs (13). One possible explanation for this contrasting result may reside in the parallel increase we observed of Veillonella, Akkermansia, and Clostridium genera, which are known to produce acetate/propionate and butyrate. The enrichment of these bacteria may have caused a shift in the ﬁnal levels of SCFAs, mirroring the balance between these taxa. Moreover, differences in the cohort studied, in terms of the number of participants (we considered a larger cohort) and other variables, including ethnic origins and host genetics, may also explain these discrepancies. BMI, coffee, and smoking), it did not match even with our data. However, in PD patients, the results showed a signiﬁcant decrease in the abundance of taxa belonging to Firmicutes, particularly in Lachnospiraceae and key members therein, such as Blautia, Coprococcus, and Butyrivibrio. Our data are in agreement with other studies that showed a reduction of the Lachnospiraceae family and related genera in fecal samples from PD patients (12, 13, 18). Several members of the Lachnospiraceae family have progressively captured attention due to their ability to produce short-chain fatty acids (SCFAs) (28) (Table 4). Acetate, propionate, and butyrate are the primary SCFA mole- cules produced from gut bacteria fermentation and are endowed with anti- inﬂammatory properties. These metabolites appear to play an important role in or- chestrating the function of the enteric nervous system and in promoting gastrointestinal integrity and motility (29). msystems.asm.org 10 DISCUSSION on September 15, 2020 at Universita Cagliari http://msystems.asm.org/ Downloaded from Finally, an evaluation of the fecal metabolic proﬁle was made, and it highlighted interesting differences between PD patients and HCs. In particular, our data from direct analysis of fecal metabolites revealed that the metabolism of several amino acids, such as phenylalanine, leucine, and isoleucine, was signiﬁcantly increased in PD patients. A recent paper showed that the levels of phenylalanine were increased in the plasma of subjects with PD (32). Other work has reported that amino acid-fermenting bacteria could modulate the distribution of amino acids in the gastrointestinal tract (33, 34) and that altered amino acid concentrations may reveal changes in energy metabolism (35). Accordingly, the data reported in the present study indicate a signiﬁcant depletion of energy metabolism in PD. Notably, PD patients revealed a signiﬁcant reduction of the glutamic acid derivative pyroglutamic acid, whereas glutamic acid is a neurotransmitter implicated in PD pathogenesis (36). Although the results concerning the levels of glutamic acid associated with PD are rather discordant among studies, some studies have suggested that a reduction of glutamic acid, which is also a precursor of glutathione, may reﬂect an increase in oxidative stress in the disease progression (37). In addition to modiﬁcation in amino acid metabolism, our ﬁndings highlighted an alteration of lipid metabolism. Interestingly, the PD group was characterized by a reduction of linoleic acid and oleic acid. In particular, linoleic acid is an omega-6 polyunsaturated fatty acid (PUFA), and it has been associated with protective effects. Previous studies have proposed that a reduction of PUFA in PD models may reﬂect an excess of oxidative stress (38). In line with our data, some authors reported that the serum of PD patients showed decreased levels of several long-chain PUFAs, including linoleic acid (39). In addition to the above reported metabolic changes, we found a reduction of B vitamins, such as nicotinic acid (vitamin B3) and pantothenic acid (vitamin B5). Both these vitamins can be directly produced and secreted by commensal bacteria in the gut; they can also elicit anti-inﬂammatory and antioxidant activity and show protective effects against neurodegenerative mechanisms (40, 41). The reported decrease in the levels of both B3 and B5 vitamins strongly correlated with Lachnospira, Pseudobutyriv- ibrio, and Roseburia genera. DISCUSSION A reduction of SCFAs may also contribute to the development of gastrointestinal motility dysfunctions, thus highlighting the po- tential role of SCFA-producing bacteria in the pathogenesis of PD. We have investigated whether a reduction of the aforementioned SCFA producers correlates with the deple- tion of these metabolites in fecal samples from PDs. Although the levels of butyric acid, propionic acid, and acetic acid, in line with the microbiota proﬁle relative to Lachno- spiraceae members, were decreased in PD patients, no statistically signiﬁcant variations were observed after FDR correction (P 0.05). This ﬁnding is in contrast with the results by another research group that found a reduction in the fecal SCFAs (13). One possible explanation for this contrasting result may reside in the parallel increase we observed of Veillonella, Akkermansia, and Clostridium genera, which are known to produce acetate/propionate and butyrate. The enrichment of these bacteria may have caused a shift in the ﬁnal levels of SCFAs, mirroring the balance between these taxa. Moreover, differences in the cohort studied, in terms of the number of participants (we considered a larger cohort) and other variables, including ethnic origins and host genetics, may also explain these discrepancies. As mentioned earlier, we found an increase of Veillonella in the PD group. Our result extended, at the genus level, a previous report that showed an increase of the msystems.asm.org 10 September/October 2020 Volume 5 Issue 5 e00561-20 Gut Microbiota, Metabolomics, and PD Veillonellaceae family in PD patients (14). In agreement and extending the results of another study (30) reporting a reduction of the Brevibacteriaceae family, we also found a decrease of the Brevibacterium genus. Among the different changes reported, the reduction in “Candidatus Blochmannia” and Dolichospermum genera in PD patients is of particular interest. It should be noted that to date, none of the studies carried out on the microbiota in PDs showed changes in Cyanobacteria, to which the Dolichospermum genus belongs. Members of Cyanobac- teria produce a series of neurotoxins that are implicated in the protein misfolding and aggregation phenomenon that is seen in PD and other neurodegenerative disorders (31). However, the role of Dolichospermum is not clear, and the decrease in the abundance of Dolichospermum in PD patients deserves further investigation concerning its potential pathophysiological role and the effects of this reduction. September/October 2020 Volume 5 Issue 5 e00561-20 msystems.asm.org 11 DISCUSSION Several genera of intestinal Firmicutes bacteria express crucial factors for vitamin B3 synthesis (42, 43), suggesting that these bacteria might affect the metabolism and bioavailability of these vitamins in the gut. Vitamin B5 is the primary precursor of coenzyme A, and its deﬁciency might be involved in the alteration of the citric acid cycle, causing defective energy levels, a ﬁnding shared by several neurodegenerative disorders, such as PD, Huntington’s disease, and Alzheimer’s disease (44). Concerning vitamin B3, other investigations have found a chronic vitamin B3 deﬁcit in PD patients (45). A toxic effect is instead ascribed to the polyamine cadaverine (46), a product of bacterial and human cometabolism (47), which we found to be increased in PD patients. A recent study revealed that cadaverine is involved in the inhibition of intestinal motility in a mouse model (48). We reported that an increased level of msystems.asm.org 11 Vascellari et al. cadaverine positively correlated with the Streptococcaceae family and the related genus Streptococcus, which are known to express cadaverine biosynthetic enzymes (49). An increase of cadaverine may also contribute to promoting a proinﬂammatory environ- ment and motility dysfunctions of the gastrointestinal tract in PD. Overall, our data highlight that microbiota modiﬁcation correlated with numerous fecal metabolites, which is suggestive of the fact that PD is associated with gut dysregulation. Moreover, the present ﬁndings highlight that there is a mutualistic relationship between gut microbes and several bacterial metabolites that favor altered gut homeostasis. on September 15, 2020 at Universita Cagliari http://msystems.asm.org/ Downloaded from on September 15, 2020 at Universita Cagliari http://msystems.asm.org/ Downloaded from In addition, we revealed alterations of several speciﬁc microbial taxa, including the reduction of the Lachnospiraceae family and genera therein (SCFA-producing bacteria), whose anti-inﬂammatory and protective role is well known within the organism. Our study provides an overview of the complex alterations associated with PD. Since the interaction between gut microbiota and dopaminergic medication as well as anticholin- ergics has only recently been recognized (18, 50), more detailed investigations are needed and are in progress in order to establish the potential role played by gut bacteria on the therapeutic drugs in use (51) or, vice versa, a direct inﬂuence of the drugs themselves on microbiota modiﬁcations, as the drugs may hide the real microbiota composition in naive untreated PD patients. DISCUSSION In addition, such studies might lead to the identiﬁcation of novel candidate biomarkers for PD diagnosis and treatment and may provide a rationale for the development of new complementary therapeutic strategies for PD. September/October 2020 Volume 5 Issue 5 e00561-20 MATERIALS AND METHODS Patients and samples. The institutional review boards and human subject committees at the participating institutions approved the study (protocol PG/2017/17817). Written or verbal informed consent was obtained from all enrolled participants: 64 patients with diagnosed PD and 51 healthy controls. Patient inclusion criteria were as follows: diagnosis of idiopathic PD according to the UK Brain Bank criteria, Hoehn and Yahr stage I to IV, age between 45 and 85 years, and stable doses of dopaminergic treatment for at least 4 weeks before enrollment. The exclusion criteria were as follows: atypical or secondary Parkinsonism; the use of probiotic or antibiotic supplements for the 3 months before enrollment; the presence of a primary gastrointestinal disease; the concomitant presence of internal medicine, neurological, or unstable psychiatric illness together with severe cognitive impairment. Patients were evaluated by the Movement Disorder Society-sponsored revision of the Uniﬁed Parkinson’s Disease Rating Scale (MDS-UPDRS) III and IV (motor part and motor ﬂuctuations/dyskinesia) (52), and the following battery of clinical scales/questionnaires: nonmotor symptom scale (NMSS) (53), Scale for Outcome in Parkinson’s disease-autonomic (SCOPA-AUT) (53), and Cognitive Assessment Montreal (MoCA) (54). Patients were classiﬁed as either tremor dominant (TD), with postural instability and gait difﬁculty (rigid-akinetic) (PIGD), or as dyskinetic patients. Stool samples from each subject were collected at outpatient facilities of the AO Brotzu and AOU Cagliari hospitals (Cagliari, Sardinia, Italy) and delivered to the laboratory within 3 h. The control group was composed of healthy participants, selected among spouses and family members of study patients. Sample and library preparation and sequencing. DNA extraction and puriﬁcation were performed as previously described (46). In particular, DNA extraction from thawed fecal samples was performed using the QIAamp DNA stool minikit following the manufacturer’s instructions (Qiagen). A total of 115 samples were sequenced using an Illumina MiSeq system. Sequences were assigned to operational taxonomic units (OTUs) using Quantitative Insights into Microbial Ecology (QIIME) (55). We performed a closed-reference OTU assignment using the “uclust” software (56) with a 97% sequence similarity threshold against Greengenes_13.8 97% OTU cluster (57) as a reference. Barcoded amplicon libraries for sequencing on the Illumina MiSeq platform were generated using degenerate primers targeting the bacterial V3-V4 16S rRNA region with the Nextera index kit (Illumina), as previously described (46). Data and statistical analysis. Analysis of the data generated on the Miseq system was carried out using the BaseSpace 16S Metagenomics app (Illumina). msystems.asm.org 12 ACKNOWLEDGMENTS This research was partially supported by a Fondazione di Sardegna grant F71I17000220002 to N.S. and grant BDS 2013.1325 to A.M. For this study, S.V. was funded by Regione Sardegna-POR FSE 2014-2020. The funding agencies had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript. on September 15, 2020 at Universita Cagliari sm.org/ We thank the Associazione Sarda Malati Parkinson (ASAMPA) for contributing to patient enrollment and sample collection. S. Vascellari (Data curation; Methodology; Formal Analysis; Visualization, Writing – original draft; Writing – review & editing), V. Palmas (Data curation; Formal Analysis; Methodology), M. Melis (Data curation; Formal Analysis), S. Pisano (Formal Analysis), D. Perra (Formal Analysis), V. Madau (Formal Analysis), M. L. Santoru (Formal Analysis), R. Cusano (Methodology), P. Uva (Data curation), M. Sarchioto (Data curation), V. Oppo (Data curation), N. Simola (Conceptualization; Writing – review & editing), M. Morelli (Conceptualization; Writing – review & editing), L. Atzori (Conceptualization; Writing – review & editing), M. Melis (Conceptualization; Writing – review & editing), G. Cossu (Conceptualization; Data curation; Writing – review & editing), A. Manzin (Project administration; Conceptualization; Supervision; Writing – original draft; Writing – review & editing). Gut Microbiota, Metabolomics, and PD Gut Microbiota, Metabolomics, and PD The GLM was implemented, followed by Bonferroni correction for multiple comparisons using Statistical Package for the Social Sciences version (SPSS) 25.0 for Windows (60), to test for confounding factors. Only bacteria that were found to be signiﬁcant at the univariate level after FDR correction were considered. No normally distributed variables had been normalized using their logarithmic value before performing GLM. The differences in microbiota composition between cases and controls were adjusted for sex, age, BMI, coffee consumption, and smoking status covariates. Microbiota and metabolome analyses. Fecal microbiota analysis was investigated as previously described (46). For metabolomics, frozen feces were mixed in methanol solution and sonicated. After centrifugation, the supernatants were dried and derivatized with methoxyamine dissolved in pyridine (Sigma-Aldrich, St. Louis, MO, USA). N-Methyl-N-(trimethylsilyl)-triﬂuoroacetamide (Sigma-Aldrich, St. Louis, MO, USA) was added, and the samples were resuspended in hexane and ﬁltered. Then, 20 l from each sample was used to create a pool for quality control. on September 15, 2020 at Universita Cagliari http://msystems.asm.org/ Downloaded from on September 15, 2020 at Universita Cagliari http://msystems.asm.org/ Downloaded from For gas chromatography-mass spectrometry (GC-MS) analysis, 1 l of the derivatized sample was injected splitless into a 7890A gas chromatography coupled with a 5975C network mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) equipped with a fused silica capillary column. The gas ﬂow rate through the column was 1 ml/min. Identiﬁcation of metabolites was performed using the standard National Institute of Standards and Technology NIST 08 standard and Golm Metabolome Database (GMD) mass spectra libraries and by comparison with authentic standards. Data processing was performed using a pipeline in KNIME. The multivariate statistical analysis, PLS-DA, and OPLS-DA were performed using SIMCA-P software (ver. 14.0; Umetrics, Sweden). GraphPad Prism software (version 7.01; GraphPad Software, CA, USA) was used to perform the Mann-Whitney U test with Holm-Bonferroni corrected P values and Spearman correlations between the microbiome and the metabolome. Data availability. Sequencing data have been deposited in the European Nucleotide Archive (ENA) under the accession number PRJEB30401. Metadata have been deposited under the accession number PRJEB36138. MATERIALS AND METHODS Operational taxonomic unit mapping to the Greengenes database (V.13.8) was performed using the QIIME platform (V.1.8.0). Alpha-diversity analysis (Shannon, Simpson, Fisher, Chao1, and abundance-based coverage estimator [ACE]), was performed on the Microbiome Analyst tool (58). For beta-diversity analysis, weighted and unweighted UniFrac and Bray-Curtis distances were calculated using Adonis in vegan R package (59). The Mann-Whitney U test followed by FDR correction test for multiple comparisons was used to identify bacterial taxa that were statistically different among PD patients and controls. Linear discriminant analysis effect size (LEfSE) (http://huttenhower.sph.harvard.edu/galaxy/) analysis was performed on a Galaxy computational tool to estimate the effect size of each differentially abundant feature. Results were then corrected by FDR correction test for multiple comparisons. Heat maps of gut microbiota composition were generated using STAMP software. msystems.asm.org 12 Functional implications of microbial and viral gut metagenome changes in early stage L-DOPA-naïve Parkinson’s dis- ease patients. Genome Med 9:39. https://doi.org/10.1186/s13073-017 -0428-y. on September 15, 2020 at Universita Cag http://msystems.asm.org/ m 33. Dai ZL, Wu G, Zhu WY. 2011. Amino acid metabolism in intestinal bacteria: links between gut ecology and host health. 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https://openalex.org/W2582903031	https://europepmc.org/articles/pmc5276858?pdf=render	English	null	ReaCog, a Minimal Cognitive Controller Based on Recruitment of Reactive Systems	Frontiers in neurorobotics	2,017	cc-by	22,497	ORIGINAL RESEARCH published: 30 January 2017 doi: 10.3389/fnbot.2017.00003 ReaCog, a Minimal Cognitive Controller Based on Recruitment of Reactive Systems Malte Schilling 1* and Holk Cruse 2 1 Center of Excellence Cognitive Interaction Technology, Bielefeld University, Bielefeld, Germany, 2 Department of Biological Cybernetics and Theoretical Biology, Bielefeld University, Bielefeld, Germany It has often been stated that for a neuronal system to become a cognitive one, it has to be large enough. In contrast, we argue that a basic property of a cognitive system, namely the ability to plan ahead, can already be fulﬁlled by small neuronal systems. As a proof of concept, we propose an artiﬁcial neural network, termed reaCog, that, ﬁrst, is able to deal with a speciﬁc domain of behavior (six-legged-walking). Second, we show how a minor expansion of this system enables the system to plan ahead and deploy existing behavioral elements in novel contexts in order to solve current problems. To this end, the system invents new solutions that are not possible for the reactive network. Rather these solutions result from new combinations of given memory elements. This faculty does not rely on a dedicated system being more or less independent of the reactive basis, but results from exploitation of the reactive basis by recruiting the lower-level control structures in a way that motor planning becomes possible as an internal simulation relying on internal representation being grounded in embodied experiences. Edited by: Poramate Manoonpong, University of Southern Denmark, Denmark Keywords: reactive system, cognitive system; internal model, motor planning, internal simulation, neural networks, attention Reviewed by: Yulia Sandamirskaya, University of Zurich, Switzerland Yoonsuck Choe, Texas A&M University, USA Michail Maniadakis, Foundation for Research & Technology – Hellas, Greece Keywords: reactive system, cognitive system; internal model, motor planning, internal simulation, neural networks, attention Correspondence: Malte Schilling mschilli@techfak.uni-bielefeld.de Received: 18 September 2016 Accepted: 11 January 2017 Published: 30 January 2017 A basic problem concerns what, after all, is meant by the term “cognition.” Deﬁnitions cover various ideas, reaching from Maturana and Varela (1981) “life is cognition” (which would include even bacteria to be cognitive systems), Engel et al. (2013) who note that “cognition is action.” Other authors avoid the problem of a short deﬁnition, which almost inevitably includes comparatively simple systems, by listing a collection of phenomena to characterize cognitive systems (e.g., Khlentzos and Schalley, 2007; Menzel et al., 2007). The most important faculties generally agreed as to characterize a cognitive system are attention, awareness, emotion, learning, speciﬁc aspects INTRODUCTION Over the last years more and more ﬁndings in neuroscience have shown that higher level cognitive capabilities cannot be detached from the functioning of lower level sensorimotor control systems (van Duijn et al., 2006; Barsalou, 2008) which is the core idea of embodied cognition as a ﬁeld. It is assumed that cognition recruits the underlying sensorimotor systems (Anderson, 2010). Intensively studied examples controlled by such sensorimotor, or reactive, systems are insects. Already a lot is known about their structure and properties of their sensorimotor systems (Menzel et al., 2007; Cruse et al., 2009) which allows to build well performing biologically inspired systems (Pfeifer et al., 2007; Ijspeert, 2014). But it is still unclear if all the crucial properties are understood that are required to form the basis for a cognitive system. Do the known principles allow to leverage the sensorimotor control systems toward cognition? Correspondence: Malte Schilling mschilli@techfak.uni-bielefeld.de Citation: In this article, we will not enter this discussion but focus on basic properties discussed by several authors as to be crucial for a cognitive system, namely the ability to invent new behaviors and the ability to plan ahead the latter being required to test the feasibility of the new invention. Lower level behaviors, often termed reactive or automatic, controlled by “reactive systems,” require procedural elements ensuring survival and allowing for basic behavioral abilities, e.g., locomotion, feeding, object avoidance. The combination of such controllers may also be suited to guide seemingly more complex behaviors (e.g., navigation). These controllers constitute the procedural memory of the system. Exploiting the loop through the world (Brooks, 1989) even a “hard-wired” memory system allows for adaptation to changing environments as will be illustrated in the second section (Reactive Walker). In reactive systems many of these procedures (or “action-perception circuits,” Pulvermüller and Garagnani, 2014) can be active at the same time, but they may also compete amongst each other for controlling the system (Brooks, 1989). Therefore, a crucial ability for each behaving system—including reactive systems— is the ability to select one among diﬀerent possible actions. This architecture is inspired by earlier authors as Arbib (1998), Brooks (1991b), and Minsky (1986). y Reactive systems, by deﬁnition, do not belong to the ﬁeld of cognition. However, many authors (e.g., Newell, 1994; Anderson, 2010; Glenberg and Gallese, 2012) argue that cognition in all known systems is strongly based on and is intimately connected with a functional reactive system. Even more, as proposed by Barsalou (2008) and others, reactive (or behavior-based) systems having internal states (as introduced in the second section, Reactive Walker) plus being embodied are basic requirements for a system to become a cognitive one. As already noted brieﬂy above, there is indeed strong support showing that neuronal elements forming cognitive properties are tightly intertwined with the reactive system itself and a functional separation is not possible. For example, planning of a movement is interpreted in this view as a mental enactment of the movement (Jeannerod, 2001; Hesslow, 2002). This view is supported as brain regions that formerly were assumed as being highly specialized, for example the motor area, are also activated during language processing or perception (Feldman and Narayanan, 2004; Buccino et al., 2005; Pulvermüller, 2005; Jeannerod, 2006; Pulvermüller and Garagnani, 2014). More generally, Gallese and Lakoﬀstate that “a key aspect of human cognition is. Citation: Schilling M and Cruse H (2017) ReaCog, a Minimal Cognitive Controller Based on Recruitment of Reactive Systems. January 2017 \| Volume 11 \| Article 3 Frontiers in Neurorobotics \| www.frontiersin.org ReaCog, a Minimal Cognitive Controller Schilling and Cruse reactive, or behavior-based, systems, and cognitive systems is that the former are restricted to apply their procedural memory elements (or internal representations, or internal models) only in the context in which the latter have been acquired (Wilson, 2008). For example, a speciﬁc movement (e.g., grasping a speciﬁc type of prey) is stored as a (congenital or learned) procedural memory. The content of this memory element may also be considered as a model of that movement, which can—in a reactive system— only be triggered by a speciﬁc stimulus, the speciﬁc prey. In contrast, cognitive systems are able to modify their behaviors and thereby may come up with solutions for a novel task (Glenberg and Gallese, 2012). A novel task is considered here a task in which, in the current context, none of the existing procedural memory elements can be applied to solve the problem, as none of the available procedures are able to deal with the actual situation or to predict the resulting consequences. Therefore, to approach a cognitive level, one has to search for systems that are creative, i.e., able to alter their procedural memory elements or to compose them in a new way allowing the system to handle such a novel tasks. This characterization agrees with the statement of Limongelli et al. (1995) “cognition is the ability to relate diﬀerent unconnected pieces of information in new ways and apply the resulting knowledge in an adaptive manner.” Taking a broader view, Anderson (2010), in his massive redeployment hypothesis, states that “neural reuse” is a fundamental principle not only applied in evolutionary time scales but also for solving current problems by a cognitive system. Thus, in this article we will focus on a system that is able to ﬁnd solutions for novel tasks. of memory, language as well as thinking, reasoning, planning ahead, decision making, volition, Theory of Mind or even subjective feelings and consciousness (for another list proposed by Langley et al. (2009, see Discussion). Frontiers in Neurorobotics \| www.frontiersin.org Citation: A key feature that might be suited for a distinction between January 2017 \| Volume 11 \| Article 3 2 ReaCog, a Minimal Cognitive Controller Schilling and Cruse constraints—is based on elements forming an artiﬁcial neural network. The article is structured in the following way. The second section (Methods and Material) is divided in three parts. In section Background and Previously Developed Models. Reactive Walker—the Walknet (Reactive Walker) the simple control system for a hexapod walker is introduced which is biologically inspired from studies on the walking of insects. In section Motor Planning: from Walknet to reaCog (Motor Planning) the cognitive expansion is presented including an example that illustrates how the basic reactive system is recruited for planning. This will be followed by a more detailed explanation of the control architecture and the experiment setup (section Cognitive Expansion). Simulation results will be presented, on the one hand, for an example scenario (section Results) explaining our approach. On the other hand, a series of simulations shall demonstrate how the approach deals with disturbed walking. While there is no similar robotic architecture which applies behaviors out of context and realizes recruitment as internal simulation, we will present a brief overview on related work and discuss diﬀerences and implications (section Related Work). In the Discussion we will analyze the properties of the complete system, discuss them and brieﬂy turn toward the question as to how aspects of higher-level phenomena being listed above may emerge in our system (Discussion and Conclusions). Using a system able to control autonomous behavior and using a complex, non-trivial body, we follow a whole-systems approach. We take the embodiment approach literally insofar as our system is constructed in such a way that it is currently used to control a simulated robot in a dynamical simulation environment, but will be transferred to a physical robot in a next step. Thus, we deal with really executable behaviors rather than with more abstract approaches on a dynamical systems level or systems that operate on a symbolic level. Application of such purely high-level approaches may bear the danger that serious problems occurring at a lower level may be overlooked (Brooks, 1991a; Verschure and Althaus, 2003). Taken together, we focus on a system that allows for the ability to plan ahead (McFarland and Bösser, 1993) relying on intersnal representation (Steels, 2003) that are grounded in embodied experiences (Gallese and Lakoﬀ, 2005). Citation: In this way, we follow the proposal of Feynman, who stated that we can only understand a system when we are able to create it (in Hawking, 2001; p. 83). We start with a decentralized, reactive neuronal network controller (Dürr et al., 2004) for a complex hexapod robot which is expanded by a holistic body model represented by a “hard- wired” recurrent neural network (RNN) and used for inverse kinematics (Schilling et al., 2012). Based on a reactive structure the robot allows for walking in an unpredictable environment. Citation: the adaptation of sensory- motor brain mechanisms to serve new roles in reason and language, while retaining their original function as well.” (Gallese and Lakoﬀ, 2005, p. 456). This is supported by behavioral research showing that behavioral and cognitive processes are functionally related insofar as both processes seem to apply the same structuring principles and seem to have access to memory in a structurally similar way (e.g., Jeannerod and Decety, 1995; Cross et al., 2006; Barsalou, 2008; Barsalou et al., 2012). What are the prerequisites to ﬁnd a solution to a current problem? One way to ﬁnd new solutions is to apply a search strategy based on simple trial and error. But trial and error is a risky approach and generally quite slow. As an alternative, “internal trial-and-error” could be applied. This means that in addition to the ability to modify the procedures and their composition, such systems are able to anticipate consequences of new actions which enables the agent to decide based on these predictions (Hesslow, 2002). These aspects have already been captured by McFarland and Bösser (1993) who indeed deﬁne cognition as the faculty to plan ahead. Planning ahead allows to verify the feasibility of new solutions before execution. Therefore, planning ahead is the second basic property of our system. The ability to predict requires internal models, or internal representations. Because our system is characterized here as to search for new solutions by exploiting the already existing memories (or internal models) in a ﬂexible way, i.e., not only in a speciﬁc context, but in diﬀerent contextual situations, an organizational scheme is required that allows for compositionality and modulation of speciﬁc parameters. In the third section (Motor Planning) we will provide a simple solution for this problem. Following the view proposed by Barsalou (2008), Glenberg and Gallese (2012) and others, our approach is to start with a non-trivial reactive system that is then equipped with the ability to plan ahead. To this end, we will consider a system with a complex enough body (i.e., having a considerable number of extra degrees of freedom), but an arguably simple controller, which—in order to comply with biological What distinguishes a reactive system from a cognitive one? Frontiers in Neurorobotics \| www.frontiersin.org MATERIALS AND METHODS g p We will further enable the robot to cope with situations for which the reactive system does not oﬀer a solution. In this case, a “cognitive expansion” shall allow the system to search for a new solution to this problem. The search space is not only characterized by the 18◦C of freedom (DoF) of the robot, but is expanded by the fact that the controller being embodied heavily depends on the “loop through the world,” i.e., depends on the unpredictable properties of the environment. Further, the complexity of the situation is increased as behavioral elements to be selected show various time dependencies. To cope with such situations, the system ﬁrst has to search for a behavioral element normally not used in the current context. The search space is large and not continuous. So, gradient descent methods are not applicable. The search for new solutions is based on (i) a somatotopic heuristic, (ii) noise applied to part of the cognitive expansion network as well as (iii) tests for physical feasibility of the solution proposed, ﬁrst by internal simulation, second by performing the behavior in reality. For internal simulation, we exploit the property of the body model used here, which means that the same model cannot only be used as an inverse model, but also as a predictive model. Therefore, this body model can be used for motor planning applying an internal simulation to test newly selected behavioral elements. Background and Previously Developed Models. Reactive Walker—The Walknet Biological Model of Insect Walking Background and Previously Developed Models. Reactive Walker—The Walknet Biological Model of Insect Walking In Figure 2, the shaded section is depicted in more detail. et al., 2013b). Figure 1 sketches the approximate anatomical arrangement of the controllers and the numbering of the legs. These single leg controllers are assumed to be situated in the thoracic ganglia (for a review see Bässler and Büschges, 1998). Figure 2 shows details of the controllers as used in Walknet for the left middle leg (LM_leg) and the left hind leg (LH_leg). A single leg controller mainly consists of several movement primitives that reﬂect the leg movement consisting of two phases. These are the stance movement, during which the leg maintains ground contact and is retracted to propel the body forward, while supporting the weight of the body, and the swing movement where the leg is lifted oﬀthe ground and moved in the direction of walking, to touch down at the location where the next stance should begin. The movement primitives controlling stance and swing are realized in the leg controller (Figure 2) as several modules, or procedures, each containing artiﬁcial neurons forming a local, in general, recurrent neural network (RNN). These modules might receive direct sensory input and provide output signals that can be used for driving motor elements. The two most important procedural elements in our example are the Swing-net, responsible for controlling a swing movement, and the Stance-net controlling a stance movement (Figure 2, for swing: see Dürr et al., 2004; Schumm and Cruse, 2006, for stance: Schmitz et al., 2008; Schilling et al., 2012). The end positions used during forward walking are stored in the procedures for the swing and stance movement, i.e., the Swing-net and Stance-net respectively (in Figure 2 Furthermore, a leg controller must also take into account the interaction with the other legs. Part of these interactions are mediated directly by the body and through the environment, making explicit computations superﬂuous (see, e.g., the local positive velocity feedback approach Schmitz et al., 2008). While the physical coupling through the environment is important, it is not suﬃcient. In addition, the controllers of neighboring legs are coupled via a small number of channels transmitting information concerning the actual state of that leg (e.g., swing, stance) or its position (i.e., values of joint angles). These coordination rules were derived from behavioral experiments on walking sticks (Cruse, 1990). In Figure 1 the channels are numbered 1–3. Background and Previously Developed Models. Reactive Walker—The Walknet Biological Model of Insect Walking g g The example we choose as a reactive basis and which will brieﬂy be explained in the following concerns a hexapod (insect-like) walking system (see review Schilling et al., 2013b for details). The task to walk over a non-predictable substrate—possibly cluttered with obstacles of varying size and holes—is by no means a trivial one. The walker has six legs each equipped with three joints. Therefore, the controller has to deal with 18◦C of freedom (DoF). As body position in space is deﬁned by only six DoFs (three for position in space, three for orientation) there are 12 DoFs free to be decided upon by the controller which means that the controller has to make these 12 (respectively 18) decisions in a sensible way at any moment of time while dealing with an unpredictable environment. As a ﬁrst step, the walker is only using tactile sensors situated in the legs (and possibly the antennae Schütz and Dürr, 2011) measuring contact with external objects, and with proprioceptors measuring position, torques and velocities of joints. The walking system to be described in the following is based on behavioral (and to some extent neurophysiological) studies on insects, in particular stick insects (Schilling et al., 2013b). At ﬁrst, we brieﬂy describe the essentials of the earlier version, Walknet, and will then introduce expansions. The results show that the cognitive expansion requires only a small number of neurons coupled by a quite simple connectivity. This simple network shows basic properties required for a cognitive system and can be used as a scaﬀold for later introduction of further properties. In addition, capabilities like showing attention or emotions, might be found as properties emerging from such an architecture as discussed in Cruse and Schilling (2013). Experiments on the walking stick insect have shown that the neuronal system is organized in a decentralized way (Wendler, 1968; Bässler, 1983; Cruse, 1990). Derived from these results, a model has been proposed in which each leg is attributed a separate controller (Dürr et al., 2004; for a review Schilling January 2017 \| Volume 11 \| Article 3 3 ReaCog, a Minimal Cognitive Controller Schilling and Cruse FIGURE 1 \| General architecture of the reactive controller Walknet. The complete system consists of one controller for each leg (LF/RF left/right front leg, LM/RM left/right middle leg, LH/RH left/right hind leg). Coordination rules (1,2,3) act between neighboring legs, prolonging, or shortening the stance phase. Background and Previously Developed Models. Reactive Walker—The Walknet Biological Model of Insect Walking Each leg controller contains several modules, a Swing-net and a Stance-net, to control swing and stance movement, respectively. In Figure 2, the shaded section is depicted in more detail. they are part of the gray rounded boxes called Swing-net and Stance-net. Swing is triggered when the stance-end-position is reached, Stance movement is triggered by ground contact). Following Maes (1990) the overall activation of a procedural element is controlled by a motivation unit (represented by yellow circles in the Figures) that gates to what extent the corresponding procedural element contributes to the control of the leg. In the network, these units forming rate coded, non- spiking neurons with leaky integrator, i.e., low pass, dynamics. They have a piecewise linear activation function (from 0 to 1) and control the strength of the output of the corresponding procedure (in a multiplicative way). Here we deal with a very simple motivation unit network that, initially, consists of just two units, the motivation units for the two procedural elements used in forward walking, Swing-net and Stance-net. Each motivation unit is reinforcing itself (not shown in Figure 2) and at the same time inhibiting the other motivation unit, forming a winner-take- all (WTA) net and allowing only one behavior to be active at any given time (Figure 2). Secondly, sensory signals control the behavior selection by inﬂuencing the motivation units and thus initiate behavioral transitions. When the leg touches the ground toward the end of a swing movement, the ground contact causes switching to stance movement by activating the motivation unit Stance. Correspondingly, during forward walking, reaching a given posterior position activates the motivation unit Swing. As an extension, we introduced backward walking. In this case, new swing and stance procedures are introduced including their motivation units (Figure 3). Swing_toBack behavior stores the target for the swing movement to the back. As for forward walking, a memory element is required representing the stance end position (for details see Schilling et al. (2013a) and explanation of the Stance movement below). FIGURE 1 \| General architecture of the reactive controller Walknet. The complete system consists of one controller for each leg (LF/RF left/right front leg, LM/RM left/right middle leg, LH/RH left/right hind leg). Coordination rules (1,2,3) act between neighboring legs, prolonging, or shortening the stance phase. Each leg controller contains several modules, a Swing-net and a Stance-net, to control swing and stance movement, respectively. Background and Previously Developed Models. Reactive Walker—The Walknet Biological Model of Insect Walking Each leg controller contains several modules: a Swing-net and a Stance-net to control swing and stance movement, respectively, each equipped with a motivation unit (depicted by yellow circles). Connections with an arrow indicate positive (“excitatory”) inﬂuences, connections ending with a T-shaped ending indicate negative (“inhibitory”) inﬂuences. On the right, one sub-module (Swing-net) is shown in more detail, as it is implemented as a neural network (numbers refer to weights). Target angles serve as an input to the neural network and are stored in the component. Each of the three neural units inside the Swing-net controls the movement of one leg joint. Only one coordination inﬂuence is shown in the diagram. In this case, coordination inﬂuence 1 (see Figure 1) is acting between the hind and the middle leg. While the hind leg is in swing, the posterior extreme position (PEP) of the anterior leg is shifted backwards and therefore the stance movement is prolonged (1-PEP). For further details see Schilling et al. (2013b). the case for all six legs (only two legs are depicted in Figure 3). These six “leg units” are in turn connected to a unit termed “walk” in Figure 3. This unit serves the function of arousing all units possibly required when the behavior “walk” is activated. higher levels, further internal states could be distinguished, as for example walking, standing still or feeding (for a more detailed discussion on how such a heterarchical network can be structured and learned see Cruse and Schilling, 2010). The heterarchical structure sketched in Figure 3 comprises a simple realization of neural reuse as proposed in Anderson’s massive redeployment hypothesis (Anderson, 2010) as speciﬁc procedures are used in diﬀerent behavioral contexts. In the case considered here, the motivation unit network, a recurrent neural network, can adopt diﬀerent stable states, or attractors, forming diﬀerent overlapping ensembles. For example, all “leg” units and “walk” are activated during backward walking and during forward walking, but only one of the two units termed “forward” and “backward” and only 12 of the 24 end position memories are active in either case. The network is therefore best described as forming a heterarchical structure (for details see Schilling et al., 2013a). Such an “internal state” adopted by the network protects the system to respond to inappropriate sensory input. Background and Previously Developed Models. Reactive Walker—The Walknet Biological Model of Insect Walking These coordination rules inﬂuence the length of the stance movement by inﬂuencing the transition from stance to swing movement, i.e., they change the value of the PEP. In Figure 2 only one connection is shown, inﬂuence # 1, which suppresses the start of a swing movement of the anterior leg during the swing movement of the posterior leg (for details see Schilling et al., 2013b). Beyond the motivation units that are directly controlling a procedural element, there are also motivation units (Figure 3, yellow circles) that are arranged to form some kind of hierarchical structure. Units which belong to the procedural nets controlling the left middle leg show positive connections to a motivation unit termed Leg_LM and this is correspondingly Frontiers in Neurorobotics \| www.frontiersin.org January 2017 \| Volume 11 \| Article 3 Frontiers in Neurorobotics \| www.frontiersin.org 4 ReaCog, a Minimal Cognitive Controller Schilling and Cruse FIGURE 2 \| Interactions between two leg controllers, left middle leg and left hind leg. This ﬁgure details the shaded area of Figure 1. The left side indicates the interaction with the environment mediated through the body. Each leg controller contains several modules: a Swing-net and a Stance-net to control swing and stance movement, respectively, each equipped with a motivation unit (depicted by yellow circles). Connections with an arrow indicate positive (“excitatory”) inﬂuences, connections ending with a T-shaped ending indicate negative (“inhibitory”) inﬂuences. On the right, one sub-module (Swing-net) is shown in more detail, as it is implemented as a neural network (numbers refer to weights). Target angles serve as an input to the neural network and are stored in the component. Each of the three neural units inside the Swing-net controls the movement of one leg joint. Only one coordination inﬂuence is shown in the diagram. In this case, coordination inﬂuence 1 (see Figure 1) is acting between the hind and the middle leg. While the hind leg is in swing, the posterior extreme position (PEP) of the anterior leg is shifted backwards and therefore the stance movement is prolonged (1-PEP). For further details see Schilling et al. (2013b). FIGURE 2 \| Interactions between two leg controllers, left middle leg and left hind leg. This ﬁgure details the shaded area of Figure 1. The left side indicates the interaction with the environment mediated through the body. Frontiers in Neurorobotics \| www.frontiersin.org Background and Previously Developed Models. Reactive Walker—The Walknet Biological Model of Insect Walking For instance, as a lower-level example, depending on whether a leg is in swing state, or in stance state, a given sensory input can be treated diﬀerently: stimulation of a speciﬁc sense organ (not depicted in Figures 2–3, but see Schilling et al., 2013b) leads to a levator reﬂex when in swing, but not during stance. In other words, the motivation unit network can be considered to act as a top-down attention controller. On The system as described so far is a slightly expanded version of the earlier Walknet that represents a typical case of an embodied controller (1st order embodiment, c.f. Metzinger, 2006, 2014): Kinematic and dynamic simulations as well as tests on robots have shown that this network can control walking at diﬀerent velocities, producing diﬀerent insect gaits including the continuous transitions between the so called wave gait, tetrapod gait and the tripod gait, negotiating curves (Kindermann, 2002), climbing over obstacles (Kindermann, 2002; Dürr et al., 2004), and over very large gaps (Bläsing, 2006), and coping with leg loss (Schilling et al., 2007). Thus, Walknet exhibits a free gait controller where the gaits emerge from a strictly decentralized architecture. Application of this January 2017 \| Volume 11 \| Article 3 5 Schilling and Cruse ReaCog, a Minimal Cognitive Controller FIGURE 3 \| The extended Walknet. Compared to the version shown in Figure 2, the ability to walk backwards has been introduced (not all details are shown here.). Each procedural element is equipped with a motivation unit (yellow circle). In addition, the coordination inﬂuences (only rule # 1 is depicted) can now be modulated by a motivation unit (yellow circle, coordination Rule # 1). Further motivation units are introduced (red connections and units) being arranged in a heterarchy—again only a fraction of the network is shown (see also Figure 2). decentralized approach allows for a dramatic simpliﬁcation of the are not explicitly given, but result from the cooperation between FIGURE 3 \| The extended Walknet. Compared to the version shown in Figure 2, the ability to walk backwards has been introduced (not all details are shown here.). Each procedural element is equipped with a motivation unit (yellow circle). In addition, the coordination inﬂuences (only rule # 1 is depicted) can now be modulated by a motivation unit (yellow circle, coordination Rule # 1). Walknet with a Body Model The control of the stance movement is a complex task which requires the coordination of multiple legs and joints. While local embodied approaches can deal with quite complex walking scenarios and disturbances (Schmitz et al., 2008), a purely embodied approach relying on the coupling through the body itself and local leg controllers has shown to become insuﬃcient in other cases (Schilling et al., 2012). For example, stick insects are able to negotiate curves which can be very tight (Dürr, 2005; Dürr and Ebeling, 2005). In the case of curve walking, the diﬀerent legs are producing quite diﬀerent movements and are taking over diﬀerent roles as there is, for example, a diﬀerentiation between inner and outer legs. To better cope with such problems, we apply an internal model of the body for the control of the stance movement (Schilling et al., 2012). The complexity of the six-legged walker is distributed in the body model into interacting submodels (see Figure 4, Schilling and Cruse, 2007). On the lowest level, each leg is represented as a detailed model of all the leg segments and connecting joints [Figure 4B, right; for details see (Schilling, 2011; Schilling et al., 2012)]. These leg models are integrated on a higher level in a model of the central body, where each leg is only represented by a vector pointing from the body segment toward the tip of the leg (Figure 4B, left; for details see Schilling and Cruse, 2012; Schilling et al., 2013a). As this network is based on the principle of pattern completion, any input vector given to the network— may it correspond to the input required for a forward model, an inverse model, or a sensor fusion model—provides an output that, after relaxation, leads to a coherent body state. This means that in any case the kinematics represent a geometrically correct body position. Next, we will explain how this body model can be integrated into the architecture of Walknet. Body models are used for three diﬀerent purposes [for a recent, comprehensive review see Morasso et al. (2015)]. First, inverse models have been applied (e.g., Wolpert and Kawato, 1998) to compute motor commands for given goal positions of an end-eﬀector. The second task concerns the ability to predict the position of the end-eﬀector when motor commands are known but not yet executed (Wolpert and Flanagan, 2001; Webb, 2004). Background and Previously Developed Models. Reactive Walker—The Walknet Biological Model of Insect Walking Further motivation units are introduced (red connections and units) being arranged in a heterarchy—again only a fraction of the network is shown (see also Figure 2). decentralized approach allows for a dramatic simpliﬁcation of the computation by exploiting the loop through the world (including the own body). For example, trajectories of swing movements are not explicitly given, but result from the cooperation between the Swing-net and the “loop through the world,” i.e., the sensor readings describing the current position of the leg joints. This January 2017 \| Volume 11 \| Article 3 Frontiers in Neurorobotics \| www.frontiersin.org 6 ReaCog, a Minimal Cognitive Controller Schilling and Cruse simple animals as insects use a high number of sensors, for example to measure joint positions or load. In order to exploit this redundancy (e.g., to improve inexact or even missing sensor data), the diﬀerent sensory inputs have to be fused which requires a body model (Makin et al, 2008). Used for visual perception, the body model, mirroring the observed movement, is strongly related to mirror systems as found in animals (Rizzolatti et al., 1996) and in humans (Rizzolatti, 2005), and might be linked to the understanding of others (Loula et al., 2005). structure allows for immediate adaptation of swing trajectories to unpredictable disturbances. Similarly, the spatio-temporal patterns of leg movement (“gaits”) are not explicitly speciﬁed but result from decentralized local coordination rules and the coupling of the legs via the substrate (see review Schilling et al., 2013b). This network has been tested in dynamic simulation (Schilling et al., 2013a,b) and applied to the robot Hector (Schneider et al., 2011; Paskarbeit et al., 2015). As will be shown in section Motor Planning: from Walknet to reaCog (Motor Planning), this modular structure is a crucial condition to allow recombination of procedural elements as required by a cognitive system. Whereas, in other approaches usually an individual model has been required for each task and each behavioral element (Wolpert and Kawato, 1998), we use one simple holistic recurrent neural network that can cope with all three tasks. The body model used copes with the at least 18◦C of freedom of the insect body (six legs of 3◦C of freedom each). Walknet with a Body Model In this case the body model is used as a forward model, for instance to overcome sensory delays. Third, even Figure 5 illustrates how the body model is integrated into the network. As depicted in this ﬁgure, the internal body model comprises an independent system, which may receive sensory input and/or motor commands. In turn, it provides sensory FIGURE 4 \| The body model. (A) illustrates how the body model (black) represents the body of the robot (gray). (B) The Mean of Multiple Computation (MMC) body model for the six-legged walker is divided into two layers. The lower layer contains six networks, each representing one leg (for details see Schilling et al., 2012). The upper layer represents the body and the six legs, which are only represented by bold vectors pointing toward the tip of each leg as shown in (B), left. On this level the leg is described with reference to the respective body segment. Both layers are connected via the shared leg vectors (marked by the double-lined vectors of the left front leg) and are implemented as recurrent neural networks. FIGURE 4 \| The body model. (A) illustrates how the body model (black) represents the body of the robot (gray). (B) The Mean of Multiple Computation (MMC) body model for the six-legged walker is divided into two layers. The lower layer contains six networks, each representing one leg (for details see Schilling et al., 2012). The upper layer represents the body and the six legs, which are only represented by bold vectors pointing toward the tip of each leg as shown in (B), left. On this level the leg is described with reference to the respective body segment. Both layers are connected via the shared leg vectors (marked by the double-lined vectors of the left front leg) and are implemented as recurrent neural networks. January 2017 \| Volume 11 \| Article 3 Frontiers in Neurorobotics \| www.frontiersin.org 7 ReaCog, a Minimal Cognitive Controller Schilling and Cruse FIGURE 5 \| The ﬁrst step to reaCog: Walknet expanded by an internal body model. Only a part of Walknet as shown in Figures 1, 2 is depicted (the left middle leg). During normal behavior, the Internal Body Model (upper left) serves perception. The body provides proprioceptive input (e.g., joint angles from the legs) that is integrated within the body model to form a coherent sensory experience. Motor Planning: from Walknet to Reacog The General Idea signals or motor commands to the reactive structure Walknet. The body model can be used for controlling the motor output of the stance behavior in complex walking scenarios. In this case it is part of the reactive controller (in Figure 5 the switch has to take position 1). Using the body model as an inverse model, movement of the legs during stance can easily be controlled by applying the passive motion paradigm (Mussa-Ivaldi et al., 1988). Like a simulated puppet, the internally simulated body is pulled by its head in the direction of desired body movement (Figure 5, sensory input). As a consequence, the stance legs of the puppet follow that movement in an appropriate way and the changes of the simulated joint angles can be used as commands to control the actual joints. Therefore, if such a body model is given, that represents the kinematical constraints of the real body, we obtain an easy solution of the inverse kinematic problem, i.e., for the question how the joints of legs standing on the ground have to be moved in concert to propel the body (for details and application for the control of curve walking see Schilling et al., 2012, 2013a). To be able to implement the faculty to plan ahead, the neuronal system has to be equipped with a representation of parts of the environment (Schilling and Cruse, 2008; Marques and Holland, 2009). As it has been argued that, as seen from the brain’s point of view, the body is the most important part of the environment (Cruse, 2003), a neural representation of the own body is the ﬁrst step to take. Later, this body model of course has to be extended to include aspects of the environment as are tools extending the body, objects to be handled or an environment to interact with, for example obstacles to be climbed over or to be circumvented. As mentioned the body model introduced in the previous section can be also used for prediction. Therefore, the body model will be applied to allow the system for being capable of planning ahead through internal simulation. The basic idea that will be detailed in this section is simple. In short, we will apply the following two-step procedure. If a problem occurs, which means that the ongoing behavior cannot be continued when using only the existing reactive controller, the behavior will be interrupted. Walknet with a Body Model With the switch in position 1, the network represents a reactive controller. If the system runs into a problem, the switch is ﬂipped from position 1 to position 2 and the motor control (double-lined arrows entering the switch on the right) is routed not to the body anymore, but instead to the body model (dashed double line). This circuit is used for internal simulation and predicts the sensory consequences of the action. The body model is now driven by the motor commands predicting the sensory consequences instead of integrating them. For further explanations see text. FIGURE 5 \| The ﬁrst step to reaCog: Walknet expanded by an internal body model. Only a part of Walknet as shown in Figures 1, 2 is depicted (the left middle leg). During normal behavior, the Internal Body Model (upper left) serves perception. The body provides proprioceptive input (e.g., joint angles from the legs) that is integrated within the body model to form a coherent sensory experience. With the switch in position 1, the network represents a reactive controller. If the system runs into a problem, the switch is ﬂipped from position 1 to position 2 and the motor control (double-lined arrows entering the switch on the right) is routed not to the body anymore, but instead to the body model (dashed double line). This circuit is used for internal simulation and predicts the sensory consequences of the action. The body model is now driven by the motor commands predicting the sensory consequences instead of integrating them. For further explanations see text. Frontiers in Neurorobotics \| www.frontiersin.org Motor Planning: from Walknet to Reacog The General Idea The system will then try to come up with new behaviors by recombining the existing procedural elements in a new way, i.e., not envisaged in the current context. A procedural element is characterized by a section of the network In the next section we will introduce a fundamental expansion termed “cognitive expansion.” The complete network, as we will argue, shows how cognitive properties can emerge from a system heavily relying on reactive structures, why we will call this network reaCog. January 2017 \| Volume 11 \| Article 3 8 ReaCog, a Minimal Cognitive Controller Schilling and Cruse that can be controlled by a motivation unit (as shown in Figure 3, red and yellow circles). The properties of the new combination will then be tested by using the internal body model instead of the real body, the former now exploiting its faculty to serve as a forward model. If the new combination turns out to be successful, it will be applied to control the behavior. If not, the system will search for another new combination. that can be controlled by a motivation unit (as shown in Figure 3, red and yellow circles). The properties of the new combination will then be tested by using the internal body model instead of the real body, the former now exploiting its faculty to serve as a forward model. If the new combination turns out to be successful, it will be applied to control the behavior. If not, the system will search for another new combination. becomes activated immediately after the motivation unit swing of the hind left leg becomes activated, i.e., before the animal would fall backwards onto the lifted leg. g If a problem has been detected by any detector the system must (i) interrupt the ongoing behavior and (ii) be able to change from the state “perform behavior” to the state “simulate behavior.” To this end, we have introduced a switch as shown in Figure 5. By moving the switch from position 1 to position 2, the output of the leg controller—which is normally (position 1 of the switch) routed to the motor output to inﬂuence the body— is now instead routed directly to the body model. Motor Planning: from Walknet to Reacog The General Idea Thereby the position of the real body is kept ﬁxed, i.e., the ongoing behavior is interrupted (Hesslow, 2002) is providing a biological account for this decoupling which has also been found in insects (Bläsing and Cruse, 2004), but the internal body model can perform the movements determined by the reactive controller. As in the case of actively moving the body, the output signals of the body model, in particular the angular values describing the position of the leg joints, are given to the reactive procedures. In this way the loop is closed and the system can internally simulate the behavior by moving the body model instead of the real body. Note that modules of the reactive procedures as are Swing-net and Stance- net are still active as is the case in Walknet. 2.2.3 Coming up with a new solution. For better illustration, we will use the following example: Imagine the case that one—say the left hind leg—has been moved far to the rear and now receives the signal to start a swing movement, i.e., to lift the leg oﬀthe ground. If the two neighboring legs—the left middle leg and the other, right, hind leg—accidentally are positioned far to the front, lifting the left hind leg might lead the body falling to the rear (Figures 6A,B). Interruption of Behavior To avoid tumbling over backwards, the system must be able to detect that it is running into trouble. Therefore, one or several systems are necessary that are able to detect that there is a problem. While there are diﬀerent biologically plausible solutions (e.g., using load sensors as found in the insects), we chose as a simple approach a stability sensor which is activated in case the leg would be lifted,. In the example scenario this detector FIGURE 6 \| A problem and a possible solution. (A) shows a posture in which the animal would fall over when trying to lift the left hind leg (dashed red arrow), because the anterior, middle, leg and the other hind leg are too far to the front. The result is depicted in (B). However, the problem detector detects a problem before the left hind leg is actually lifted, the cognitive system should start searching for a solution through mental simulation (C). The system might come up with the idea to perform a backward swing with its left middle leg and afterwards proceed walking. After successful testing in simulation (C), the plan can be executed in reality (D), i.e., ﬁrst swinging the middle leg backwards and then swinging the hind leg to the front while continuing normal walking. FIGURE 6 \| A problem and a possible solution. (A) shows a posture in which the animal would fall over when trying to lift the left hind leg (dashed red arrow), because the anterior, middle, leg and the other hind leg are too far to the front. The result is depicted in (B). However, the problem detector detects a problem before the left hind leg is actually lifted, the cognitive system should start searching for a solution through mental simulation (C). The system might come up with the idea to perform a backward swing with its left middle leg and afterwards proceed walking. After successful testing in simulation (C), the plan can be executed in reality (D), i.e., ﬁrst swinging the middle leg backwards and then swinging the hind leg to the front while continuing normal walking. January 2017 \| Volume 11 \| Article 3 Frontiers in Neurorobotics \| www.frontiersin.org 9 ReaCog, a Minimal Cognitive Controller Schilling and Cruse simpliﬁed version of the network as presented in Figure 5. Interruption of Behavior The expansion depicted at the right side enables the system ﬁnding “new solutions” and then testing their qualiﬁcation to solve the problem. This expansion—that we will call “cognitive expansion” or, as will be motivated in Section Discussion and Conclusions), “attention system”—contains three additional layers, a spreading activation layer (SAL, red circles), a winner-take-all layer (WTA, green circles) and a remember-tested-behavior (RTB, blue circles) layer with identical number of units each. In addition, there is a small network termed Global Phases (lower part of Figure 7). This switch given, it appears of course not very interesting to simulate exactly the behavior which has just led to the problem. Instead, it is necessary to test new, currently not available solutions. Therefore, the signal from the problem detectors is not only used to move the switch, but also to start the search for a new solution. To allow for this faculty, reaCog requires a further fundamental expansion. The main idea is that for internal simulation a new behavioral element has to be selected. This new behavioral element may be selected also from procedures not belonging to the current context. How is this solved by reaCog? In Figure 7, the upper, left part (i.e., without SAL net, WTA net, and RTB net) shows a p g At the bottom, Global Phases, the structure is illustrated that organizes the temporal sequence of ﬁnding a behavior as FIGURE 7 \| ReaCog: Walknet plus cognitive expansion. This ﬁgure shows an extension of the Walknet structure presented in Figure 5. The motivation unit structure (yellow, e.g., Swing, Swing_toFront) is replicated on the right side, termed attention system, in three ways. There is a Spreading-Activation-Layer (SAL, red circles), the WTA layer (green circles), and the remember-tested-behavior (RTB units, blue circles) layer. The problem detector (red and yellow, the latter for the internal model) not only activates the switch, but also the spreading activation layer (SAL; red arrows) The activated spreading activation layer units activate their partner units in the WTA network. The winner of the WTA is activating the corresponding motivation unit (dashed black arrows) and the corresponding motor program will be carried out using internal simulation. Note that the connections within the WTA layer are not completely depicted. FIGURE 7 \| ReaCog: Walknet plus cognitive expansion. This ﬁgure shows an extension of the Walknet structure presented in Figure 5. Cognitive Expansion In the following we will explain the function of the cognitive expansion as depicted in Figure 7. The goal of the cognitive expansion network is to search for a new procedural element that allows for a solution of the current problem. The ﬁrst step is to look for behavioral elements existing in the memory, which are, however, not activated in the current context. As will be explained, only such procedural elements can be selected that can be activated by a motivation unit. Second, the possible contribution of this additional memory element will be tested by internal simulation. What is the functional role of these three additional layers forming an expansion that we will call “cognitive expansion” or, as will be motivated later in the discussion (Section Discussion and Conclusions), “attention system”? Assume that in our example (Figure 5) the problem detector situated in the left hind leg has been activated (Figure 7, bold red arrow, starting at the left). As noted earlier, this signal moves the switch from position 1 to position 2 to route the motor output to the body model instead of the body itself. Thereby the ongoing behavior is interrupted. In addition this signal activates one (or several) neighboring units of the Spreading Activation layer. Figure 8 illustrates the sequential activation of WTA layer, and RTB layer. How is this done? The units of the SAL (Figure 7, red circles) receive input from morphologically neighboring problem detectors (in Figure 7, one example is depicted by a bold, red circle). Neighboring units are connected by positive weights. In this way, an activation arising from a problem detector is spread over the SAL roughly corresponding to a circular wave starting at the position of the unit excited by the problem detector. Further, there is noise added to the units of the spreading activation layer. The middle layer is representing a winner-take- all network. The units of the WTA layer (Figure 7, green circles) are activated by the corresponding partner units in the SAL layer. In addition, already active behavioral elements, i.e., their active motivation units, are inhibiting their counterparts in the WTA-layer (Figure 7, black solid line with T-shaped end). In this way, currently active behaviors are prevented from being selected for testing in internal simulation. Through the winner-take-all process the units are inhibiting each other in a way that only one unit remains active when the network settles. Cognitive Expansion For the third, the right hand layer, there is again a one-to-one connection to the WTA-layer. These RTB units (Figure 7, blue circles) store which of the WTA units have already been tested in an earlier internal simulation run. The winning WTA unit activates its motivation unit and as a consequence, the corresponding—new—procedural element. After the WTA net has made its decision and has activated the motivation unit of a procedure normally not used in the actual context, simulation using the internal body model will be started to test the contribution of this new procedure. Note that therefore a problem detector is also required inside the internal model which functions in the same way, i.e., it observes static stability of the (internally simulated) body (Figure 7, bold yellow arrows). If during the internal simulation no problem detector becomes active, the procedure appears to be a suitable solution for the given problem. Thus, the solution is found following a search The winning WTA unit activates its motivation unit and as a consequence, the corresponding—new—procedural element. After the WTA net has made its decision and has activated the motivation unit of a procedure normally not used in the actual context, simulation using the internal body model will be started to test the contribution of this new procedure. Note that therefore a problem detector is also required inside the internal model which functions in the same way, i.e., it observes static stability of the (internally simulated) body (Figure 7, bold yellow arrows). If during the internal simulation no problem detector becomes active, the procedure appears to be a suitable solution for the given problem. Thus, the solution is found following a search FIGURE 8 \| Illustration of the sequential changes of activation of SAL, WTA, and RTB units. When a problem occurs, the problem detector, on the one hand stops the execution of current behavior (not shown). On the other hand, it induces activity in the spreading activation layer (SAL, red) which indicates where the problem occurred. The activation is spreading vertically in the SAL. Each SAL unit excites its corresponding WTA unit. Importantly, currently active motivation units (yellow) inhibit the WTA units (green units). The WTA units compete among each other producing one winning unit which in turn activates the corresponding motivation unit and behavior. Interruption of Behavior The motivation unit structure (yellow, e.g., Swing, Swing_toFront) is replicated on the right side, termed attention system, in three ways. There is a Spreading-Activation-Layer (SAL, red circles), the WTA layer (green circles), and the remember-tested-behavior (RTB units, blue circles) layer. The problem detector (red and yellow, the latter for the internal model) not only activates the switch, but also the spreading activation layer (SAL; red arrows) The activated spreading activation layer units activate their partner units in the WTA network. The winner of the WTA is activating the corresponding motivation unit (dashed black arrows) and the corresponding motor program will be carried out using internal simulation. Note that the connections within the WTA layer are not completely depicted. January 2017 \| Volume 11 \| Article 3 Frontiers in Neurorobotics \| www.frontiersin.org Frontiers in Neurorobotics \| www.frontiersin.org 10 ReaCog, a Minimal Cognitive Controller Schilling and Cruse The diﬀerent procedural elements of Walknet and their motivation units are anatomically arranged in a way that this arrangement coarsely reﬂects the morphological ordering of the legs (Figure 1, left). Consequently, the motivation units of neighboring legs as well as the partner units of the Spreading Activation layer (SAL) and of the winner-take-all (WTA) layer are neighboring, too, and thus form some kind of somatotopical map. Thus, the problem detector is not only signaling the problem, but in addition also carries some information where the problem occurred. In this way, the search for a new behavior is not purely random, but follows some heuristics,—there is some probability that a solution may be found morphologically near the cause of the problem—which may accelerate the searching process. a solution to a novel problem. Additional units (gray circles) show temporal properties and are used to organize the switching between stages as explained in the text. Units “count” represent a speciﬁc time delay. Cognitive Expansion The units in the RTB layer (blue) represent which behavior has been active once during the simulation process and will inhibit a future activation during the WTA selection process. FIGURE 8 \| Illustration of the sequential changes of activation of SAL, WTA, and RTB units. W FIGURE 8 \| Illustration of the sequential changes of activation of SAL, WTA, and RTB units. When a problem occurs, the problem detector, on the one hand stops the execution of current behavior (not shown). On the other hand, it induces activity in the spreading activation layer (SAL, red) which indicates where the problem occurred. The activation is spreading vertically in the SAL. Each SAL unit excites its corresponding WTA unit. Importantly, currently active motivation units (yellow) inhibit the WTA units (green units). The WTA units compete among each other producing one winning unit which in turn activates the corresponding motivation unit and behavior. The units in the RTB layer (blue) represent which behavior has been active once during the simulation process and will inhibit a future activation during the WTA selection process. FIGURE 8 \| Illustration of the sequential changes of activation of SAL, WTA, and RTB units. When a problem occurs, the problem detector, on the one hand stops the execution of current behavior (not shown). On the other hand, it induces activity in the spreading activation layer (SAL, red) which indicates where the problem occurred. The activation is spreading vertically in the SAL. Each SAL unit excites its corresponding WTA unit. Importantly, currently active motivation units (yellow) inhibit the WTA units (green units). The WTA units compete among each other producing one winning unit which in turn activates the corresponding motivation unit and behavior. The units in the RTB layer (blue) represent which behavior has been active once during the simulation process and will inhibit a future activation during the WTA selection process. GURE 8 \| Illustration of the sequential changes of activation of SAL, WTA, and RTB units. When a problem occurs, the pro January 2017 \| Volume 11 \| Article 3 11 Frontiers in Neurorobotics \| www.frontiersin.org ReaCog, a Minimal Cognitive Controller Schilling and Cruse driven by a heuristic including noise (given to the SAL units). As a next step, this solution is tested for being mechanically appropriate. In this case the switch is set back to position 1 and the corresponding behavior will then be applied in reality. Cognitive Expansion By setting back the switch the real body will provide the sensory input. As the winning WTA unit is still active (thus representing a short term memory), the newly selected procedure will be executed. If, however, already during the internal simulation this “new solution” has proven not to be a solution—deﬁned by a problem detector of the internal model becoming active—the search for a solution will be continued further. To this end, the internal model will be reset to the current state of the body. Then, the SAL net will continue the spreading of its activations and a new behavior will be selected by the WTA-net. In this way the procedure will be repeated until a solution is found. simulation (we use 400 iterations which equals 4 s or about three to four step cycles), no problem occurred, the motivation unit “Test” will be activated instead to start the real behavior. If during the test of the real behavior the problem occurs again or a new problem is detected (in contrast to the situation during simulation), the behavior is inhibited and the “SAL” motivation unit is activated again. If however the behavioral test is successful, too, the motivation unit “Beh” is activated (and the motivation unit “Test” inhibited) to allow continuation of the normal behavior. In contrast, if during simulation a problem is detected, the simulation is interrupted (motivation unit “SIM” is inhibited) and instead the motivation unit “SAL” is excited again to search for a new “idea.” The temporal order of activation of the diﬀerent motivation units of the Global Phases network is controlled by dedicated connections running in parallel to the mutual inhibitory connections (Figure 7, gray) of all these units, When the new solution is tested in reality, there are still two possibilities to be considered. If the realization of the proposed solution is successful, behavior continues. However, the solution may also turn out not to be realizable. This might for example happen because the body model does not simulate the physical properties of the body (and the environment) well enough. In this case a—possibly diﬀerent—problem detector will be activated by starting again a new search procedure, with the internal body model being reset to the current real state of the body as given through the sensors. Importantly, each internal simulation has to start from the real situation, i.e., the situation that led to the problem. Cognitive Expansion Therefore, the internal body model as well as the control system have to be reset to this state before a new internal simulation is started. This reset is triggered during the “SAL” stage. As the body did not actively move during internal simulation, the current posture and sensor readings can be used to reset the internal body model. It takes the reactive part of the control system only a couple (one or two) iterations to converge to the original state. It turned out that the internal state does not have to be stored explicitly. In the remainder of this section, the structure that controls the temporal sequences sketched above is explained in detail. As indicated in the lower part of Figure 7, the complete procedure is controlled by ﬁve speciﬁc motivation units, Beh, SAL, WTA, SIM, and Test forming the center of the Global Phases network. These units are coupled via mutual inhibition (not depicted in Figure 7) and in part by transient, i.e., high-pass like, units (Figure 7, gray units and connections in the lower part). The complete procedure controlled by the Global Phase network corresponds to what has been termed “incubation” and “veriﬁcation” (Helie and Sun, 2010), and is similar to the “note- assess-guide” strategy or “metacognitive loop” as introduced by Anderson et al. (2006). In a mathematical analysis applied for example to logic reasoning systems the latter authors could show that introduction of such a strategy indeed improves the behavior of the complete system. The complete period, during which the body is ﬁxed and the body model is used for internal simulation, may correspond to what Redish (2016), referring to Tolman, has termed “vicarious trial an error.” During normal, i.e., reactively controlled walking the motivation unit “Beh” is active, thereby inhibiting the other four motivation units. If a problem is detected, the problem detector is activated which in turn inhibits the ongoing behavior (motivation unit “Beh”) and activates the “SAL” motivation unit. In addition, the switch is moved to bypass the physical body (the switch might be realized by further mutually coupled motivation units, not shown in Figure 7) and the current forward movement of the robot is inhibited for some time that corresponds to duration of about one step of the leg (i.e., 100 iterations). This allows suﬃcient time to test movements before starting to continue forward walking. Cognitive Expansion After a given time required for sensible spreading of activations (e.g., two iterations, triggered by the “Delay” unit shown in gray in Figure 7), the SAL motivation unit is inhibited and the WTA motivation unit is activated instead. The relaxation of the WTA net may require a variable number of iterations. A simple solution is to introduce one unit observing the convergence of the WTA- network (“Relax”). This unit is activated as soon as the ﬁrst unit of the WTA layer has reached a given threshold, representing the winning unit. Frontiers in Neurorobotics \| www.frontiersin.org Simulation Results for the Example Scenario In this section, we will show a dynamic simulation of the reaCog system. The example illustrates the faculty of reaCog to ﬁnd new solutions to a current problem using its capabilities for planning ahead. (In this study there is no physical robot used yet, but it is represented by a dynamic simulation.). Figure 6 shows an awkward posture. This conﬁguration can become problematic as the left hind leg is already very far to the back and cannot move further back. Therefore, in this situation the left hind leg has to produce a swing movement. If the position of the left middle leg and right hind leg are positioned very far to the front, lifting the left hind leg can lead to instability, because the center of mass is placed quite far to the rear, between the hind legs. A sensible solution in our paradigm (Figure 6) might be the activation of the Swing_toBack module of the left middle leg: A backward step Only after a winner is detected (“Relax” in Figure 7), the “WTA” motivation unit is inhibited and the simulation is started (motivation unit “SIM”). If, after a given time of internal Frontiers in Neurorobotics \| www.frontiersin.org January 2017 \| Volume 11 \| Article 3 12 ReaCog, a Minimal Cognitive Controller Schilling and Cruse of the anterior middle leg might allow this leg to take over the body weight, and—as a consequence—afterwards allow lifting of the left hind leg. Thereby, continuation of walking may become possible. (middle panel) shows a footfall pattern which illustrates the swing movements of the legs over time. A leg which is in swing phase is marked as a black (or red) bar. For the medium velocity chosen a gait is emerging that can be seen in the stepping pattern in the left part of the ﬁgure. From a tripod-like starting posture the robot converges more toward a fast tetrapod-like gait (at about 500 iterations). The lower part of Figure 9 shows still images of the dynamic simulation (see Supplementary Material Videos 1, 2), whereas the upper part provides a top view of the robots’ (or internal models’) conﬁguration. The upper part shows four speciﬁc snapshots of the posture of the walker (top view) facing to the right. Only legs in stance phase, i.e., legs which support stability are depicted. Simulation Results for the Example Scenario In normal walking the reactive part of the controller is not ending up in such a strange posture. Therefore, we had to introduce an external disturbance to make the system tumble. To this end, the placements of the left middle leg and right hind leg will be changed in a way that during swing movement the target position is pushed further to the front (by a third of a step length). Such a strong change might occur in insects when climbing over irregular ground. When there is no foothold, the insects are starting searching movements to the anterior in order to ﬁnd a foothold (Dürr and Krause, 2001; Bläsing and Cruse, 2004; Schütz and Dürr, 2011) which can be quite far to the front. This does not pose a problem for the stick insect as stability is strongly supported through the ability to attach the feet to the ground. As the robot cannot use this method, he has to ﬁnd another solution (for example the one sketched in Figure 6). For the same run, Figure 10 illustrates the position of each leg over time. The position is plotted on the ordinate showing the movement of the leg (green lines, swing movements during forward walking are pointing upwards; stance movements are going into the opposite direction). The jumps in the position of the legs are due to the switching from the real robot to the internal model required to reset the internal model. Colors are used as in Figure 9. For further explanations see text. In the following, with help from Figures 9, 10, we will explain how the system deals with this intervention. Figure 9 FIGURE 9 \| Solving the problem illustrated in Figure 6: Foot fall patterns. The middle panel shows the footfall pattern of the hexapod over time (black/red bars indicate swing movement of the leg). The upper panel shows some critical conﬁgurations of the robot (or, during internal simulation, the conﬁguration of the internal model). The robot is walking from left to right. In three cases, the left hind leg is shown as a dashed line indicating that it is supposed to start a swing movement. The lower panel illustrates the behavior by screen shots taken from the Supplementary Material Videos 1, 2. The robot starts with a tripod-like leg conﬁguration and converges to a fast tetrapod gait. The problem is detected at (#2). Simulation Results for the Example Scenario The section highlighted red represents an unsuccessful internal simulation [ending in an unstable conﬁguration again as shown in (#3)]. The second internal simulation, highlighted green [starting at (#3)], turns out to be successful and solves the problem (backswing of the left middle leg, depicted by red bars, (#4) shows the new posture before the start of the forward swing movement of the left hind leg). Highlighted blue is the application of this solution to the robot (starting at (#5) which shows the robot posture at the beginning of the backward swing movement of the left middle leg). This ﬁnal test is successful, too, and the robot continues to walk (N indicates center of mass). FIGURE 9 \| Solving the problem illustrated in Figure 6: Foot fall patterns. The middle panel shows the footfall pattern of the hexapod over time (black/red bars indicate swing movement of the leg). The upper panel shows some critical conﬁgurations of the robot (or, during internal simulation, the conﬁguration of the internal model). The robot is walking from left to right. In three cases, the left hind leg is shown as a dashed line indicating that it is supposed to start a swing movement. The lower panel illustrates the behavior by screen shots taken from the Supplementary Material Videos 1, 2. The robot starts with a tripod-like leg conﬁguration and converges to a fast tetrapod gait. The problem is detected at (#2). The section highlighted red represents an unsuccessful internal simulation [ending in an unstable conﬁguration again as shown in (#3)]. The second internal simulation, highlighted green [starting at (#3)], turns out to be successful and solves the problem (backswing of the left middle leg, depicted by red bars, (#4) shows the new posture before the start of the forward swing movement of the left hind leg). Highlighted blue is the application of this solution to the robot (starting at (#5) which shows the robot posture at the beginning of the backward swing movement of the left middle leg). This ﬁnal test is successful, too, and the robot continues to walk (N indicates center of mass). January 2017 \| Volume 11 \| Article 3 Frontiers in Neurorobotics \| www.frontiersin.org 13 ReaCog, a Minimal Cognitive Controller Schilling and Cruse FIGURE 10 \| Solving the problem: Position of the individual legs over time. Simulation Results for the Example Scenario Shortly after the left middle and right hind leg performed swing movements that point very far to the front of the working range (#1), the walker becomes unstable (#2) when trying to lift the left hind leg. Therefore, internal simulations are started (highlighted in green and red) during which motor commands are routed to the internal body model, the leg positions of which are shown. First (highlighted red), an unsuccessful behavior is tested: a stance movement which has initially no effect as the agent is stopped. But when the agent accelerates again (after 100 iterations) the problem is still present and the agent becomes instable (#3). As a second trial, a backward swing movement of the middle left leg is tested via internal simulation (green highlighted area; the swing movement in the unusual direction is plotted in red). Afterwards (#5) the solution found is tested on the real robot (highlighted in blue) showing that walking continues successfully. As mentioned, we forced the robot into an awkward posture in such a way that the swing movement of the left middle and right hind leg moved very far to the front of their working range, i.e., beyond their normal AEP. Next, the left hind leg marked by a dashed line in Figure 9 is supposed to start a swing movement. The center of mass would then not be supported anymore by the left middle leg and right hind leg [Figures 9, 10 (2), after 580 iterations]. Therefore, the system would tumble backwards. As a consequence, a second iteration of the cognitive expansion is invoked (this section is highlighted green, as it turns out to be successful): First, activation is further spread in the SAL layer. Second, a behavior is selected in the WTA layer which has not yet been tested. And third, the behavior is applied as internal simulation. For this second internal simulation, the internal body model and control system have to be reset initially. To this end, it turned out to be suﬃcient to update, ﬁrst, the internal model with the values from the real robot structures (this is the starting condition required for the internal simulations; see Figure 10, at the border of the red and green section, the position of the leg in the internal model jumps back to the original position of the robot leg). Simulation Results for the Example Scenario Green lines show the position of each leg over time—positive values are toward the front of the walker. Ordinate is given in cm with the origin ﬁxed to the COM of the robot. The blue dashed lines indicate the average extreme positions: The Anterior Extreme Position (AEP) is the target position for the swing movement and is ﬁxed during forward walking. The Posterior Extreme Position (PEP) indicates the position at which a leg controller initiates a swing movement on average and switches from stance to swing (note that the coordination rules act on the PEP and shift the PEP forward or rearward to organize the overall behavior which is not shown in the ﬁgure). Shortly after the left middle and right hind leg performed swing movements that point very far to the front of the working range (#1), the walker becomes unstable (#2) when trying to lift the left hind leg. Therefore, internal simulations are started (highlighted in green and red) during which motor commands are routed to the internal body model, the leg positions of which are shown. First (highlighted red), an unsuccessful behavior is tested: a stance movement which has initially no effect as the agent is stopped. But when the agent accelerates again (after 100 iterations) the problem is still present and the agent becomes instable (#3). As a second trial, a backward swing movement of the middle left leg is tested via internal simulation (green highlighted area; the swing movement in the unusual direction is plotted in red). Afterwards (#5) the solution found is tested on the real robot (highlighted in blue) showing that walking continues successfully. FIGURE 10 \| Solving the problem: Position of the individual legs over time. Green lines show the position of each leg over time—positive values are toward the front of the walker. Ordinate is given in cm with the origin ﬁxed to the COM of the robot. The blue dashed lines indicate the average extreme positions: The Anterior Extreme Position (AEP) is the target position for the swing movement and is ﬁxed during forward walking. The Posterior Extreme Position (PEP) indicates the position at which a leg controller initiates a swing movement on average and switches from stance to swing (note that the coordination rules act on the PEP and shift the PEP forward or rearward to organize the overall behavior which is not shown in the ﬁgure). Frontiers in Neurorobotics \| www.frontiersin.org Simulation Results for the Example Scenario But in the case of the robot (highlighted blue), a small passive movement would be suﬃcient to initiate a swing movement. Nonetheless, as can be seen from the footfall pattern, the application on the robot is also successful and the system converges to a stable gait pattern. This stresses the robustness of the underlying control approach and highlights how important it is that planning and control are tightly intertwined. In the blue area and beyond, Figure 10 shows the movements of the leg of the real robot. Immediately after the new behavior has been induced, one can observe how the phases of the individual leg controllers are rearranged. For example, the right front leg is forced to make an early swing movement after the right middle leg has ﬁnished its swing movement (see Schilling et al., 2013b). But already after a very short time, a single step of the robot, a stable tetrapod-like gait emerges (as can be seen in Figure 9). As a more severe disturbance, we performed a series of simulations in which two legs were targeted. Again, after a randomly chosen point in time (during the ﬁrst 10 s of walking) two legs are selected randomly for which the next swing movement is shifted to the front (about 5 cm which equals a third of a complete step length). We performed 100 simulation runs with all kind of combinations between legs multiple times. As already discussed for the example shown above (Section Simulation Results for the Example Scenario), in this case the reactive biologically inspired control system may run into unstable situations that require to stop the walking behavior to avoid that the robot would topple over. In the following we provide results on for how many cases the system struggled with stability and how the cognitive expansion was able to deal with those situations. Overall, there are eight instable situations which were caused by a disturbance of a middle and the diagonal hind leg (a case as described in detail above, Section Simulation Results for the Example Scenario). For these eight simulation runs the cognitive expansion had to take over and has found a solution in all instances. The system always became instable when the other (non-disturbed) hind leg tried to initiate a swing movement. Interestingly, diﬀerent solutions have been found. Simulation Results for the Example Scenario As a consequence, insects perform searching movements that may shift the end position of the swing movements further to the front. As a ﬁrst series of simulations, after a randomly chosen point in time (during the ﬁrst 10 s of walking) one leg is selected randomly for which the next swing movement is shifted to the front (about 5 cm which equals a third of a complete step length). In this way, diﬀerent legs are aﬀected in diﬀerent walking situations. We ran 100 diﬀerent simulations, therefore each leg was targeted multiple times and in the diﬀerent stages of the 10 s of walking. As a result, when only one leg is targeted the reactive control system showed to be suﬃcient and the walker never got unstable independent of which leg was shifted. For all simulations, walking continued for at least 5 more seconds after the disturbance. In most cases, already after one subsequent step the control system has established again a stable walking pattern. Only for an early change in a front leg this requires two stepping cycles. Stability is accomplished mainly through compensating the leg shift. While the shifting of the target position would prolong the next step for the respective leg, the local coordination inﬂuences force the leg into an earlier lift- oﬀin order to compensate. Detailed results are provided as Supplemental Data 1 in Supplementary Material which show for each of the diﬀerent legs (front, middle, and hind leg) a single run as an example. As can also be seen in the data, the walking pattern emerges quite early in the ﬁrst or the second step. g g When the internal simulation was successful the behavior selected (which is still stored in the WTA layer) will be applied on the (simulated) physical system (see #5 and blue area in Figures 9, 10). This part is still regarded as a test of the behavior. This test is necessary because internal simulation and robot can of course lead to slightly diﬀerent results which over time might accumulate. For example, in Figure 9 the behavior of the right middle leg diﬀers between internal simulation and testing the behavior on the robot. The right middle leg is very close to its posterior extreme position and on the verge of starting a swing movement. In both cases, the robot is standing still and not supposed to move further forward. Simulation Results for the Example Scenario Second, as the control system is behavior-based it depends on the sensor state represented by the current position of the robot. This state can be enforced onto the control system so that the system converges back to its behavioral state. As a consequence, the problem detector is activated, which stops the overall movement of the robot and triggers the cognitive expansion which then starts motor planning. In the example shown in Figures 9, 10 the robot ﬁrst selects a stance movement in the left hind leg (due to the somatotopical neighborhood, see Figure 7, in SAL layer). This stance movement is then applied in internal simulation. As a result, an unsuccessful internal simulation can be observed (highlighted in red) (2)–(3), which is interrupted when the left hind leg should be lifted, because this action would again lead to an instable conﬁguration of the internal body model [see upper panel, (3)]. Note that during the time highlighted in red (and green, see below) the robot is not moving. Only the internal model is used to provide predictions of the movements. In the simulation run shown, the behavior selected next is a backward swing movement of the left middle leg (depicted in Figure 10 by a red line for the position of the left middle leg; correspondingly, in Figure 9 the swing movement backwards is shown as a red bar). As illustrated in the parts highlighted in green, again the forward movement of the body is interrupted for January 2017 \| Volume 11 \| Article 3 14 ReaCog, a Minimal Cognitive Controller Schilling and Cruse some time. During this time the newly selected behavior is tested by internal simulation. When the system starts to accelerate again, the left middle leg now being placed further to the rear helps to support the robot. When the left hind leg starts to swing, the left middle leg is ready to take over and to support the body (shown in Figure 9 in the upper panel in the body posture at #4 at around 800 iterations). The internal simulation runs further for a given time (here we used additional 300 iterations) in order to guarantee that normal walking can be continued. disturbance as the insects are often climbing through twigs that do not provide many footholds. Frontiers in Neurorobotics \| www.frontiersin.org Simulation Results for the Example Scenario On the one hand, a rearrangement of the legs could be observed in a way that one leg was moved backwards and unload the non-disturbed hind leg which afterwards was able to initiate a swing movement. This was accomplished either through moving backwards the anterior middle leg or the contra lateral hind leg. On the other hand, we observed two cases in which the slowing down of the walking speed of the complete system was already suﬃcient to solve the problem as during the slowing down a swing movement The example illustrates the faculty of reaCog to activate behavioral elements out of context in order to ﬁnd a solution to a current problem. As illustrated, the system (reaCog plus robot) manifests an impressive stable behavior, although various deviations from normal walking behavior can be observed during the complete process. Models of Cognitive Systems Models of cognitive systems generally address selected aspects of cognition and often focus on speciﬁc ﬁndings from cognitive experiments (e.g., with respect to memory, attention, spatial imagery; review see Langley et al. (2009), Wintermute (2012). Duch et al. (2008) introduced a distinction between diﬀerent cognitive architectures. First, these authors identiﬁed symbolic approaches. As an example, the original SOAR (State, Operator, and Result; Laird, 2008) has to be noted, a rule-based system in which knowledge is encoded in production rules that allow to state information or derive new knowledge through application of the rules. Second, emergent approaches follow a general bottom-up approach and often start from a connectionist representation. As one example, following a bottom-up approach, Verschure et al. (2003) introduced the DAC (Distributed Adaptive Control) series of robot architectures (Verschure et al., 2003; Verschure and Althaus, 2003). These authors initiated a sequence of experiments in simulation and in real implementation. Verschure started from a reﬂex-like system and introduced higher levels of control on top of the existing ones which modulated the lower levels and which were subsequently in charge on longer timespans (also introducing memory into the system) and were integrating additional sensory information. The experiments showed that the robots became more adapted to their environment exploiting visual cues for orienting and navigation etc., (Verschure et al., 2003). Many other approaches in emergent systems concentrate on perception, for example, the Neurally Organized Mobile Adaptive Device (NOMAD) which is based on Edelman (1993) Neural Darwinism approach and demonstrates pattern recognition in a mobile robot platform (Krichmar and Snook, 2002). Recently, this has gained broader support in the area of autonomous mental development (Weng et al., 2001) and has established the ﬁeld of developmental robotics (Cangelosi and Schlesinger, 2015). A particular focus in g Many models of cognition take, quite in contrast to our approach, as a starting point the anatomy of the human brain. A prominent example is the GNOSIS project (Taylor and Zwaan, 2009). It deals with comparatively ﬁne-grained assumptions on functional properties of brain modules, relying on imaging studies as well as on speciﬁc neurophysiological data. While GNOSIS concentrates mainly on perceptual, in particular visual input, the motor aspect is somewhat underrepresented. GNOSIS shows the ability to ﬁnd new solutions to a problem, including the introduction of intermediate goals. Simulation Series on Disturbed Walking g For a more quantitative evaluation of the performance of the reaCog architecture we performed two additional series of simulations to illustrate the contributions of the diﬀerent parts of the system. On the one hand, there is the underlying reactive and biological inspired control system (based on Walknet Schilling et al., 2013a). On the other hand, when running into stability problems the cognitive expansion has been introduced which can take over in order to reconﬁgure the posture in a way that allows to continue stable walking. Following the approach presented above in detail, we again used the repositioning of a leg during swing movement which means that, for a selected swing movement, the target position is shifted to the front. This represents a quite natural example Frontiers in Neurorobotics \| www.frontiersin.org January 2017 \| Volume 11 \| Article 3 15 ReaCog, a Minimal Cognitive Controller Schilling and Cruse such architectures concerning learning is currently not covered in reaCog. In general, as pointed out by Langley et al. (2009), these kinds of approaches have not yet demonstrated the broad functionality associated with cognitive architectures (and—as in addition mentioned by Duch et al. (2008)—many of such models are not realized and are often not detailed enough to be implemented as a cognitive system). ReaCog realizes such an emergent system but with focus on a complex behaving system that, in particular, aims at higher cognitive abilities currently not reached by such emergent systems. The third type concerns hybrid approaches which try to bring together the advantages of the other two paradigms, for example ACT-R (Adaptive Components of Thought-Rational, Anderson, 2003). The, in our view, most impressive and comprehensive model of such a cognitive system is presented by the CLARION system (review see Sun et al., 2005; Helie and Sun, 2010) being applied to creative problem solving. This system is detailed enough so that it can be implemented computationally. Applying the so called Explicit- Implicit Interaction (EII) theory and being implemented in the CLARION framework, this system can deal with a number of quantitatively and qualitatively known human data, by far more than can be simulated by our approach as reaCog, in contrast, does not deal with symbolic/verbal information. Apart from this aspect, the basic diﬀerence is that the EII/CLARION system comprises a hybrid system as it consists of two modules, the explicit knowledge module and the implicit knowledge module. Simulation Series on Disturbed Walking Applying the so called Explicit- Implicit Interaction (EII) theory and being implemented in the CLARION framework, this system can deal with a number of quantitatively and qualitatively known human data, by far more than can be simulated by our approach as reaCog, in contrast, does not deal with symbolic/verbal information. Apart from this aspect, the basic diﬀerence is that the EII/CLARION system comprises a hybrid system as it consists of two modules, the explicit knowledge module and the implicit knowledge module. Whereas, the latter contains knowledge that is not “consciously accessible” in principle, the explicit network contains knowledge that may be accessible. Information may be redundantly stored in both subsystems. Mutual coupling between both modules allows for mutual support when looking for a solution to a problem. In our approach, instead of using representational diﬀerences for implicit and explicit knowledge to cope with the diﬀerent accessibility, we use only one type of representation, that, however, can be diﬀerently activated, either being in the reactive mode or in the “attended” mode. In our case, the localist information (motivational units) and the distributed information (procedural networks) are not separated into two modules, but form a common, decentralized structure. In this way, the reaCog system realizes the idea of recruitment as the same clusters are used in motor tasks and cognitive tasks. Whereas, we need an explicit attention system, as given in the spreading activation and winner-take-all layer, in the CLARION model decisions result from the recurrent network ﬁnding an attractor state. These results show that the cognitive expansion is able to ﬁnd diﬀerent suitable solutions. Note, that the solution disrupts the coordination pattern of all the legs. Only together with the reactive system and the coordination rules the system is able to select a movement which enables stable ongoing walking. In some instances the system discarded solutions which we, on a ﬁrst guess, would have assessed as possible solutions, but which later-on run into conﬂicts. RELATED WORK In this section, we will compare reaCog as a system with related recent approaches in order to point out diﬀerences. While there are many approaches toward cognitive systems and many proposals concerning cognitive architectures, we will concentrate on models that, like reaCog, consider a whole systems approach. First, we will deal with cognitive architectures in general. Second, we will brieﬂy present relevant literature concerning comparable approaches in robotics, because a crucial property of reaCog is that it uses an embodied control structure to run a robot. Frontiers in Neurorobotics \| www.frontiersin.org Simulation Series on Disturbed Walking Whereas, the latter contains knowledge that is not “consciously accessible” in principle, the explicit network contains knowledge that may be accessible. Information may be redundantly stored in both subsystems. Mutual coupling between both modules allows for mutual support when looking for a solution to a problem. In our approach, instead of using representational diﬀerences for implicit and explicit knowledge to cope with the diﬀerent accessibility, we use only one type of representation, that, however, can be diﬀerently activated, either being in the reactive mode or in the “attended” mode. In our case, the localist information (motivational units) and the distributed information (procedural networks) are not separated into two modules, but form a common, decentralized structure. In this way, the reaCog system realizes the idea of recruitment as the same clusters are used in motor tasks and cognitive tasks. Whereas, we need an explicit attention system, as given in the spreading activation and winner-take-all layer, in the CLARION model decisions result from the recurrent network ﬁnding an attractor state. Many models of cognition take, quite in contrast to our could be terminated which provided enough support for the walker. such architectures concerning learning is currently not covered in reaCog. In general, as pointed out by Langley et al. (2009), these kinds of approaches have not yet demonstrated the broad functionality associated with cognitive architectures (and—as in addition mentioned by Duch et al. (2008)—many of such models are not realized and are often not detailed enough to be implemented as a cognitive system). ReaCog realizes such an emergent system but with focus on a complex behaving system that, in particular, aims at higher cognitive abilities currently not reached by such emergent systems. The third type concerns hybrid approaches which try to bring together the advantages of the other two paradigms, for example ACT-R (Adaptive Components of Thought-Rational, Anderson, 2003). The, in our view, most impressive and comprehensive model of such a cognitive system is presented by the CLARION system (review see Sun et al., 2005; Helie and Sun, 2010) being applied to creative problem solving. This system is detailed enough so that it can be implemented computationally. Models of Cognitive Systems With respect to reaCog the most similar approach has been pursued by Bongard et al. (2006). These authors use a four-legged, eight DoFs robot which, through motor babbling—i.e., randomly selected motor commands— learns the relation between motor output and the sensory consequences. This information is used to distinguish between a limited number of given hypotheses concerning the possible structure of the body. Finding the best ﬁtting solution, one body model is selected. After the body model has been learned, in a second step the robot learns to move. To this end, the body model was used to perform diﬀerent simulated behaviors and was only used as a forward model. Based on a reward given by an external supervisor and an optimizing algorithm, the best controller (sequence of moving the eight joints) was then realized to run the robot. Continuous learning allows the robot to register changes in the body morphology and to update its body model correspondingly. As the most important diﬀerence, Bongard et al. (2006) distinguish between the reactive system and the internal predictive body model. The central idea of their approach is that both are learned in distinct phases one after another. In reaCog the body model is part of the reactive system and required for the control of behavior. This allows diﬀerent controllers driving the same body part and using the same body model for diﬀerent functions (e.g., using a limb as a leg or as a gripper, Schilling et al., 2013a, Figure 10). In addition, diﬀerent from our approach, Bongard et al. (2006) do not use artiﬁcial neural networks (ANN) for the body model and for the controller, but an explicit representation because application of ANN would make it “diﬃcult to assess the correctness of the model” (Bongard et al., 2006, p. 1119). ReaCog deals with a much more complex structure as it deals with 18 DoFs instead of the only eight DoFs used by Bongard et al. (2006) which makes an explicit representation even more problematic. ReaCog as a system is clearly embodied. The procedures cannot by themselves instantiate the behavior, but require a body. The body is a constitutive part of the computational system, because the sensory feedback from the body is crucially required to activate the procedural memories in the appropriate way. The overall behavior emerges from the interaction between controller, body and environment. Models of Cognitive Systems Although an attention system is applied, this is used for controlling perception, not for supporting the search, as is the case in reaCog. Related to this, the search procedure—termed non-linguistic reasoning—in January 2017 \| Volume 11 \| Article 3 16 ReaCog, a Minimal Cognitive Controller Schilling and Cruse GNOSIS appears to be less open as the corresponding network is tailored to the actual problem to avoid a too large search space. In our approach, using the attention system, the complete memory can be used as substrate for ﬁnding a solution.4.2 Cognitive Robotic Approaches (Mouret and Clune, 2015). This pre-computed behaviors are exploited when a new situation or problem is encountered. As the behavioral space is continuous, the pre-computed behavior can be used to adapt for ﬁnding a new behavior. Further, there is no explicit body model that is shared between diﬀerent behaviors. Instead, the memory approximates an incomplete body model, as it contains only a limited range of those movements which are geometrically possible. In contrast, reaCog, using its internal body model, allows to exploit all geometrically possible solutions and is not constraint to search in a continuous space, as illustrated by our example case, where a single leg is selected to perform completely out of context. The approaches introduced in the previous section are not embodied and it appears diﬃcult to envision how they could be embodied (Duch et al., 2008). Following the basic idea of embodied cognition (Brooks, 1989; Barsalou, 2008; Barsalou et al., 2012) embodiment is assumed as being necessary for any cognitive system. Our approach toward a minimal cognitive system is based on this core assumption. Robotic approaches have been proposed as ideal tools for research on cognition as the focus cannot narrowed down to a singular cognitive phenomenon, but it is required to put a uniﬁed system into the full context of diﬀerent control processes and in interaction with the environment (Pezzulo et al., 2012). p y While there is only a small number of robotic approaches dealing with explicit internal simulation, most of these are using very simple robotic architectures with only a very small number of degrees of freedom [for example see Svensson et al. (2009) or Chersi et al. (2013)]. It should further be mentioned that predictive models are also used to anticipate the visual eﬀects of the robot’s movements (e.g., Hoﬀmann, 2007; Möller and Schenck, 2008). Frontiers in Neurorobotics \| www.frontiersin.org DISCUSSION AND CONCLUSION A network termed reaCog has been proposed that is based on the reactive controller Walknet equipped with decentrally organized behavioral modules, or procedures, all connected to motivation units, and a body model. The motivation units form a network that represents a heterarchical architecture allowing for the realization of various internal states. These states result from parallel activation of elements as well as competitive selection between elements. When discussing the properties of a network like reaCog, a crucial aspect concerns the notion of emergence. The rational behind searching for emergent properties is the assumption that many “higher level” properties are not based on dedicated neuronal systems speciﬁcally responsible for the respective properties. Rather, emergent properties arise from the cooperation of lower-level elements and are characterized as to require levels of description other than those used to describe the properties of the elements. In the remainder, such emergent properties will, where appropriate, be related to the requirements posed by Langley et al. (2009) supporting the idea that reaCog provides a minimal functional description for some of those requirements. The body model can be used as an inverse model for controlling motor output, as a forward model for internal simulation of behavior, and it can be exploited to improve erroneous sensor data (“sensor fusion”). Whereas, the reactive part uses the ability of the body model to function as an inverse model, the cognitive expansion exploits the internal body model to be used as a forward model and thereby as a tool for internal simulation of behavior. Internal simulation is used for ﬁnding a new solution for a problem detected by a problem detector. To this end, a three-layered network has been introduced that selects a new, currently not used module of the procedural memory, the contribution of which will then be tested through internal simulation. If this simulation turns out to be successful, i.e., shows a solution for the current problem, the corresponding behavior will be executed in reality. Thus, motor planning is possible using an extremely small expansion, a network consisting of essentially six units plus three parallel layers of units connected in a simple way. According to Langley et al. Models of Cognitive Systems While the notion of innate body representations is controversial (de Vignemont, 2010), there is at least a general consensus about that there is some form January 2017 \| Volume 11 \| Article 3 17 ReaCog, a Minimal Cognitive Controller Schilling and Cruse of innate body model (often referred to as the body schema) reﬂecting general structural and dynamic properties of the body (Carruthers, 2008) which is shaped and develops further during maturation. This aspect is captured by our body model that encodes general structural relations of the body in service for motor control, but may adapt to developmental changes. While currently only kinematic properties are applied, dynamic inﬂuences can be integrated in the model as has been shown in Schilling (2009). for pattern completion that is used in reactive behavior and can be recruited for planning ahead, and (4) a small network, called cognitive expansion, that enables the otherwise reactive system to become—in the sense of McFarland and Bösser (1993)— a cognitive one. We are not aware of any other neuronal approach that covers all these properties. Although the network represents a simple architecture, in the following we will argue that properties often attributed to “higher” brains can be found in reaCog, too, thereby approaching the question concerning the basic neuronal requirements of such higher level phenomena. A further important diﬀerence concerns the structure of the memory. Whereas, in Bongard’s approach one monolithic controller is learned to deal with eight DoFs and producing one speciﬁc behavior, in reaCog the controller consists of modularized procedural memories. This memory architecture allows for selection between diﬀerent states and therefore between diﬀerent behaviors. Before entering into this discussion, one important aspect missing in the current version of reaCog has to be noted. There is long term memory represented by the procedures in the form of “species memory” (Fuster, 1995). There is short term memory as a new solution is stored until the corresponding behavior is executed. There is however no faculty yet to transform the content of this short term memory into a long term memory. The ability to store such a newly acquired procedure as a long term memory would of course be an advantageous property. To gain this capability, the sensory situation accompanying the occurrence of a “problem” should be able to directly elicit activation of the procedure found to solve the problem. Models of Cognitive Systems In the following, we will review relevant embodied robotic approaches. Today, many robotic approaches deal with the task of learning behaviors. In particular, behaviors should be adaptive. This means, a learned behavior should be transferable to similar movements and applicable in a broader context. Deep learning approaches have proven quite successful in such tasks e.g., Lenz et al. (2015), but many require large datasets for learning. Only recently Levine et al. (2015) presented a powerful reinforcement learning approach in this area. In this approach, the robot uses trial-and-error during online learning to explore possible behaviors. This allows the robot to quickly learn control policies for manipulation skills and has shown to be eﬀective for quite diﬃcult manipulation tasks. When using deep learning methods it is generally diﬃcult to access the learned model. In contrast to reaCog such internal models are therefore not well suited for recruitment in higher-level tasks and planning ahead. In particular, there is no explicit internal body model which could be recruited. Rather, only implicit models are learned and have to be completely acquired anew for every single behavior. In the following, two exciting robotic examples tightly related to our approach will be addressed in more detail. The approach by Cully et al. (2015) aims at solving similar tasks as reaCog for a hexapod robot. It also applies as a general mechanism the idea of trial-and-error learning when the robot encounters a novel situation. In their case these new situations are walking up a slope or losing a leg. There are some diﬀerences compared to reaCog. Most notably, the testing of novel behaviors is done on the real robot. This is possible as the trial-and-error method is not applying discrete behaviors. Instead, central to the approach by Cully et al. (2015) is the idea of a behavioral parametrization which allows to characterize the currently experienced situation in a continuous, low dimensional space. A complete mapping toward optimal behaviors has been constructed in advance oﬄine Diﬀerent from their approach, we do not consider how the body model and the basic controllers will be learned, but take both as given (or “innate”). Frontiers in Neurorobotics \| www.frontiersin.org DISCUSSION AND CONCLUSION (2009) is concerned with action selection on lower levels and “choice” of behavior on a higher level. Action selection is indeed a crucial property of the network. On a lower level, within a given behavioral context—in our case walking—speciﬁc procedures compete via local WTA connections. For instance, a leg controller has to decide when to perform swing or stance movements. On an intermediate level, a decision can be made between, for example, forward walking and backward walking. On an even higher level, reaCog, exploiting the cognitive expansion, can select one speciﬁc behavioral element to be activated in addition to the currently active units. Therefore, Langley et al.’s requirement (5) is covered, too. Although the structure of reaCog is far away from any morphological similarity to mammalian brains, functionally reaCog shows some similarity and may, therefore, in spite of its simplicity, be considered as a scaﬀold helpful for the understanding of properties of higher brains. To this end, taking a more abstract view, one might ask whether higher level properties characterized by using psychological terminology might be attributed to reaCog. As noted earlier, in reaCog emergent properties can already be observed at lower levels (e.g., production of diﬀerent gaits) but they can also be found at higher levels, thereby supporting Langley et al.’s second requirement. Above, we had already used one such higher level term, attention. It has been argued (Cruse and Schilling, 2013, 2015) that further emergent properties as are intentions and emotions might be attributed to a system as reaCog, too, at least on the functional level. When adding some further procedures, reaCog might even be equipped with basic aspects required for Access Consciousness as well as Reﬂexive Consciousness (Cruse and Schilling, 2013, 2015). Thus, reaCog shows action selection not only on the reactive level, but also on the cognitive level, whereby the decisions based on internal simulation (or imagined action, “mental” action, or “probehandeln” according to Freud (1911) are not determined strictly by the sensorily given situation. Even if an external observer had the ability to monitor the internal states of the agent controlled by reaCog, the behavior of the agent could not be predicted by this observer. This is the case because, due to the noise in the SAL network, there is a stochastic element contributing to the decision. DISCUSSION AND CONCLUSION The cognitive expansion network does not prohibit parallel activation of procedures. This requirement is in line with current developments in the area of cognitive systems research as pointed out by Duch et al. (2008). Inspired by the way how brains are organized, these authors propose, ﬁrst, that cognitive systems in the future should incorporate a mechanism to focus attention, which is realized in reaCog through simple local competition as realized in the WTA structures. And second, that a neural network-like spreading activation mechanism is required in order to broaden search and follow associations, which is given in the spreading activation layer. independently, but are always represented within their context. Nonetheless, the functioning of the cognitive expansion allows to integrate them in another context. In other words, in reaCog, the procedures are globally available. Therefore, global availability may not require procedures being stored independent of any context (or “amodally”). Thus, reaCog represents a concrete architecture showing how global availability might be established in a neural system without requiring independent representation. There is a group of related terms addressing a fundamental principle of brains. These are the “massive redeployment hypothesis” (Anderson, 2010), the “neural recycling theory” (Dehaene, 2005), the “shared circuits model” of Hurley (2008) and Gallese’s “neural exploitation” (Gallese and Lakoﬀ, 2005), summarized by Anderson (2010) by the term “neural reuse.” Neural reuse means that a system is able to exploit existing components that do something useful to support a new task, either in the evolutionary time frame or by learning (Anderson, 2010, p. 250). In other words, neural reuse states that existing elements are used for other purposes. ReaCog models neural reuse of two kinds as listed by Anderson. One type, already applied in the current version of reaCog, corresponds to the use of the same procedural elements for both motor control and planning. Here reuse corresponds to the case of having been installed in evolutionary time scales. The second type addressable in reaCog concerns the reuse of procedural elements as a result of learning the integration of a given procedure in a new context as described above, which is, in reaCog, currently only given in the form of short term memory. But the ability to transfer this information into long term memory is a major focus for future work. The ﬁfth aspect of Langley et al. DISCUSSION AND CONCLUSION (2009), a cognitive system should show the following properties: (1) Storing motor skills and covering the continuum from fully reactive, closed-loop behavior to (automatic) open-loop behavior; (2) Emergent properties resulting from the cooperation between diﬀerent independent modules; (3) Long term memory and short term memory; (4) Attention to select sensory input; (5) Decisions on the lower level and “choice” on the higher level; (6) Predictions of possible actions; (7) Problem solving and planning of actions in the world; (8) Recognition and categorization of sensory input; (9) Remembering and episodic memory; (10) Application of symbols and reasoning; (11) To support reasoning, relationships between beliefs have to be realized; (12) Interaction and communication, including representation of verbal symbols; (13) Reﬂection and explanation (metareasoning); (14) Confronting the interactions between body and mind. In reaCog, there is no explicit, separate planner as used in hybrid systems. Rather, the ability to plan ahead relies on exploiting the reactive basis by operating on it much like a parasite operates on its host, that is, by only controlling the functioning of the reactive part. In other words, the cognitive expansion does not represent a separate planner, but organizes the activity of the reactive part, which is, during planning, not connected with the motor output. Requirements (1) and (2) are properties of Walknet. Above, we already argued that requirements (3), (6), and (7) can be found in reaCog, too. Below we will argue that also requirements (4) and (5) are fulﬁlled, but not aspects (8)–(14). Thus, constitutive elements of reaCog are (1) embodiment, (2) a decentralized organization of various procedures arranged in a heterarchical architecture, (3) a holistic body model allowing As the cognitive expansion of the reactive network allows the complete system—using psychological terms to describe January 2017 \| Volume 11 \| Article 3 18 ReaCog, a Minimal Cognitive Controller Schilling and Cruse its function—to “focus” or “concentrate” or “attend” on a speciﬁc behavior, we have already earlier termed this expansion “attention system” supporting Langley et al.’s issue (4). Its ability to focus on speciﬁc memory elements may correspond to what sometimes has been termed “spot light” (Baars and Franklin, 2007) referring to the observation that the content of only one memory element becomes aware at a given moment in time. Recall, that selection of a speciﬁc procedure via the WTA network of the attention system does not mean that the other procedures are suppressed. Frontiers in Neurorobotics \| www.frontiersin.org DISCUSSION AND CONCLUSION On the other hand, the ﬁnal decision is not purely random, because the proposals made by the attention system are tested for feasibility via the internal simulation and are to some extent guided by the somatotopic structure of the SAL network. The proposal is further tested by performing the behavior in reality. In this way, this process of ﬁnding a new solution may be viewed as to be based on a Darwinian procedure, starting with an, in part, stochastic “mutation,” followed by a selection testing the proposal for “ﬁtness.” Taken together, Langley et al.’s (Langley et al., 2009) requirements for cognitive systems (1)–(7) are well covered by reaCog. To conclude, we will brieﬂy address the remaining issues (some of which have already been mentioned above): The capability to categorize sensory input, [Langley et al.’s issue (8)] is not given in reaCog as we focus mainly on the motor aspects. As mentioned, learning will be the focus of future work and It has been stated that in a cognitive system, in order to address memory elements out of context (“global availability,” e.g., Dehaene and Changeux, 2011), these elements have to be represented independently, i.e., not embedded in reactive structures. In reaCog, procedures are not represented January 2017 \| Volume 11 \| Article 3 19 ReaCog, a Minimal Cognitive Controller Schilling and Cruse model plus the corresponding circuit to control both models independently. will address episodic memory (9). Other aspects would require further extension: Langley et al.’s issues (10)–(12) refer to the ability to use (verbal) symbols, a property not given in reaCog. However, a way has been sketched how this might be possible (for a ﬁrst step toward this property see (Schilling and Spranger, 2010; Cruse and Schilling, 2013; Schilling and Narayanan, 2013), based on ideas of Steels and Belpaeme (2005) and Narayanan (1999). The term “cognition” as used here, is based on the simple deﬁnition proposed by McFarland and Bösser (1993), i.e., the faculty of being able to plan ahead. This faculty is achieved here by using a reactive system plus introduction of a “cognitive expansion.” As discussed above, such a system appears to be suited to form a basis on which further emergent properties may be realized, properties that are often listed as being required for a system termed cognitive as are Langley et al’s requirements (8)–(14), for example. ACKNOWLEDGMENTS This research/work was supported by the Cluster of Excellence Cognitive Interaction Technology “CITEC” (EXC 277) at Bielefeld University, which is funded by the German Research Foundation (DFG), by the EC project EMICAB, the Wissenschaftskolleg zu Berlin, the ICSI, and the DAAD (PostDoc grant to MS). This research/work was supported by the Cluster of Excellence Cognitive Interaction Technology “CITEC” (EXC 277) at Bielefeld University, which is funded by the German Research Foundation (DFG), by the EC project EMICAB, the Wissenschaftskolleg zu Berlin, the ICSI, and the DAAD (PostDoc grant to MS). DISCUSSION AND CONCLUSION If this view is correct, these properties need not necessarily be explicitly included in such a deﬁnition, but appear to result from a system based on reactive structures plus the critical capability of planning ahead, underlining the power of McFarland and Bösser (1993) clear-cut deﬁnition of cognition. Langley et al.’s issue (13) concerns Reﬂection and explanation. This property is not realized in reaCog and may also depend on the ability to apply symbolic knowledge. Issue (14), the property of cognitive systems to “confront the interactions between body and mind” addresses the property of having phenomenal experience, and is not found in reaCog, too (for a discussion of this matter see Cruse and Schilling, 2013, 2015). In summary, a number of emergent properties can be observed in reaCog, including Langley et al.’s issues (1)–(7). Issues (8)–(13) require further expansions. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fnbot. 2017.00003/full#supplementary-material Supplemental Data 1 \| Showing example runs from additional simulations. Supplemental Data 1 \| Showing example runs from additional simulations. Supplementary Video 1 \| AwkwardPosture_InternalSimulation.mp4. Video showing the example case of testing different behaviors out of context in internal simulation and afterwards applying a successful solution to the robot. Supplementary Video 1 \| AwkwardPosture_InternalSimulation.mp4. Video showing the example case of testing different behaviors out of context in internal Supplementary Video 2 \| AwkwardPosture_InternalSimulation_ LegPositions.mp4. Video showing the example case of testing different behaviors out of context in internal simulation and afterwards applying a successful solution to the robot. Showing the leg positions over time. AUTHOR CONTRIBUTIONS In this article we focus on the situation that there is a problem which requires immediate solution and as a consequence, immediate internal simulation. As in our situation the body model is needed for simulation, the former cannot be used for controlling other behaviors at the same time. In other words, the body position has to be kept constant during internal simulation. In the following we brieﬂy mention three cases which do not comply with this situation. In the ﬁrst case internal simulation is not required. In this simple case the network is equipped with reactive procedures that allow for unspeciﬁc, general responses in case a problem detector is activated. An ubiquitous example is given by freezing behavior without active search for a solution, hoping that the problem will disappear on its own. Another example might be a procedure that allows emitting a general alarm signal that activates conspeciﬁcs. As a second case, one might think of situations that allow to postpone the search for a solution. In this case the normal behavior can be continued until a situation is given that allows to use the internal model without getting into conﬂict with current behavior. This case would at least require a short term memory to store the problem situation so that this could later be reactivated to start internal simulation, an expansion not yet implemented in the current version of reaCog. As a third, more complex case there might be a network that is able to control any behavior and at the same time, run an internal simulation. Whereas, for the second case a comparatively simple expansion of reaCog would suﬃce, the latter case appears to be much more demanding. It might, for example, require a second internal Conceptualization, methodology and writing—MS and HC. Software, investigation and simulation—MS. Conceptualization, methodology and writing—MS and HC. Software, investigation and simulation—MS. REFERENCES (1990). What mechanisms coordinate leg movement in walking arthropods? Trends Neurosci. 13, 15–21. doi: 10.1016/0166-2236(90)90057-H Hesslow, G. (2002). Conscious thought as simulation of behaviour and perception. Trends Cogn. Sci. (Regul. Ed). 6, 242–247. doi: 10.1016/S1364-6613(02)01913-7 Cruse, H. (2003). The evolution of cognition: a hypothesis. Cogn. Sci. 27, 135–155. doi: 10.1016/S0364-0213(02)00110-6 Hoﬀmann, H. (2007). 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https://openalex.org/W4280625805	https://economyandsociety.in.ua/index.php/journal/article/download/1123/1081	Ukrainian	null	РЕМЕСЛА ЯК ОСНОВА РОЗВИТКУ КРЕАТИВНИХ ТУРИСТИЧНИХ ДЕСТИНАЦІЙ	Ekonomìka ta suspìlʹstvo	2,022	cc-by	6,054	Senkiv Mariana, Mokh Romana Lviv Polytechnic National University У статті проаналізовано сучасні підходи до розуміння креативного туризму, креативних туристичних ресурсів і креативних туристичних дестинацій. Визначено роль народних ремесел для розвитку креатив- ного туризму. Охарактеризовано і картографічно відображено географію народних ремесел Львівської області як однієї з найперспективніших у контексті розвитку креативного туризму. Виділено основні класи ремесел та коротко охарактеризовано їхній сучасний стан на Львівщині. На прикладі села Гавареччина проаналізовано досвід формування креативної туристичної дестинації. На основі даних про кількість ремісників та туристичну діяльність Гавареччини у різні часові періоди, побудовано циклічну модель кре- ативної туристичної дестинації. Запропоновано шляхи формування та розвитку креативних туристичних дестинацій в Україні. д ц р Ключові слова: ремесло, креативний туризм, дестинація, спадщина, Львівська область. Now tourism in the Lviv region is a significant sector of the economy and one of the priority areas of devel- opment. This factor, as well as the presence of unique crafts and problems of their preservation, the conditions caused by the COVID-19 pandemic, the main concentration of tourists in Lviv, the need for sustainable develop- ment of territories, as well as the appeal of tourist needs to authenticity, have necessitated the diversification of tourism offer in the Lviv region. Crafts can become the basis for the formation of creative tourism destinations, providing benefits to all participants in tourism activities, including preservation of unique resources, economic benefits for craftsmen and the satisfaction of new tourist needs. Purpose of the article is a development of practi- cal recommendations on the use of crafts of the Lviv region for the formation and development of creative tourism destinations. The following methods are used: the study of literary sources – to reveal the theoretical and meth- odological foundations of creative tourism, approaches to its definition; analysis, systematization and comparison – to identify successful examples of effective management of creative tourism resources in the countries of the world and the Lviv region; cartographic method – for studying the geography of crafts in the Lviv region; Butler’s model – to highlight the stages of formation and development of a tourism destination. The article analyzes mod- ern approaches to understanding creative tourism, creative tourism resources and creative tourism destinations. The role of folk crafts in the development of creative tourism is determined. The geography of folk crafts of the Lviv regionis characterized and reflected cartographically as one of the most promising in the context of the develop- ment of creative tourism. Випуск # 36 / 2022 Випуск # 36 / 2022 ЕКОНОМІКА ТА СУСПІЛЬСТВО DOI: https://doi.org/10.32782/2524-0072/2022-36-5 CRAFTS AS A BASIS FOR THE DEVELOPMENT OF CREATIVE TOURISM DESTINATIONS Сеньків Мар’яна Ігорівна кандидат географічних наук, асистент, Національний університет «Львівська політехніка» ORCID: https://orcid.org/0000-0002-2146-3456 Мох Романа Назарівна студентка, Національний університет «Львівська політехніка» ORCID: https://orcid.org/0000-0001-6397-0520 Senkiv Mariana, Mokh Romana Lviv Polytechnic National University Senkiv Mariana, Mokh Romana Lviv Polytechnic National University The main classes of crafts are singled out and their condition in the Lviv region is briefly characterized. On the example of the village of Gavarechchyna in the Lviv region, the experience of creative tourism destination formation is analyzed. On the basis of data on the number of craftsmen and tourism activity of Gavarechchyna in different time periods, a cyclic model of a creative tourism destination is built. The ways of formation and development of creative tourism destinations in Ukraine are proposed. ТУРИЗМ p Keywords: craft, creative tourism, destination, heritage, Lviv region. 41 © Сеньків М.І., Мох Р.Н. ЕКОНОМІКА ТА СУСПІЛЬСТВО Випуск # 36 / 2022 Випуск # 36 / 2022 Постановка проблеми. Туристична інду- стрія, що до початку пандемії COVID-19 стабільно стрімко розвивалася, за даними World Travel & Tourism Council у 2019 році становила 10,4% світового ВВП, з 334 млн. зайнятих осіб [1]. Одним із трендів туризму XXI століття є збільшення кількості подорожу- ючих серед середнього класу, що демонструє загальне поширення туризму серед насе- лення світу. Туризм став невід’ємною части- ною культури та економіки світу. Пандемія COVID-19 нанесла значної шкоди сектору туризму у 2020 році. Ремесла України, зокрема, і у Львівській області переживають переломний момент в історії свого існування, оскільки чимало з них вже є забутими або такими, що майже не роз- виваються, проте, все ж залишаються такими, зникненню яких можна запобігти, та завдяки креативному туризму використати як основу для сталого розвитку території. Аналіз останніх досліджень і публіка- цій. Підґрунтям для формування креативного туризму був культурний туризм. Основна ідея концепції креативного туризму включає залу- чення туриста до творчої діяльності та набуття автентичного досвіду. З цього випливає осно- вна відмінність культурного та креативного туризму: у культурному туризмі громада віді- грає незначну роль, оскільки залучаються найпопулярніші місця; креативний туризм заохочує відійти від масового туризму і спря- мований на збереження старих традицій та автентичної культури. Проте зміни, викликані пандемією, зумо- вили пошуки нових, креативних шляхів роз- витку туристичної сфери. Закриття кордонів багатьох країн призвело до активного розви- тку внутрішнього туризму. Збільшення кіль- кості внутрішніх туристів спричинило розвиток багатьох дестинацій, зокрема, через форму- вання нових туристичних маршрутів, ство- рення нових проектів розвитку туризму тери- торії тощо. Під час дебатів ЮНЕСКО у вересні 2020 року була визначена необхідність роз- робки нових моделей та підходів до сталого відновлення туризму, що підтримує громади, створює робочі місця, сприяє захисту спад- щини [27]. Senkiv Mariana, Mokh Romana Lviv Polytechnic National University Культура та креативність посідають центральну роль у таких дебатах, а туризм став важливим інструментом у реалізації кре- ативних стратегій. Саме тому креативність стала стратегією позиціонування багатьох місць. Зокрема, практика позиціонування території як «креативна» набула свого вті- лення в концепції креативних міст, котрі були об’єднані в мережу креативних міст ЮНЕСКО [28]. Проте, слід зауважити, що не всі креа- тивні території є креативними туристичними дестинаціями: окрім володіння хоча б одним креативним туристичним ресурсом, такі тери- торії повинні ним ефективно управляти та розвивати. Під креативним туризмом найчастіше розу- міють подорож, під час якої відбувається спо- стереження або безпосередня участь у твор- чому процесі, притаманному певній місцевості (регіону, містечку, селу) для проникнення культурою, ознайомлення з традиціями, мис- тецтвом чи ремеслами. Креативність може виражатися як в матеріальній формі (при- лад, картина), так і в нематеріальній (музика, ідея, наукова теорія). Тому в світі існують різні підходи до визначення поняття «креативний туризм». Причиною різних підходів до визна- чення поняття креативного туризму (табл. 1) є також відмінності у культурному середовищі як в різних країнах, так і в межах однієї країни, а часто вони можуть спостерігатися навіть в межах однієї області. За останні десятиліття багато сіл, міст та навіть регіонів почали позиціонувати себе як «креативні», тож ЮНЕСКО визнала їх та в 2004 році ввела поняття «креативних міст». За останні десятиліття багато сіл, міст та навіть регіонів почали позиціонувати себе як «креативні», тож ЮНЕСКО визнала їх та в 2004 році ввела поняття «креативних міст». Креативний туризм є однією з форм креа- тивних індустрій, значення та роль у форму- ванні економіки яких з кожним роком зростає. Це ті галузі, які походять від індивідуаль- ної творчості, навичок і талантів і які мають потенціал для створення робочих місць через використання інтелектуальної власності [14]. У різних країнах світу індустрії, що належать до креативних, відрізняються [14–20]. В Україні, починаючи з 2017 року діє сектор розвитку кре- ативних індустрій [2], а у 2018 році почав функ- ціонувати Директорат креативних індустрій [3]. Національне бюро програми ЄС «Креа- ТУРИЗМ Необхідність та світова тенденція до ста- лого розвитку зумовлюють потребу в збере- женні унікальних креативних ресурсів тери- торій, зокрема ремесел, оскільки механізація усіх процесів виробництва та глобалізація практично витіснили ремісництво. Його збе- реження завдяки туризму відіграє ключову роль для збереження ідентичності народу, його культури та традицій. Окрім того, сучасні туристи все частіше звертаються до локаль- ного, автентичного досвіду, що робить подо- рож персоналізованою, об’єднує населення та сприяє мультикультурному обміну в межах однієї країни чи між країнами. Таблиця 1 Таблиця 1 Підходи до визначення креативного туризму Автор Визначення Хана Білікова та Зузана Паленчікова «Креативний туризм – це нішевий туризм, який пропонує відвідувачам можливість саморозвитку через участь у творчих заходах (майстер- класи, курси), характерних для місця, де вони проводяться» [5] Айсегуль Кесімоглу «Креативний туризм – це вид туризму, який спирається на креативні ресурси та креативні кластери; і забезпечує творчий досвід» [6] Соня Морейра Кабеса та ін. «Креативний туризм – це вид туризму, який розвиває креативність туристів через їх активну участь у досвіді, характерному для дестинації, яку вони відвідують» [7] Мануела Пішітеллі «Креативний туризм – це туризм, який дає можливість розвинути власний творчий потенціал через досвід, пов’язаний з культурою місць у багатьох сферах, таких як кулінарія, живопис, фотографія, ремесла та свята» [8] Франсіско Барбоза Гонсалвес і Карлос Коста «Креативний туризм – це феномен, що виникає в результаті подорожей, спрямованих на справжній досвід, із залученням вивчення мистецтва, спадщини, забезпечення зв’язку з тими, хто живе в цьому місці і створює цю культуру» [9] Руй Мігель Феррейра Карвалью та ін. «Креативний туризм –це туризм особливого інтересу, альтернатива масовим формам культурного споживання туризму, де туристи мають можливість розвивати свій творчий потенціал через спілкування з місцевими жителями» [10] Есекьель Азеведу Сантуш і Graça Joaquim «Креативний туризм – це тип туризму, каталізатором якого є творчість у формі особистої участі у творенні мистецтва» [11] Марта Ізабель Амарал та Ана Ізабель Родрігеш «Креативний туризм – це прогресивний напрям культурного туризму, в якому споживачі шукають інтерактивні та динамічні враження» [12] Мелтем Алтинай Оздемір та Емре Ергун «Креативний туризм – це вид туризму, який дає можливість туристам дізнатися про культуру регіону, який вони відвідують, спілкуючись з місцевим населенням шляхом активної участі» [13] Джерело: розроблено авторами Підходи до визначення креативного туризму «Креативний туризм –це туризм особливого інтересу, альтернатива масовим формам культурного споживання туризму, де туристи мають можливість розвивати свій творчий потенціал через спілкування з місцевими жителями» [10] «Креативний туризм – це тип туризму, каталізатором якого є творчість у формі особистої участі у творенні мистецтва» [11] «Креативний туризм – це прогресивний напрям культурного туризму, в якому споживачі шукають інтерактивні та динамічні враження» [12] Одним з головних ресурсів креативного туризму є ремесло, що найкращим чином відображає місцевий побут та культуру, оскільки розвивалося в залежності від куль- тури, релігії, а іноді – політичних поглядів. Народне ремесло – це виготовлення вручну предметів за допомогою звичайних підручних матеріалів і простих конструкцій. Важливо зазначити, що народні ремесла належать до нематеріальної культурної спадщини. Senkiv Mariana, Mokh Romana Lviv Polytechnic National University Креативний туризм є однією з форм креа- тивних індустрій, значення та роль у форму- ванні економіки яких з кожним роком зростає. Це ті галузі, які походять від індивідуаль- ної творчості, навичок і талантів і які мають потенціал для створення робочих місць через використання інтелектуальної власності [14]. У різних країнах світу індустрії, що належать до креативних, відрізняються [14–20]. В Україні, починаючи з 2017 року діє сектор розвитку кре- ативних індустрій [2], а у 2018 році почав функ- ціонувати Директорат креативних індустрій [3]. ТУРИЗМ Національне бюро програми ЄС «Креа- тивна Європа» [4] в рамках нової програми Випуск # 36 / 2022 ЕКОНОМІКА ТА СУСПІЛЬСТВО Таблиця 1 Ремесло може бути важливим аспектом розвитку креативного туризму в різних кон- текстах і може стати міцною основою для ініціатив (проектів) з позиціонування місця (території). Проте в останні десятиліття ста- тус ремесла опинився під загрозою масового виробництва та усвідомленої переваги про- мислового виробництва (дизайну) над тради- ційною технікою. Виділення невирішених раніше частин загальної проблеми. Зараз туризм у Львів- ській області є значною сферою економіки та одним з пріоритетних напрямків розвитку. Цей фактор, а також наявність унікальних ремесел та проблеми їх збереження, умови, спричинені пандемією COVID-19, основна концентрація туристів у місті Львові, потреба у сталому розвитку територій, а також звер- нення потреб туриста до автентичності зумо- вили необхідність диверсифікації туристичної пропозиції Львівщини. Розвиток креативного туризму є части- ною створення привабливих місць для про- живання, роботи та туризму, коли села, міста чи регіони прагнуть підвищити свою прива- бливість для креативного класу, підтримати креативні індустрії чи стати креативними міс- тами. Креативні простори включають зрос- таючу кількість просторових кластерів, які об’єднують креативних виробників і реміс- ників, щоб створити творче середовище для туристичного споживання. У Китаї такі клас- тери, як 798 Art Zone в Пекіні, приваблюють мільйони відвідувачів щороку, включаючи багатьох міжнародних туристів [29]. р ц щ Окрім напрямків туризму, що вже роз- виваються, Львівська область має високий потенціал розвитку креативного туризму. Про потенціал та важливість креативних індустрій загалом зазначається у Стратегії розвитку Львівської області на період 2021–2027 років [35]. Перелік видів економічної діяльності, що належать до креативних, змінюється. До 2019 року до цього переліку не входили народні художні промисли. Це свідчить про те, що увага до ремесел, як перспективного ресурсу для розвитку креативного туризму на Львівщині, зростатиме. Ремесла можуть стати основою формування креативних туристич- них дестинацій, забезпечуючи вигоду усім учасникам туристичної діяльності, зокрема збереження унікальних ресурсів, економічну вигоду носіям ремесла та задоволення нових потреб туристів. Але досі в Україні і на Львів- щині зокрема, бракує ефективного досвіду формування і розвитку креативних туристич- них дестинацій. Дуже часто стратегії, що засновані на при- вабливості території, пов’язані з джентрифіка- цією та серійним виробництвом, що призводить до того, що місця втрачають свою унікальність [30]. Це вказує на необхідність краще проекту- вати креативні місця, таким чином, щоб вони зберігали свої унікальні риси. Отже, креативний розвиток можна розгля- дати як систему спільної творчості, яка вима- гає співпраці всіх тих, хто бере участь у від- відуванні, використанні та проживанні в місці [31]. Таблиця 1 Хоча у результаті діяльності ремісника створюється річ, тобто матеріальний об’єкт, їхнє створення є неможливим без спеціального знання, нави- чки, техніки та досвіду, а також, що не менш важливо, творчості окремого майстра, що зберігаються та передаються роками. Укра- їна є учасником Конвенції про охорону нема- теріальної культурної спадщини [23]. Згідно з нею, нематеріальна культурна спадщина втілена в: усних традиціях та формах вира- ження, зокрема в мові, виконавському мис- тецтві, звичаях, обрядах, святкуваннях, зна- ннях та практиці, що стосуються природи та ЄС «Креативна Європа» 2021–2027 в Україні визначає 12 секторів культурних та креативних індустрій, зокрема, також ремесла (текстиль, кераміка, дерево, метал, скло, графіка). ЄС «Креативна Європа» 2021–2027 в Україні визначає 12 секторів культурних та креативних індустрій, зокрема, також ремесла (текстиль, кераміка, дерево, метал, скло, графіка). Ресурси креативного туризму, основою яких є нематеріальна культурна спадщина, можуть варіюватися залежно від дестина- ції (сільська місцевість, малі чи великі міста, агломерації), країни, в межах якої знаходиться дестинація та візії креативного туризму тієї чи іншої країни. ЮНЕСКО у своїй Конвенції про збереження нематеріальної культурної спад- щини [21] зазначає, що нематеріальна куль- турна спадщина – це звичаї, форми представ- лення і вираження, знання і навички, а також пов’язані з ними інструменти, предмети, артефакти і культурні простори, визнані спів- товариствами, групами і, в деяких випадках, особами як частина їхньої культурної спад- щини. Станом на зараз Національний перелік нематеріальної культурної спадщини України налічував 26 елементів (наприклад, традиції косівської мальованої кераміки) [22]. ЕКОНОМІКА ТА СУСПІЛЬСТВО Випуск # 36 / 2022 Креативність не має прив’язки до розмірів території, а тому може стати пріоритетним напрямком як для сільської місцевості, так і для міст та регіонів. Країна Таїланд позиціонує себе як «перша креативна дестинація в Азії». На національному рівні Управління туризму Таїланду розробило туристичну програму під назвою «Discover the Other You», метою якої є показати Таїланд з іншої сторони, а саме дес- тинацією для втілення креативного потенціалу. Зразковим є приклад розвитку села Бан Дон Кай Ді (фарфорове село Бенджаронг) завдяки креативному туризму. Ремесло створення кераміки привезли до села китайські торговці у XV столітті, багато місцевих перейняло це ремесло. У 2001 році, коли ремісники в Бан Дон Кай Ді створили ремісничу кооперацію, що збіглося з впровадженням нового урядо- вого проекту – One Tambon One Product, який популяризує місцеві продукти, продаючи їх, що сприяє збільшенню надходжень до місцевої економіки та доходів громадян. всесвіту, знаннях та практиці, що пов’язані з традиційними ремеслами. Таблиця 1 Делла Лусія та Трунфіо [32] ілюструють цей принцип на прикладі культурного парку «The Farm» у Фавері, Італія, який охоплює художні виставки, коворкінг та кулінарні май- стер-класи. ТУРИЗМ Випуск # 36 / 2022 ЕКОНОМІКА ТА СУСПІЛЬСТВО Формулювання цілей статті (постановка завдання). Мета наукової роботи: розро- блення практичних рекомендацій щодо вико- ристання ремесел Львівської області для фор- мування та розвитку креативних туристичних дестинацій. Завдання: розкрити теоретичні та методологічні засади розвитку креатив- ного туризму; визначити ресурси креативного туризму та місце ремесел серед них; проана- лізувати географію та сучасний стан ремесел у Львівській області; запропонувати шляхи фор- мування та розвитку креативних туристичних дестинацій Львівщини. Використовувалися методи: дослідження літературних джерел – для розкриття теоретичних та методологічних засад креативного туризму, підходів до його визначення; аналіз, систематизація та порів- няння – для виявлення успішних прикладів ефективного управління ресурсами креатив- ного туризму в країнах світу та у Львівській області; картографічний метод – для вивчення географії ремесел у Львівській області; модель Батлера – для виділення стадій формування та розвитку туристичної дестинації. були важливим засобом заробітку і забезпе- чення життя для багатьох людей. В Україні є багато ремісничих осередків з виразними регі- ональними особливостями, Львівська область не є виключенням. У табл. 2 зображено кла- сифікацію ремесел, поширених на території України та Львівської області зокрема. Незважаючи на те, що у сучасному світі ручну працю стає все легше замінити, її цін- ність невпинно зростає. Багато з ремесл залишилися тільки як згадка, про деякі з них свідчать лише предмети, що були виготов- лені ремісниками, а саме ремесло забуте, а деякі з ремесел знайшли своїх наступників і у сучасному світі (рис. 1). Гончарство є одним із найстаріших видів ремесел, що зародилися на території сучас- ної України. У Львівській області основними осередками були м. Сокаль, с. Гавареччина, с. Потелич, с. Глинськ, смт Немирів, с. Гонча- рівка, смт Стара Сіль, м. Самбір. Зараз посе- лень, що зберегли своє ремесло гончарства лишилося вкрай мало, зокрема, село Гава- реччина – осередок традиційної чорнодимле- ної гаварецької кераміки. Зараз в селі зали- шилося лише 5 гончарів, що продовжують займатися ремеслом. Виклад основного матеріалу дослі- дження. Виклад основного матеріалу дослі- дження. Географія і сучасний стан ремесел Львів- ської області Недалеко від Червонограда є поклади глини з великим вмістом окису заліза, що слу- жить сировиною. За словами майстрів, тради- ція сокальської чорнодимленої кераміки част- ково була втрачена, так як за радянських часів промисли не розвивалися. Тож сьогодні тра- дицію виготовлення доводиться відстежувати за тим, що залишилося від гончарних виробів Львівська область географічно розташована в межах трьох природніх зона: лісовій, лісосте- повій, передгірних і гірських районах Карпат, а, отже, така унікальна геологічна різноманіт- ність спричинила на цих теренах виникнення та розміщення різноманітних промислів та ремесел. Протягом багатьох століть ремесла Табли Класифікація ремесел, поширених на території України Таблиця 2 Класифікація ремесел, поширених на території України Ремесла України Шкіряно-хутряна галузь Деревообробна галузь Гончарство Чинбарство * Ложкарство Посудництво Шорництво* Токарство Архітектурна кераміка Кушнірство* Гребінництво* Кераміка малих форм Кожухарство* Бондарство Шевське ремесло Лозоплетіння Шапкарство Художнє різьблення Текстильна галузь Металообробна галузь Окремі види ремесел Килимарство Ковальство Виготовлення масок Вишивання Мосяжництво Декоративний розпис Виготовлення музичних інструментів Писанкарство * забуте ремесло Джерело: розроблено авторами Таблиця 2 Класифікація ремесел, поширених на території України Ремесла України Шкіряно-хутряна галузь Деревообробна галузь Гончарство Чинбарство * Ложкарство Посудництво Шорництво* Токарство Архітектурна кераміка Кушнірство* Гребінництво* Кераміка малих форм Кожухарство* Бондарство Шевське ремесло Лозоплетіння Шапкарство Художнє різьблення Текстильна галузь Металообробна галузь Окремі види ремесел Килимарство Ковальство Виготовлення масок Вишивання Мосяжництво Декоративний розпис Виготовлення музичних інструментів Писанкарство * забуте ремесло Джерело: розроблено авторами Класифікація ремесел, поширених на території України ТУРИЗМ ЕКОНОМІКА ТА СУСПІЛЬСТВО Випуск # 36 / 2022 вишнева палітра кольорів була властива Жидачівщині, Сокальщині – чорно-білі узори, Дорогобиччині – чорно-червоні. Однією з найбільш впізнаваних традицій вишивки на Львівщині є сокальська вишивка. Сьогодні у м. Сокаль діє музей «Людина. Земля. Всес- віт», у фондах якого збергіаються зразки унікальної сокальської вишивки, також в історико-краєзнавчому музеї «Сокальщина» зберігаються сотні експонатів сокальської вишивки. На базі cокальської вишивки було створено культурну подію ETNO Тартаків, що проводилася в Тартаківському замку. ХІХ–ХХ століття, багато з яких представлено у художньому музеї «Людина. Земля. Всесвіт» у Сокалі. В селі Стара Скварява на Жовківщині відкрили унікальну для цієї місцевості дитячу літню школу гончарства, де викладають про- фесійні гончарі із приватної львівської школи. Місто Глиняни має велику історію кили- марства. Зараз у місті намагаються відро- дити традицію ткацтва глинянського килима. У 2016 році в місті відкрили відновлену будівлю навчально-виробничого комплексу «Мозаїка», реконструкція якої була зроблена за фінанси Європейського Союзу в рамках програми «Підтримка політики регіонального розвитку в Україні». Виклад основного матеріалу дослі- дження. Базуючись на аналізі географії пош та сучасного стану ремесел на Ль можна припустити, що тут є туристи тинації, які можуть позиціонувати креативні на основі ремесел. Одним прикладів є село Гавареччина. На моделі Батлера, можемо виділити пе дії розвитку цієї туристичної дестина мої завдяки унікальній кераміці (р Зокрема, зі стадією становлення Гава як туристичної дестинації пов’язана ція нового туристичного маршруту « Біле» по території Золочівського та Б районів за маршрутом Львів – Гаваре Свята гора – Білий камінь – Підлисс гора – Кути – Львів у 2013 році. Стаді тку почалася у 2019 році, коли в село прибувати туристи, здебільшого ук поодинокі випадки – туристи з По Німеччини, проводитися майстер-кл Випуск # 36 / 2022 ЕКОНОМІКА ТА СУСПІЛЬСТВО і пошиття з неї одягу. Таке ремесло, що осно- вним чином пов’язане з галуззю мисливства, хутровим промислом, успішно розвивалося на території України. Основним осередком кушнір- ства на Львівщині було місто Старий Самбір. Формування і розвиток креативної турис- тичної дестинації неможливі без ефективного управління туристичним ресурсом. Цінність управління полягає не тільки в досягненні поставлених цілей, але й у взаємозв’язках. Приклад Таїланду, зокрема, ілюструє необхід- ність і конкретні завдання для залучення усіх зацікавлених сторін, включаючи державні, приватні, громадянські та наукові установи, а також місцевих громад і туристів до процесу створення місця [34]. Наприкінці ХІХ – поч. ХХ ст. в поселеннях Західної України почали відкривати школи лозоплетіння, зокрема у селах Джурів та Іза, містах Львів, Сторожинець, Яворів. Діяльність шкіл згодом стала початком для формування лозомеблевих артілей. Базуючись на аналізі географії поширення та сучасного стану ремесел на Львівщині, можна припустити, що тут є туристичні дес- тинації, які можуть позиціонувати себе як креативні на основі ремесел. Одним з таких прикладів є село Гавареччина. На основі моделі Батлера, можемо виділити певні ста- дії розвитку цієї туристичної дестинації, відо- мої завдяки унікальній кераміці (рис. 2.). Зокрема, зі стадією становлення Гавареччини як туристичної дестинації пов’язана апроба- ція нового туристичного маршруту «Чорне і Біле» по території Золочівського та Буського районів за маршрутом Львів – Гавареччина – Свята гора – Білий камінь – Підлисся – Біла гора – Кути – Львів у 2013 році. Стадія розви- тку почалася у 2019 році, коли в село почали прибувати туристи, здебільшого українські, поодинокі випадки – туристи з Польщі та Німеччини, проводитися майстер-класи для Досвід формування креативної турис- тичної дестинації у Львівській області ц у Незалежно від розмірів території, існує загальна послідовність управління креативним туризмом. Виклад основного матеріалу дослі- дження. Були створені майстерні, виставкові зали, музей килимарства та тка- цтва Глинян, конференц-зали. В музеї також є три ткалі, котрі переймають досвід створення килимів. На основі глинянського килимарства був розроблений туристичний маршрут. р д р у у У Львівській області основним осередком створення дерев’яної іграшки було м. Яво- рів. До сьогоднішніх часів збереглися зде- більшого дерев’яні та керамічні екземпляри XIX століття: ляльки, птахи, візочки, коники, меблі, дзиґи, вітряки, механічні іграшки, голо- воломки [36; 37]. Окрім того, Яворів – давній осередок таких ремесел, як різьблення на деревині, декоративний розпис, вишивка, тка- цтво, виробництво сувенірів. Значна кількість осередків вишивання окреслилася на Львівщині. Кожному з осе- редку були властиві свої кольори, візерунки, крій та тканина сорочки. На Яворівщині використовували яскраві кольори, червоно- Кушнірство – це традиційне українське ремесло, що включало вичинку шкіри з хутром ТУРИЗМ Рис. 1. Географія поширення ремесел у Львівській області Джерело: розроблено авторами ТУРИЗМ Рис. 1. Географія поширення ремесел у Львівській області Джерело: розроблено авторами Випуск # 36 / 2022 ЕКОНОМІКА ТА СУСП і пошиття з неї одягу. Таке ремесло, що осно- вним чином пов’язане з галуззю мисливства, хутровим промислом, успішно розвивалося на території України. Основним осередком кушнір- ства на Львівщині було місто Старий Самбір. Наприкінці ХІХ – поч. ХХ ст. в поселеннях Західної України почали відкривати школи лозоплетіння, зокрема у селах Джурів та Іза, містах Львів, Сторожинець, Яворів. Діяльність шкіл згодом стала початком для формування лозомеблевих артілей. Досвід формування креативної турис- тичної дестинації у Львівській області Незалежно від розмірів території, існує загальна послідовність управління креативним туризмом. Вона включає два етапи: підготов- чий та етап впровадження, що, в свою чергу, містять певну кількість кроків. На першому, під- готовчому етапі слід виділити головний ресурс креативного туризму, на основі якого можна будувати стратегію, після виявлення ресурсу необхідно підібрати підхід та такі інструменти управління, що забезпечать ефективну реалі- зацію стратегії, та, останній крок на підготов- чому етапі включає саме управління. Другий етап – етап впровадження залучає інші, допо- міжні ресурси, дії, чи будь-які каталізатори, що сприятимуть реалізації стратегії. Наступним кроком етапу впровадження є виявлення та розуміння значення такого напрямку розвитку для різних сторін та отримання результатів [33]. Формування і розвиток креативно тичної дестинації неможливі без ефе управління туристичним ресурсом. управління полягає не тільки в до поставлених цілей, але й у взаємоз Приклад Таїланду, зокрема, ілюструє ність і конкретні завдання для залуче зацікавлених сторін, включаючи д приватні, громадянські та наукові уст також місцевих громад і туристів до створення місця [34]. Виклад основного матеріалу дослі- дження. Вона включає два етапи: підготов- чий та етап впровадження, що, в свою чергу, містять певну кількість кроків. На першому, під- готовчому етапі слід виділити головний ресурс креативного туризму, на основі якого можна будувати стратегію, після виявлення ресурсу необхідно підібрати підхід та такі інструменти управління, що забезпечать ефективну реалі- зацію стратегії, та, останній крок на підготов- чому етапі включає саме управління. Другий етап – етап впровадження залучає інші, допо- міжні ресурси, дії, чи будь-які каталізатори, що сприятимуть реалізації стратегії. Наступним кроком етапу впровадження є виявлення та розуміння значення такого напрямку розвитку для різних сторін та отримання результатів [33]. ТУРИЗМ Рис. 2. Стадії розвитку Гавареччини як креативної туристичної дестинації Джерело: розроблено авторами Рис. 2. Стадії розвитку Гавареччини як креативної туристичної дестинації Джерело: розроблено авторами ЕКОНОМІКА ТА СУСПІЛЬСТВО Випуск # 36 / 2022 також застосовувати нові способи комунікації не лише з туристами, а й з іншими суб’єктами туристичної діяльності. дітей та дорослих. Про туристичний контекст розвитку села йдеться у Програмі соціально- економічного та культурного розвитку Золо- чівського району на 2022 рік. Поєднання ремесла та туризму, перш за все, створює додаткову вигоду: відкриває новий напрямок розвитку туризму, створює додану вартість на продукти ремісничого виробни- цтва. По-друге, сприяє сталому розвитку тери- торії, зберігаючи місцеве заняття, приваблює туристів, котрі готові залишати гроші в тій чи іншій громаді, тим самим сприяє її розвитку (як у контексті туристичної інфраструктури, так і в контексті вигоди для місцевого населення), оживляє простори. По-третє, створює робочі місця серед місцевого населення, а туризм, в свою чергу, забезпечує прибутковість від заняття тим чи іншим ремеслом. По-четверте, обрання ремесла як основи для креативного туризму сприяє його збереженню, що стало гострим питанням для багатьох країн, оскільки ремесло слугує відображенням історії народу та країни, її особливих традицій та культури. Отже, село Гавареччина демонструє успіх на підготовчому етапі формування та роз- витку креативної туристичної дестинації на основі місцевого ремесла, проте поки бракує розробленої стратегії розвитку туризму та ефективних заходів для зростання популяр- ності ремесла. Ефективним кроком може бути організація ярмарків та ринків, а також співп- раця місцевих ремісників з інформаційними центрами, готелями та садибами зеленого туризму, місцевими гідами, спеціалістами з туризму місцевої ради тощо [38]. Висновки. Отже, Львівська область багата на ремесла, які знаходяться на різних етапах свого існування. Досвід країн світу та окремих поселень (Гавареччина) демонструє можли- вість використання ремесла як основи для формування креативних туристичних дести- націй. СПИСОК ВИКОРИСТАНИХ ДЖЕРЕЛ 1. The World Travel & Tourism Council. Available at: https://wttc.org/Research/Economic-Impact (accessed 17 January 2022). 2. Ministerstvo ekonomichnoho rozvytku i torhivli Ukrainy. Sektor rozvytku kreatyvnykh industrii. Available at: https://regulation.gov.ua/catalogue/department/id4415 (accessed 17 January 2022). 3. Dyrektorat kreatyvnykh industrii. 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https://openalex.org/W1916770830	https://vbn.aau.dk/ws/files/248893601/energies_10_00050.pdf	English	null	Thermal impact analysis of circulating current in high power modular online uninterruptible power supplies application	null	2,015	cc-by	11,184	Thermal Impact Analysis of Circulating Current in High Power Modular Online Uninterruptible Power Supplies Application Thermal Impact Analysis of Circulating Current in High Power Modular Online Uninterruptible Power Supplies Application Published in: Energies DOI (link to publication from Publisher): 10.3390/en10010050 DOI (link to publication from Publisher): 10.3390/en10010050 Citation for published version (APA): Zhang, C., Guerrero, J. M., & Quintero, J. C. V. (2017). Thermal Impact Analysis of Circulating Current in High Power Modular Online Uninterruptible Power Supplies Application. Energies, 10(1), Article 50. https://doi.org/10.3390/en10010050 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Citation for published version (APA): Zhang, C., Guerrero, J. M., & Quintero, J. C. V. (2017). Thermal Impact Analysis of Circulating Current in High Power Modular Online Uninterruptible Power Supplies Application. Energies, 10(1), Article 50. https://doi.org/10.3390/en10010050 Thermal Impact Analysis of Circulating Current in High Power Modular Online Uninterruptible Power Supplies Application Zhang, Chi; Guerrero, Josep M.; Quintero, Juan Carlos Vasquez Published in: Energies DOI (link to publication from Publisher): 10.3390/en10010050 Publication date: 2017 Document Version Publisher's PDF, also known as Version of record Link to publication from Aalborg University Citation for published version (APA): Zhang, C., Guerrero, J. M., & Quintero, J. C. V. (2017). Thermal Impact Analysis of Circulating Current in High Power Modular Online Uninterruptible Power Supplies Application. Energies, 10(1), Article 50. https://doi.org/10.3390/en10010050 Thermal Impact Analysis of Circulating Current in High Power Modular Online Uninterruptible Power Supplies Application Zhang, Chi; Guerrero, Josep M.; Quintero, Juan Carlos Vasquez Published in: Energies DOI (link to publication from Publisher): 10.3390/en10010050 Publication date: 2017 Document Version Publisher's PDF, also known as Version of record Link to publication from Aalborg University Citation for published version (APA): Zhang, C., Guerrero, J. M., & Quintero, J. C. V. (2017). Thermal Impact Analysis of Circulating Current in High Power Modular Online Uninterruptible Power Supplies Application. Energies, 10(1), Article 50. https://doi.org/10.3390/en10010050 Aalborg Universitet Thermal Impact Analysis of Circulating Current in High Power Modular Online Uninterruptible Power Supplies Application Zhang, Chi; Guerrero, Josep M.; Quintero, Juan Carlos Vasquez Published in: Energies DOI (link to publication from Publisher): 10.3390/en10010050 Publication date: 2017 Document Version Publisher's PDF, also known as Version of record Link to publication from Aalborg University Citation for published version (APA): Zhang, C., Guerrero, J. M., & Quintero, J. C. V. (2017). Thermal Impact Analysis of Circulating Current in High Power Modular Online Uninterruptible Power Supplies Application. Energies, 10(1), Article 50. https://doi.org/10.3390/en10010050 Aalborg Universitet Thermal Impact Analysis of Circulating Current in High Power Modular Online Uninterruptible Power Supplies Application Chi Zhang, Josep M. Guerrero * and Juan C. Vasquez Department of Energy Technology, Aalborg University, Pontoppidanstraede 111, 9220 Aalborg, Denmark; qantoo.zc@gmail.com (C.Z.); juq@et.aau.dk (J.C.V.) * Correspondence: joz@et.aau.dk; Tel.: +45-20378262 Chi Zhang, Josep M. Guerrero * and Juan C. Vasquez Department of Energy Technology, Aalborg University, Pontoppidanstraede 111, 9220 Aalborg, Denmark; qantoo.zc@gmail.com (C.Z.); juq@et.aau.dk (J.C.V.) * Correspondence: joz@et.aau.dk; Tel.: +45-20378262 Academic Editor: Chunhua Liu Received: 17 October 2016; Accepted: 26 December 2016; Published: 4 January 2017 Academic Editor: Chunhua Liu Received: 17 October 2016; Accepted: 26 December 2016; Published: 4 January 2017 Academic Editor: Chunhua Liu Received: 17 October 2016; Accepted: 26 December 2016; Published: 4 January 2017 Abstract: In modular uninterruptible power supplies (UPSs), several DC/AC modules are required to work in parallel. This structure allows the system to be more reliable and ﬂexible. These DC/AC modules share the same DC bus and AC critical bus. Module differences, such as ﬁlter inductor, ﬁlter capacitor, control parameters, and so on, will make it possible for the potential zero sequence current to ﬂow among the modules. This undesired type of circulating current will bring extra losses to the power semiconductor devices in the system, which should be paid special attention in high power application scenarios. In this paper, plug’n’play modules and cycle control are discussed and validated through experimental results. Moreover, potential zero sequence circulating current impact on power semiconductor devices thermal performance is also analyzed in this paper. Keywords: modular; online uninterruptible power supplies (UPSs) system; thermal evaluation Energies 2017, 10, 50; doi:10.3390/en10010050 General rights C i h d - Users may download and print one copy of any publication from the public portal for the purpose of private study or research. - You may not further distribute the material or use it for any profit-making activity or commercial gain - Users may download and print one copy of any publication from the public portal for the purpose of private study or research. - You may not further distribute the material or use it for any profit-making activity or commercial gain - Users may download and print one copy of any publication from the public portal for the purpose of private study or re You may not further distribute the material or use it for any profit making activity or commercial gain You may not further distribute the material or use it for any profit making activity o - You may freely distribute the URL identifying the publication in the public portal - y y p g y - You may freely distribute the URL identifying the publication in the public portal - Take down policy If you believe that this document breaches copyright please contact us at vbn@aub.aau.dk providing details, and w the work immediately and investigate your claim. y this document breaches copyright please contact us at vbn@aub.aau.dk providing details, and we will remove access to ely and investigate your claim. www.mdpi.com/journal/energies energies energies 1. Introduction Recently, more and more advanced technologies are entering human beings everyday life. A large number of modern electrical facilities, such as communication equipment, IT facilities, medical equipment, and so on, are widely used in both the industrial ﬁeld and daily life [1]. As a result, the utility suffers great challenges in order to provide a clean, continuous, and reliable electrical energy. On the other hand, with the prosperity of the renewable energy sources, more and more factories and ofﬁces tend to use a local smart utility system [2]. However, renewable energy sources are unpredictable. This will have negative impacts on the power quality that they provide. Consequently, uninterruptible power supply (UPS) system is receiving more and more attention from both researchers and industrial application engineers [3]. Based on IEC 62040-3, UPS system is able to be divided into three categories, namely ofﬂine UPS system, line interactive UPS system, and online UPS system. Compared with the other two, online UPS system, also known as double conversion UPS system, mainly aims at high power application scenarios due to its outstanding capability in output voltage control and decoupling of the utility and the load [4,5]. In order to improve UPS performance, a series of enhanced different online UPS structure is proposed in [6–8]. Among them, the modular concept is drawing more and more attention due to its attractive ﬂexibility and expansibility. Consequently, when the system is required to be maintained or has module failure, a modular online UPS system is still able to operate normally by replacing to stop the damaged module. g Normally a number of DC/AC modules operates in parallel and shares the same DC bus and AC bus (as shown in Figure 1a) in a typical modular online UPS system. Thus, possible paths for potential zero sequence circulating current exist (which is marked by red in Figure 1b). It still has negative Energies 2017, 10, 50; doi:10.3390/en10010050 www.mdpi.com/journal/energies 2 of 11 Energies 2017, 10, 50 impacts on losses and thermal distribution of the power semiconductor devices. The unbalanced losses and thermal distribution performances, especially in high power modular scenarios, are required to be investigated further, which can be a guidance concerning heat problems in designing the modular online UPS systems. In order to avoid such kinds of circulating current, a number of approaches are proposed, namely isolation [9–11], high impedance [12,13], synchronized issues [14–17], and active issues [18–20]. 1. Introduction Isolation and high impedance issues will add extra devices into the system, such as extra DC sources, transformers, or inter-phase reactors and these will increase the UPS system cost. As such, it is not a cost effective and volume effective issue. As a result, synchronization and active issues are considered hereby. Energies 2017, 10, 50 2 of 11 required to be investigated further, which can be a guidance concerning heat problems in designing the modular online UPS systems. In order to avoid such kinds of circulating current, a number of approaches are proposed, namely isolation [9–11], high impedance [12,13], synchronized issues [14– 17], and active issues [18–20]. Isolation and high impedance issues will add extra devices into the system, such as extra DC sources, transformers, or inter-phase reactors and these will increase the UPS system cost. As such, it is not a cost effective and volume effective issue. As a result, synchronization and active issues are considered hereby. Figure 1. Modular uninterruptible power supplies (UPSs) system architecture and circulating current path: (a) modular UPS system structure; and (b) circulating current flowing path. DC bus AC bus idc1 idc2 ia2 ib2 ic2 ia1 ib1 ic1 va1 vb1 vc1 va2 vb2 vc2 T1 D1 T2 D2 AC/DC DC/AC #1 Isolated Transformer Utility side Static Bypass Switch Load side Normal DC/AC #2 Central Controller CAN Bus Battery Pack Emergency Mode DC/DC AC Bus DC Bus DC/AC #n …… Figure 1. Modular uninterruptible power supplies (UPSs) system architecture and circulating current path: (a) modular UPS system structure; and (b) circulating current ﬂowing path. DC bus AC bus idc1 idc2 ia2 ib2 ic2 ia1 ib1 ic1 va1 vb1 vc1 va2 vb2 vc2 T1 D1 T2 D2 s AC/DC DC/AC #1 Isolated Transformer Utility side Static Bypass Switch Load side Normal DC/AC #2 Central Controller CAN Bus Battery Pack Emergency Mode DC/DC AC B DC Bus DC/AC #n …… Figure 1. Modular uninterruptible power supplies (UPSs) system architecture and circulating current path: (a) modular UPS system structure; and (b) circulating current flowing path. Figure 1. Modular uninterruptible power supplies (UPSs) system architecture and circulating current path: (a) modular UPS system structure; and (b) circulating current ﬂowing path. On the other hand, active power and reactive power of each of the DC/AC modules should be equally shared in order to minimize the active power circulating current. 1. Introduction A number of parallel technologies have been proposed in [21–26], which can be categorized into two main types, namely communication based and communication-less. Since the mature digital signal processor (DSP) technology provides a smaller data transmit time delay, a communication based parallel technology is used here. By using virtual resistor and phase regulating methods, both active and reactive power are shared among all the modules. In this paper, unsynchronized pulse width modulation (PWM) condition and physical On the other hand, active power and reactive power of each of the DC/AC modules should be equally shared in order to minimize the active power circulating current. A number of parallel technologies have been proposed in [21–26], which can be categorized into two main types, namely communication based and communication-less. Since the mature digital signal processor (DSP) technology provides a smaller data transmit time delay, a communication based parallel technology is used here. By using virtual resistor and phase regulating methods, both active and reactive power are shared among all the modules. In this paper, unsynchronized pulse width modulation (PWM) condition and physical parameters mismatch is considered since these two are more common in real applications. With the power devices thermal model proposed in [27], the thermal impedance of devices is edited in the simulation software PLECS. Thus, the temperature and losses of devices are able to be monitored to investigate the zero circulating current impacts on the losses and temperature of different power semiconductor devices In this paper, unsynchronized pulse width modulation (PWM) condition and physical parameters mismatch is considered since these two are more common in real applications. With the power devices thermal model proposed in [27], the thermal impedance of devices is edited in the simulation software PLECS. Thus, the temperature and losses of devices are able to be monitored to investigate the zero circulating current impacts on the losses and temperature of different power semiconductor devices. semiconductor devices. This paper is organized as follows: Section 2 depicts the system control mechanism; Section 3 presents experimental results while the thermal analysis results are presented in Section 4; and Section 5 gives the final conclusion This paper is organized as follows: Section 2 depicts the system control mechanism; Section 3 presents experimental results while the thermal analysis results are presented in Section 4; and Section 5 gives the ﬁnal conclusion. 2.1. Single Module Control DC/AC modules inner loop uses conventional double control structure (voltage and current), which is considered in αβ frame: Gv(s) = kpv + krvs s2 + ωo2 + ∑ h=5,7,11 khrvs s2 + (ωoh)2 (1) Gc(s) = kpc + krcs s2 + ωo2 + ∑ h=5,7,11 khrcs s2 + (ωoh)2 (2) (1) Gc(s) = kpc + krcs s2 + ωo2 + ∑ h=5,7,11 khrcs s2 + (ωoh)2 (2) (2) with kpv, krv, ωo, khrv, h, kpc, krc, and khrc being the voltage proportional term, fundamental frequency voltage resonant term, fundamental frequency, the hth harmonic voltage compensation term, harmonic order, current proportional term, fundamental frequency current resonant term, and the hth harmonic current compensation term, respectively. Two typical PRs are used due to its harmonic compensation capability. 2.2. Power Balance among Modules As mentioned in [24], if the output impedance of the DC/AC modules is designated to be mainly resistive, the active power and reactive power that each DC/AC module injects into the AC bus are able to be regulated by voltage amplitude and phase, respectively, thus a virtual resistor and phase regulating loop are inserted into the control loop: δnk = δnkre f −kphQnk (3) vnk = vnkre f −iLabcZvir (4) (3) (4) vnk = vnkre f −iLabcZvir (4) Here n is the number of DC/AC module (1, 2, 3, . . . , N), k is the phase order (a, b, c), Vnkref is the nominal voltage reference, Zvir is the virtual resistor, δnkref is the nominal phase reference, kph is the phase regulating coefﬁcients, and Qnk is the reactive power of each phase of each DC/AC module. 2.3. Voltage Amplitude and Phase Synchronized 2 System Control Mechanism 2. System Control Mechanism As shown in Figure 1a, one three-phase AC/DC creates the DC bus for the DC/AC modules. Its control is considered in the conventional dq frame, which is descripted in [28] in detail. Several DC/AC modules are working in parallel and the detailed information for the modules is listed in As shown in Figure 1a, one three-phase AC/DC creates the DC bus for the DC/AC modules. Its control is considered in the conventional dq frame, which is descripted in [28] in detail. Several DC/AC modules are working in parallel and the detailed information for the modules is listed in Table 1. 3 of 11 Energies 2017, 10, 50 Table 1. Module characteristics. Nominal Power Switch Frequency DC Side Bus Voltage Bus Current 200 kW 5 kHz 700 V 230 V 290 A Phase Number Topology Filter Inductor Capacitance 3 H_bridge LC ﬁlter 1.8 mH 27 µF Single Module Control 2.1. Single Module Control 2.4. Cycle and Plug’n’Play Control The modular online UPS system is designed to be ﬂexible, which means that any of the DC/AC modules can be plugged into or out of the system randomly due to module failure or maintenance reasons. Thus, some voltage overshoot or sag will occur in the AC critical bus, and this requires a control that can suppress the voltage overshoot or sag in a short transient time. In the standard IEC 62040-3, detailed requirements are included for the transient performance for an online UPS system, which is shown in Table 2. Energies 2017, 10, 50 4 of 11 62040-3, detailed requirements are included for the transient performance for an online UPS system, which is shown in Table 2. +- Vref Vbus δref δbus +- + - Gv(s) Gc(s) PWM+Filter vc vref iL Zvir iL kph Q Gv_rec(s) Gδ_rec(s) Vref δg + +- vc + + + - + Synchronization Loop vrec δrec CAN Bus Power Sharing Loop Inner Loop V δ sin( ) V t ω δ + Figure 2. Control mechanism for modular UPS system: (a) overall control; and (b) plug’n’play control mechanism. PWM: pulse width modulation. +- Vref Vbus δref δbus +- + - Gv(s) Gc(s) PWM+Filter vc vref iL Zvir iL kph Q Gv_rec(s) Gδ_rec(s) Vref δg + +- vc + + + - + Synchronization Loop vrec δrec CAN Bus Power Sharing Loop Inner Loop V δ sin( ) V t ω δ + …… Figure 2. Control mechanism for modular UPS system: (a) overall control; and (b) plug’n’play control mechanism. PWM: pulse width modulation. …… Figure 2. Control mechanism for modular UPS system: (a) overall control; and (b) plug’n’play control mechanism. PWM: pulse width modulation. Figure 2. Control mechanism for modular UPS system: (a) overall control; and (b) plug’n’play control mechanism. PWM: pulse width modulation. Table 2. Transient time requirements. Volta e Va iatio (%) Dy a i Ti e Table 2. Transient time requirements. ab e a ie i e equi e e Load Type Voltage Variation (%) Dynamic Time Requirement (ms) Linear Load 14 20–40 12 40–60 11 60–100 10 100–1000 Nonlinear Load 12 40–60 11 60–100 10 100–1000 t can be seen that with a smaller voltage overshoot or sag, the transient time for the sys Table 2. Transient time requirements. 2.4. Cycle and Plug’n’Play Control Load Type Voltage Variation (%) Dynamic Time Requirement (ms) Linear Load 14 20–40 12 40–60 11 60–100 10 100–1000 Nonlinear Load 12 40–60 11 60–100 10 100–1000 be a little longer. While designing the system, the overshoot or sag should be guaranteed that its amplitude should be controlled small as 10% compared with the nominal critical bus voltage amplitude. Thus, less stress will be put on the transient performance of the controller. On the other hand, another reason for using modular UPS structure is that modules can operate in turns in order to allow all modules to be maintained or repaired equally as shown in Figure 2b It can be seen that with a smaller voltage overshoot or sag, the transient time for the system can be a little longer. While designing the system, the overshoot or sag should be guaranteed that its amplitude should be controlled small as 10% compared with the nominal critical bus voltage amplitude. Thus, less stress will be put on the transient performance of the controller. in turns in order to allow all modules to be maintained or repaired equally, as shown in Figure 2b. When one of the modules disconnects with the AC critical bus, another module that has already finished the maintenance will start to connect with the AC critical bus at the same time in a fixed sequence, called Normal Cycle (NC). As a result, there is voltage transient during this process, which should be paid specific attention. Normally, such a cycle duration time is quite long in real applications, for instance, routinely numbering in the hours. However, in case of an emergency condition, such as module failures, the fixed sequence is interrupted and it will have a much shorter cycle duration time, which is called Emergency Cycle (EC). Such kinds of plug’n’play modules will result in PWM unsynchronized problems, which will cause strong potential zero sequence circulating current In addition although each module is On the other hand, another reason for using modular UPS structure is that modules can operate in turns in order to allow all modules to be maintained or repaired equally, as shown in Figure 2b. When one of the modules disconnects with the AC critical bus, another module that has already ﬁnished the maintenance will start to connect with the AC critical bus at the same time in a ﬁxed sequence, called Normal Cycle (NC). 2.3. Voltage Amplitude and Phase Synchronized 2.3. Voltage Amplitude and Phase Synchronized By measuring AC critical bus voltage, two typical Proportional Integrator (PI) controllers are used to keep online UPS output voltage tightly synchronized with the utility: Gv_rec(s) = kpv_rec + kiv_rec s (5) Gδ_rec(s) = kpδ_rec + kiδ_rec s (6) (5) Gδ_rec(s) = kpδ_rec + kiδ_rec s (6) (6) with kpv_rec, kiv_rec, kpδ_rec, and kiδ_rec being voltage amplitude recovery proportional term, voltage amplitude recovery integral term, phase recover proportional term, and phase recover integral term, respectively. The overall control diagram is shown in Figure 2a. with kpv_rec, kiv_rec, kpδ_rec, and kiδ_rec being voltage amplitude recovery proportional term, voltage amplitude recovery integral term, phase recover proportional term, and phase recover integral term, respectively. The overall control diagram is shown in Figure 2a. 4 of 11 Energies 2017, 10, 50 2.4. Cycle and Plug’n’Play Control As a result, there is voltage transient during this process, which should be paid speciﬁc attention. Normally, such a cycle duration time is quite long in real applications, for instance, routinely numbering in the hours. However, in case of an emergency condition, such as module failures, the ﬁxed sequence is interrupted and it will have a much shorter cycle duration time, which is called Emergency Cycle (EC). cause strong potential zero sequence circulating current. In addition, although each module is designated to be the same, they still have many differences, such as filter inductors, capacitance, control parameters, and so on. These things will also provide the possibility for the zero sequence Such kinds of plug’n’play modules will result in PWM unsynchronized problems, which will cause strong potential zero sequence circulating current. In addition, although each module is designated to 5 of 11 Energies 2017, 10, 50 be the same, they still have many differences, such as ﬁlter inductors, capacitance, control parameters, and so on. These things will also provide the possibility for the zero sequence circulating current to occur. be the same, they still have many differences, such as ﬁlter inductors, capacitance, control parameters, and so on. These things will also provide the possibility for the zero sequence circulating current to occur. 3. Experimental Results An experimental setup was built in the MicroGrid Intelligent Lab [28]. Four Danfoss converters were used. One of them works as AC/DC while the other three are working as the DC/AC modules. A control system was established in Matlab/Simulink and complied into the dSpace 1006 to achieve the real time control for the experimental setup. Energies 2017, 10, 50 5 of 11 A control system was established in Matlab/Simulink and complied into the dSpace 1006 to achieve the real time control for the experimental setup. 3.1. Modules Plug’n’Play Performance 3.1. Modules Plug’n’Play Performance Hereby, one of the modules connected and disconnected with the AC critical bus in order to test the proposed control transient performance. Active and reactive power performance was monitored on the control desk in PC while the voltage real time performance was observed in scope. Hereby, one of the modules connected and disconnected with the AC critical bus in order to test the proposed control transient performance. Active and reactive power performance was monitored on the control desk in PC while the voltage real time performance was observed in scope. g p p At t1, one module was plugging out of the system and both reactive and active power was well shared among the other two modules. At t2, it plugged into the system again and the power was equally shared among the three modules, as shown in Figure 3a,b. on the control desk in PC while the voltage real time performance was observed in scope. At t1, one module was plugging out of the system and both reactive and active power was well shared among the other two modules. At t2, it plugged into the system again and the power was equally shared among the three modules, as shown in Figure 3a,b. Figure 3. Power performance: (a) one module plug out; (b) module plug in; and (c) cycle control. Figure 3. Power performance: (a) one module plug out; (b) module plug in; and (c) cycle control. Figure 3. Power performance: (a) one module plug out; (b) module plug in; and (c) cycle control. Figure 3. Power performance: (a) one module plug out; (b) module plug in; and (c) cycle control. The thermal conditi 4. Thermal Analysis Results Figure 4. consideration. It is tightly related with the cooling system of each DC/AC module, especially in high power application scenarios. Based on the thermal model shown in Figure 5, the thermal impedance from junction to case Z(j-c) is able to be treated as a four layer resistor-cpacitor (RC) network. For a specific power device, the information can be found in the manufacture datasheet. Since the thermal capacitance is mainly related with the dynamic process before steady state [29], big thermal capacitance of ZT/D(c-h) and Z(h-a) can be neglected in order to achieve a faster simulation. Tj Tj IGBT Diode ZT(j ) Tj: Junction temperature Tc: Case temperature TH: Heatsink temperature TA: Ambient temperature The thermal condition of power devices is an important issue that should be taken into consideration. It is tightly related with the cooling system of each DC/AC module, especially in high power application scenarios. Based on the thermal model shown in Figure 5, the thermal impedance from junction to case Z(j-c) is able to be treated as a four layer resistor-cpacitor (RC) network. For a speciﬁc power device, the information can be found in the manufacture datasheet. Since the thermal capacitance is mainly related with the dynamic process before steady state [29], big thermal capacitance of ZT/D(c-h) and Z(h-a) can be neglected in order to achieve a faster simulation. 4. Thermal Analysis Results The thermal condition of power devices is an important issue that should be taken into consideration. It is tightly related with the cooling system of each DC/AC module, especially in high power application scenarios. Based on the thermal model shown in Figure 5, the thermal impedance from junction to case Z(j-c) is able to be treated as a four layer resistor-cpacitor (RC) network. For a specific power device, the information can be found in the manufacture datasheet. Since the thermal capacitance is mainly related with the dynamic process before steady state [29], big thermal capacitance of ZT/D(c-h) and Z(h-a) can be neglected in order to achieve a faster simulation. Figure 5. Thermal model: (a) power devices thermal model; and (b) IGBT resistor-cpacitor (RC) network of ZT/D(j-c). Based on the module information shown in Table 1, the IGBT pack 5SND-0800M170100 from ABB was chosen for the power semiconductor devices, whose thermal information is shown in Table 3 [30]. The thermal conditi 4. Thermal Analysis Results Figure 4. TA TA TA Tc Tc TH ZD(j-c) ZT(c-h) ZD(c-h) Z(h-a) Z(j-c) : Thermal impedance form junction to case Z(c-h): Thermal impedance from case to heat sink Z(h-a): Thermal impedance from heat sink to ambient Rth1 Rth2 Rth3 Rth4 τ1 τ2 τ3 τ4 Tc TA Tj ZT/D(j-c) (a) (b) Figure 5. Thermal model: (a) power devices thermal model; and (b) IGBT resistor-cpacitor (RC) network of ZT/D(j-c). B d th d l i f ti h i T bl 1 th IGBT k 5SND 0800M170100 f TA TA TA Tc Tc TH Tj Tj IGBT Diode ZT(j-c) ZD(j-c) ZT(c-h) ZD(c-h) Z(h-a) Tj: Junction temperature Tc: Case temperature TH: Heatsink temperature TA: Ambient temperature Z(j-c) : Thermal impedance form junction to case Z(c-h): Thermal impedance from case to heat sink Z(h-a): Thermal impedance from heat sink to ambient Rth1 Rth2 Rth3 Rth4 τ1 τ2 τ3 τ4 Tc TA Tj ZT/D(j-c) (a) (b) Figure 5. Thermal model: (a) power devices thermal model; and (b) IGBT resistor-cpacitor (RC) network of ZT/D(j-c). ABB was chosen for the power semiconductor devices, whose thermal information is shown in Table 3 [30]. Figure 5. Thermal model: (a) power devices thermal model; and (b) IGBT resistor-cpacitor (RC) network of ZT/D(j-c). Based on the module information shown in Table 1 the IGBT pack 5SND 0800M170100 from Figure 5. Thermal model: (a) power devices thermal model; and (b) IGBT resistor-cpacitor (RC) network of ZT/D(j-c). Thermal Impedance ZT/D(j-c) 1 2 3 4 RiIGBT (K/kW) 15.2 3.6 1.49 0.74 ABB was chosen for the power semiconductor devices, whose thermal information is shown in Table 3 [30]. Table 3 Module the al a a ete Based on the module information shown in Table 1, the IGBT pack 5SND-0800M170100 from ABB was chosen for the power semiconductor devices, whose thermal information is shown in Table 3 [30]. τiIGBT (ms) 202 20.3 2.01 0.52 RiDiode (K/kW) 25.3 5.78 2.6 2.52 ( ) 210 29 6 7 01 1 49 Thermal Impedance ZT/D(j-c) 1 2 3 4 Table 3. Module thermal parameters. τiIGBT (ms) 202 20.3 2.01 0.52 RiDiode (K/kW) 25.3 5.78 2.6 2.52 τiIGBT (ms) 210 29.6 7.01 1.49 With PLECS, the thermal impedance model of IGBT pack 5SND-0800M170100 can be edit simulation. Thus, thermal information, including losses and temperature can be monitored. 3.2. Cycle Control Transient Performance 3.2. Cycle Control Transient Performance The transient performance, when two DC/AC modules were switched, is presented in Figure 3c. In real applications, supervisory control monitors the operating condition of each of the DC/AC modules to decide operating rules. At t3, module #3 was ordered to disconnect and rest for maintenance and module #2 was required to start working again in order to avoid overloading of the other modules, and at t4, module #2 and #3 were switched for the second time. It can be observed that both active power and reactive power were well shared among the remaining modules. The voltage detail at t3 and t4 is shown in Figure 4a,b. The voltage overshoot can be calculated The transient performance, when two DC/AC modules were switched, is presented in Figure 3c. In real applications, supervisory control monitors the operating condition of each of the DC/AC modules to decide operating rules. At t3, module #3 was ordered to disconnect and rest for maintenance and module #2 was required to start working again in order to avoid overloading of the other modules, and at t4, module #2 and #3 were switched for the second time. It can be observed that both active power and reactive power were well shared among the remaining modules. 6 of 11 Energies 2017, 10, 50 Energies 2017, 10, 50 The voltage detail at t3 and t4 is shown in Figure 4a,b. The voltage overshoot can be calculated through the scope, which is ∆v = (620 −570) V/570 V = 8.77% or (610 −560) V/560 V = 8.92%. This value is below 10%, and the transient time is about three and a half utility periods (i.e., 70 ms), which is faster than 100 ms. Energies 2017, 10, 50 6 of 11 Figure 4. Real time voltage waveform: (a) details at t3; and (b) details at t4. 4. Thermal Analysis Results Figure 4. Real time voltage waveform: (a) details at t3; and (b) details at t4. Energies 2017, 10, 50 6 of 11 6 of 11 Energies 2017, 10, 50 Figure 4. Real time voltage waveform: (a) details at t3; and (b) details at t4. Figure 4. Real time voltage waveform: (a) details at t3; and (b) details at t4. 4.1. Single DC/AC Module 75%. As such, in the temperature informa In order to compare with the modular UPS system, a single DC/AC module was simulated. Figure 6a,b presents the devices temperature and losses. It can be seen that the losses and temperature are distributed nearly balanced between T1 and T2, and D1 and D2. 4.1. Single DC/AC Module In order to compare with the modular UPS system, a single DC/AC module was simulated. Figure 6a,b presents the devices temperature and losses. It can be seen that the losses and temperature Energies 2017, 10, 50 7 of 11 75% A h i th l i th l d diti i id d f 50% t 100% d l d Another critical parameter, namely the temperature ﬂuctuation amplitude of the devices, is also important for power semiconductor devices. In Figure 6c,d, it can be observed that T1 and T2 share nearly the same temperature ﬂuctuation amplitude, and a similar result can be seen for D1 and D2. Figure 6e shows the steady state temperature information in 100% load condition. g , p p p are distributed nearly balanced between T1 and T2, and D1 and D2. Another critical parameter, namely the temperature fluctuation amplitude of the devices, is also important for power semiconductor devices. In Figure 6c,d, it can be observed that T1 and T2 share nearly the same temperature fluctuation amplitude, and a similar result can be seen for D1 and D2. Figure 6e shows the steady state temperature information in 100% load condition. 75%. As such, in the analysis, the load condition is considered from 50% to 100%, and losses and temperature information of T1, T2, D1, and D2 are discussed. 4.1. Single DC/AC Module In order to compare with the modular UPS system, a single DC/AC module was simulated. Figure 6a b presents the devices temperature and losses It can be seen that the losses and temperature Figure 6. Single module result: (a) junction temperature; (b) devices loss; (c) junction temperature fluctuation; (d) temperature fluctuation difference; and (e) detailed temperature result in 100% load. 4.2. In this condition, an unsynchronized PWM was given t 4.2. DC/AC Modules with Zero Sequence Circulating Current Figure 6. Single module result: (a) junction temperature; (b) fluctuation; (d) temperature fluctuation difference; and (e) deta each module filter inductor was given a 30% difference to emulate the manufacturer difference. The control parameters were also different among different modules. Thus, an obvious circulating current can be observed, as shown in Figure 7a. It can be seen that there is some DC component; this is because both physical and control parameters differences will result in a small amount of active power circulating current. 85 90 85 90 85 90 40 T1 T1 T1 Module #1 Module #2 In this condition, an unsynchronized PWM was given to the three DC/AC modules. Moreover, each module ﬁlter inductor was given a 30% difference to emulate the manufacturer difference. The control parameters were also different among different modules. Thus, an obvious circulating current can be observed, as shown in Figure 7a. It can be seen that there is some DC component; this is because both physical and control parameters differences will result in a small amount of active power circulating current. ; ( ) p ; ( ) p 4.2. DC/AC Modules with Zero Sequence Circulating Current In this condition, an unsynchronized PWM was given to the three DC/AC modules. Moreover, each module filter inductor was given a 30% difference to emulate the manufacturer difference. The control parameters were also different among different modules. Thus, an obvious circulating current can be observed, as shown in Figure 7a. It can be seen that there is some DC component; this is because both physical and control parameters differences will result in a small amount of active power circulating current. Figure 7. Circulating current and detailed temperature result with obvious circulating current: (a) three modules circulating current; and (b) detailed temperature in 100% load. 4.8 4.9 50 55 60 65 70 75 80 4.8 4.9 50 55 60 65 70 75 80 4.8 4.9 50 55 60 65 70 75 80 Module #1 Junction Temperature ( ) (a) (b) 1.4 1.41 1.42 1.43 1.44 1.45 -60 -40 -20 0 T2 D1 D2 Module #2 Junction Temperature ( ) Module #3 Junction Temperature ( ) T2 D1 D2 T2 D1 D2 Module #3 Figure 7. Circulating current and detailed temperature result with obvious circulating current: (a) three modules circulating current; and (b) detailed temperature in 100% load. The thermal conditi 4. Thermal Analysis Results Figure 4. In it mentions that normally DC/AC modules are designed to work in the load condition from 5 Thermal Impedance ZT/D(j-c) 1 2 3 4 RiIGBT (K/kW) 15.2 3.6 1.49 0.74 τiIGBT (ms) 202 20.3 2.01 0.52 RiDiode (K/kW) 25.3 5.78 2.6 2.52 τiIGBT (ms) 210 29.6 7.01 1.49 With PLECS, the thermal impedance model of IGBT pack 5SND-0800M170100 can be edi simulation. Thus, thermal information, including losses and temperature can be monitored. In it mentions that normally DC/AC modules are designed to work in the load condition from 5 Table 3. Module thermal parameters. Thermal Impedance ZT/D(j-c) 1 2 3 4 RiIGBT (K/kW) 15.2 3.6 1.49 0.74 τiIGBT (ms) 202 20.3 2.01 0.52 RiDiode (K/kW) 25.3 5.78 2.6 2.52 τiIGBT (ms) 210 29.6 7.01 1.49 Energies 2017, 10, 50 7 of 11 Energies 2017, 10, 50 With PLECS, the thermal impedance model of IGBT pack 5SND-0800M170100 can be edited in simulation. Thus, thermal information, including losses and temperature can be monitored. In [31], it mentions that normally DC/AC modules are designed to work in the load condition from 50% to 75%. As such, in the analysis, the load condition is considered from 50% to 100%, and losses and temperature information of T1, T2, D1, and D2 are discussed. Energies 2017, 10, 50 7 of 11 4.1. Single DC/AC Module 75%. As such, in the temperature informa In Figure 6c,d, it can be observed that T1 and T2 share ctuation amplitude, and a similar result can be seen for D1 and D2. temperature information in 100% load condition. 60 65 70 75 80 85 90 Junction Temperature ( ) Devices Total Losses (W) (b) Junction Temperature Fluctuation Amplitude T1 T2 D1 D2 0 200 400 600 800 000 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 1 1,5 2 2,5 ΔT1-ΔT2 ΔD1-ΔD2 (a) Junction Temperature Fluctuation Amplitude( ) Junction Temperature ( ) 60 65 70 75 80 85 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 1 0 2 4 6 8 50% 60% 70% 80% 90% 100% ΔT1 ΔD1 ΔT2 ΔD2 (c) are distributed nearly balanced Another critical parameter important for power semicond nearly the same temperature fl Figure 6e shows the steady stat (a) Junction Temperature ( ) 60 65 70 75 80 85 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 4 6 8 ΔT1 (e) Figure 6. Single module result: (a) junction temperature; (b) devices loss; (c) junction temperature fluctuation; (d) temperature fluctuation difference; and (e) detailed temperature result in 100% load. 4 2 DC/AC Modules with Zero Sequence Circulating Current Figure 6. Single module result: (a) junction temperature; (b) devices loss; (c) junction temperature ﬂuctuation; (d) temperature ﬂuctuation difference; and (e) detailed temperature result in 100% load. 4.8 4.9 5 50 55 Junction Temperature Fluctuation Amplitude( ) Difference( ) 0 2 50% 60% 70% 80% 90% 100% ΔT2 ΔD2 0 0,5 50% 60% 70% 80% 90% 100% (c) (d) (e) 4.1. Single DC/AC Module 75%. As such, in the temperature informa DC/AC Modules with Zero Sequence Circulating Current 4.8 4.9 5 50 55 60 65 70 75 80 85 90 Junction Temperature ( ) Devices Total Losses (W) (a) (b) Junction Temperature Fluctuation Amplitude( ) Junction Temperature Fluctuation Amplitude Difference( ) T1 T2 D1 D2 Junction Temperature ( ) 60 65 70 75 80 85 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 0 200 400 600 800 1000 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 0 2 4 6 8 50% 60% 70% 80% 90% 100% ΔT1 ΔD1 ΔT2 ΔD2 0 0,5 1 1,5 2 2,5 50% 60% 70% 80% 90% 100% ΔT1-ΔT2 ΔD1-ΔD2 (c) (d) (e) Figure 6. Single module result: (a) junction temperature; (b) devices loss; (c) junction temperature ﬂuctuation; (d) temperature ﬂuctuation difference; and (e) detailed temperature result in 100% load. are distributed nearly balanced between T1 and T2, and D1 and D2. Another critical parameter, namely the temperature fluctuation amplitude of the devices, is also important for power semiconductor devices. In Figure 6c,d, it can be observed that T1 and T2 share nearly the same temperature fluctuation amplitude, and a similar result can be seen for D1 and D2. Figure 6e shows the steady state temperature information in 100% load condition. 4.8 4.9 5 50 55 60 65 70 75 80 85 90 Junction Temperature ( ) Devices Total Losses (W) (a) (b) Junction Temperature Fluctuation Amplitude( ) Junction Temperature Fluctuation Amplitude Difference( ) T1 T2 D1 D2 Junction Temperature ( ) 60 65 70 75 80 85 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 0 200 400 600 800 1000 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 0 2 4 6 8 50% 60% 70% 80% 90% 100% ΔT1 ΔD1 ΔT2 ΔD2 0 0,5 1 1,5 2 2,5 50% 60% 70% 80% 90% 100% ΔT1-ΔT2 ΔD1-ΔD2 (c) (d) (e) 4.8 4.9 5 50 55 60 65 70 75 80 85 90 Junction Temperature ( ) Devices Total Losses (W) (b) Junction Temperature Fluctuation Amplitude Difference( ) T1 T2 D1 D2 0 00 00 00 00 00 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 0 ,5 1 ,5 2 ,5 50% 60% 70% 80% 90% 100% ΔT1-ΔT2 ΔD1-ΔD2 (d) (e) between T1 and T2, and D1 and D2. namely the temperature fluctuation amplitude of the devices, is also ctor devices. In this condition, an unsynchronized PWM was given t 4.2. DC/AC Modules with Zero Sequence Circulating Current Figure 6. Single module result: (a) junction temperature; (b) fluctuation; (d) temperature fluctuation difference; and (e) deta 4.8 4.9 50 55 60 65 70 75 80 85 90 4.8 4.9 50 55 60 65 70 75 80 85 90 4.8 4.9 50 55 60 65 70 75 80 85 90 Module #1 Junction Temperature ( ) (a) (b) 1.4 1.41 1.42 1.43 1.44 1.45 -60 -40 -20 0 20 40 T1 T2 D1 D2 Module #2 Junction Temperature ( ) Module #3 Junction Temperature ( ) T1 T2 D1 D2 T1 T2 D1 D2 Module #1 Module #2 Module #3 Figure 7. Circulating current and detailed temperature result with obvious circulating current: (a) three modules circulating current; and (b) detailed temperature in 100% load. Figure 7. Circulating current and detailed temperature result with obvious circulating current: (a) three modules circulating current; and (b) detailed temperature in 100% load. Figure 7. Circulating current and detailed temperature result with obvious circulating current: (a) three modules circulating current; and (b) detailed temperature in 100% load. Energies 2017, 10, 50 Energies 2017 10 50 8 of 11 8 of 11 Energies 2017, 10, 50 Energies 2017 10 50 Figure 8a,b shows the losses and temperature information of each module. It can be seen that they are distributed unbalanced. In all the three modules, T1 has a higher loss and temperature than T2, and D2 has a higher loss and temperature than D1. A similar trend is also observed for the temperature ﬂuctuation amplitude and ﬂuctuation amplitude difference of the devices, as shown in Figure 8c. In Figure 7b, the steady state temperature for devices in 100% load condition is presented. It can be seen that the temperature is distributed in an unbalanced way. Since the thermal impedance of the heat sink is smaller in scale, the difference is not too obvious. Figure 8a,b shows the losses and temperature information of each module. It can be seen that they are distributed unbalanced. In all the three modules, T1 has a higher loss and temperature than T2, and D2 has a higher loss and temperature than D1. A similar trend is also observed for the temperature fluctuation amplitude and fluctuation amplitude difference of the devices, as shown in Figure 8c. In Figure 7b, the steady state temperature for devices in 100% load condition is presented. It can be seen that the temperature is distributed in an unbalanced way. In this condition, an unsynchronized PWM was given t 4.2. DC/AC Modules with Zero Sequence Circulating Current Figure 6. Single module result: (a) junction temperature; (b) fluctuation; (d) temperature fluctuation difference; and (e) deta Since the thermal impedance of the heat sink is smaller in scale, the difference is not too obvious. Figure 8. Thermal results of the modules with obvious circulating current flowing: (a) three module junction temperature; (b) devices losses; and (c) junction temperature differences. Module #1 device loss (W) Module #3 Junction Temperature ( ) Module #2 Junction Temperature ( ) Module #1 Junction Temperature ( ) 60 65 70 75 80 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 60 65 70 75 80 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 60 65 70 75 80 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 0 200 400 600 800 1000 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 0 200 400 600 800 1000 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 0 200 400 600 800 1000 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 0 1 2 3 4 5 6 7 50% 60% 70% 80% 90% 100% ΔT1 ΔD1 ΔT2 ΔD2 0 1 2 3 4 5 6 7 50% 60% 70% 80% 90% 100% ΔT1 ΔD1 ΔT2 ΔD2 0 1 2 3 4 5 6 7 50% 60% 70% 80% 90% 100% ΔT1 ΔD1 ΔT2 ΔD2 (c) (a) (b) Module #2 device loss (W) Module #3 device loss (W) Module #1 temperature difference ( ) Module #2 temperature difference ( ) Module #3 temperature difference ( ) Figure 8. Thermal results of the modules with obvious circulating current ﬂowing: (a) three module junction temperature; (b) devices losses; and (c) junction temperature differences. In this condition, an unsynchronized PWM was given t 4.2. DC/AC Modules with Zero Sequence Circulating Current Figure 6. Single module result: (a) junction temperature; (b) fluctuation; (d) temperature fluctuation difference; and (e) deta Module #3 Junction Temperature ( ) Module #2 Junction Temperature ( ) Module #1 Junction Temperature ( ) 60 65 70 75 80 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 60 65 70 75 80 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 60 65 70 75 80 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 (a) Module #3 Junction Temperature ( ) Module #2 Junction Temperature ( ) ) 0% 60 65 70 75 80 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 60 65 70 75 80 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 (a) Module #3 Junction Temperature ( ) 60 65 70 75 80 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 Module #1 device loss (W) 0 200 400 600 800 1000 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 0 200 400 600 800 1000 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 Module #3 device loss (W) 0 200 400 600 800 1000 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 ( ) (b) Module #2 device loss (W) (b) 0 1 2 3 4 5 6 7 50% 60% 70% 80% 90% 100% ΔT1 ΔD1 ΔT2 ΔD2 Module #1 temperature difference ( ) 0 1 2 3 4 5 6 7 50% 60% 70% 80% 90% 100% ΔT1 ΔD1 ΔT2 ΔD2 Module #3 temperature difference ( ) 0 1 2 3 4 5 6 7 50% 60% 70% 80% 90% 100% ΔT1 ΔD1 ΔT2 ΔD2 (c) ( ) Module #2 temperature difference ( ) (c) Figure 8. Thermal results of the modules with obvious circulating current flowing: (a) three module junction temperature; (b) devices losses; and (c) junction temperature differences. Figure 8. Thermal results of the modules with obvious circulating current ﬂowing: (a) three module junction temperature; (b) devices losses; and (c) junction temperature differences. 4.3. DC/AC Modules with Suppressed Zero Sequence Circulating Current 4.3. DC/AC Modules with Suppressed Zero Sequence Circulating Current 4.3. DC/AC Modules with Suppressed Zero Sequence Circulating Current b h l d 4.3. DC/AC Modules with Suppressed Zero Sequence Circulating Current It can be seen that the T2 had a higher loss and temperature than T1, and D1 had a higher loss and temperature than D2. g The detailed temperature information of the three modules at 100% load condition is shown in Figure 9b. It can be seen that the T2 had a higher loss and temperature than T1, and D1 had a higher loss and temperature than D2. g The detailed temperature information of the three modules at 100% load condition is shown in Figure 9b. It can be seen that the T2 had a higher loss and temperature than T1, and D1 had a higher loss and temperature than D2. Figure 10. Thermal results of the modules without obvious circulating current flowing: (a) three module device losses; (b) junction temperature; and (c) junction temperature differences. Conclusions 0 200 400 600 800 1000 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 0 200 400 600 800 1000 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 0 200 400 600 800 1000 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 55 60 65 70 75 80 85 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 0 1 2 3 4 5 6 7 50% 60% 70% 80% 90% 100% ΔT1 ΔD1 ΔT2 ΔD2 (a) (b) (c) Module #1 device loss (W) Module #2 device loss (W) Module #3 device loss (W) Modules Junction Temperature ( ) Modules temperature difference ( ) Figure 10. Thermal results of the modules without obvious circulating current flowing: (a) three module device losses; (b) junction temperature; and (c) junction temperature differences. 0 200 400 600 800 1000 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 0 200 400 600 800 1000 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 0 200 400 600 800 1000 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 55 60 65 70 75 80 85 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 0 1 2 3 4 5 6 7 50% 60% 70% 80% 90% 100% ΔT1 ΔD1 ΔT2 ΔD2 (a) (b) (c) Module #1 device loss (W) Module #2 device loss (W) Module #3 device loss (W) Modules Junction Temperature ( ) Modules temperature difference ( ) Figure 10. 4.3. DC/AC Modules with Suppressed Zero Sequence Circulating Current b h l d 4.3. DC/AC Modules with Suppressed Zero Sequence Circulating Current Hereby, the zero sequence circulating current was suppressed. All the PWM for the three modules were synchronized, and zero vector operating time in the space vector modulation was tuned based on the active suppression method proposed in [18]. The zero sequence circulating current is presented in Figure 9a. It can be seen that the zero sequence circulating current was suppressed to a small amount of value. Figure 10a shows the losses of each module. All three modules shared the same loss distribution condition that T2 has a higher loss and temperature than T1, and D1 had a higher loss and temperature than D2. Consequently, they had the same temperature results, as shown in Figure 10b. Hereby, the zero sequence circulating current was suppressed. All the PWM for the three modules were synchronized, and zero vector operating time in the space vector modulation was tuned based on the active suppression method proposed in [18]. The zero sequence circulating current is presented in Figure 9a. It can be seen that the zero sequence circulating current was suppressed to a small amount of value. Figure 10a shows the losses of each module. All three modules shared the same loss distribution condition that T2 has a higher loss and temperature than T1, and D1 had a higher loss and temperature than D2. Consequently, they had the same temperature results, as shown in Figure 10b. Energies 2017, 10, 50 E i 2017 10 50 9 of 11 9 f 11 Figure 9. Circulating current and detailed temperature result without circulating current: (a) three modules circulating current; and (b) detailed temperature in 100% load. 4.8 4.9 50 55 60 65 70 75 80 85 90 4.8 4.9 50 55 60 65 70 75 80 85 90 4.8 4.9 50 55 60 65 70 75 80 85 90 (a) (b) Module #1 Junction Temperature ( ) Module #2 Junction Temperature ( ) Module #3 Junction Temperature ( ) T2 T1 D1 D2 T2 T1 D1 D2 T2 T1 D1 D2 1.4 1.41 1.42 1.43 1.44 1.45 -10 -5 0 5 10 1.4 1.41 1.42 1.43 1.44 1.45 -10 -5 0 5 10 1.4 1.41 1.42 1.43 1.44 1.45 -10 -5 0 5 10 Module #1 Module #2 Module #3 Figure 9. Circulating current and detailed temperature result without circulating current: (a) three modules circulating current; and (b) detailed temperature in 100% load. 4.3. DC/AC Modules with Suppressed Zero Sequence Circulating Current b h l d 4.3. DC/AC Modules with Suppressed Zero Sequence Circulating Current Energies 2017, 10, 50 9 of 11 Figure 9. Circulating current and detailed temperature result without circulating current: (a) three modules circulating current; and (b) detailed temperature in 100% load. 4.8 4.9 50 55 60 65 70 75 80 85 90 4.8 4.9 50 55 60 65 70 75 80 85 90 4.8 4.9 50 55 60 65 70 75 80 85 90 (a) (b) Module #1 Junction Temperature ( ) Module #2 Junction Temperature ( ) Module #3 Junction Temperature ( ) T2 T1 D1 D2 T2 T1 D1 D2 T2 T1 D1 D2 1.4 1.41 1.42 1.43 1.44 1.45 -10 -5 0 5 10 1.4 1.41 1.42 1.43 1.44 1.45 -10 -5 0 5 10 1.4 1.41 1.42 1.43 1.44 1.45 -10 -5 0 5 10 Module #1 Module #2 Module #3 (b) (b) (a) ( ) 1.42 Figure 9. Circulating current and detailed temperature result without circulating current: (a) three modules circulating current; and (b) detailed temperature in 100% load. Figure 9. Circulating current and detailed temperature result without circulating current: (a) three modules circulating current; and (b) detailed temperature in 100% load. Figure 9. Circulating current and detailed temperature result without circulating current: (a) three modules circulating current; and (b) detailed temperature in 100% load. As for the temperature fluctuation amplitude, three modules shared the same changing pattern ΔT2 first had a sudden increase at 90% load and then decreased. It was higher than ΔT1. ΔD1 decreased smoothly and was higher than ΔD2. The fluctuation difference also presented a similar trend, as shown in Figure 10c. The detailed temperature information of the three modules at 100% load condition is shown in As for the temperature ﬂuctuation amplitude, three modules shared the same changing pattern ∆T2 ﬁrst had a sudden increase at 90% load and then decreased. It was higher than ∆T1. ∆D1 decreased smoothly and was higher than ∆D2. The ﬂuctuation difference also presented a similar trend, as shown in Figure 10c. As for the temperature fluctuation amplitude, three modules shared the same changing pattern ΔT2 first had a sudden increase at 90% load and then decreased. It was higher than ΔT1. ΔD1 decreased smoothly and was higher than ΔD2. The fluctuation difference also presented a similar trend, as shown in Figure 10c. The detailed temperature information of the three modules at 100% load condition is shown in Figure 9b. 4.3. DC/AC Modules with Suppressed Zero Sequence Circulating Current b h l d 4.3. DC/AC Modules with Suppressed Zero Sequence Circulating Current Thermal results of the modules without obvious circulating current ﬂowing: (a) three module device losses; (b) junction temperature; and (c) junction temperature differences. 0 200 400 600 800 1000 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 Module #1 device loss (W) 0 200 400 600 800 1000 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 Module #1 device loss (W) 0 200 400 600 800 1000 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 7 (a) Module #2 device loss (W) 0 200 400 600 800 1000 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 (a) Module #2 device loss (W) 0 200 400 600 800 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 Module #3 device loss (W) 0 200 400 600 800 1000 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 Module #3 device loss (W) 7 ( ) (a) obvious circulating current flowing 0 1 2 3 4 5 6 7 50% 60% 70% 80% 90% 100% ΔT1 ΔD1 ΔT2 ΔD2 (c) Modules temperature difference ( ) 0 1 2 3 4 5 6 7 50% 60% 70% 80% 90% 100% ΔT1 ΔD1 ΔT2 ΔD2 (c) Modules temperature difference ( ) mal results of the modules without 55 60 65 70 75 80 85 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 (b) Modules Junction Temperature ( ) 55 60 65 70 75 80 85 50% 60% 70% 80% 90% 100% T1 D1 T2 D2 (b) Modules Junction Temperature ( ) modules withou (b) % 80% 90% 100% (b) modu ( ) (b) ating (c) module device losses; (b) junction temperature; and (c) junction temperature differences. onclusions Figure 10. Thermal results of the modules without obvious circulating current flowing: (a) three module device losses; (b) junction temperature; and (c) junction temperature differences. Figure 10. Thermal results of the modules without obvious circulating current ﬂowing: (a) three module device losses; (b) junction temperature; and (c) junction temperature differences. In this pap 5. Conclusions I 5. Conclusions tested. Experimental results were provided to validate the proposed control steady and transient performance, which were able to meet the standard IEC 62040-3. Moreover, zero sequence circulating current impact on thermal and losses distribution of the power semiconductor devices was discussed. Through simulation results, it can be concluded that while operating in different kinds of conditions associated with circulating current T1 had a higher temperature than T2 while D2 had a higher In this paper, the plug’n’play capability for a modular online UPS system was proposed and tested. Experimental results were provided to validate the proposed control steady and transient performance, which were able to meet the standard IEC 62040-3. Moreover, zero sequence circulating current impact on thermal and losses distribution of the power semiconductor devices was discussed. Through simulation results, it can be concluded that while operating in different kinds of conditions In this paper, the plug’n’play capability for a modular online UPS system was proposed and tested. Experimental results were provided to validate the proposed control steady and transient performance, which were able to meet the standard IEC 62040-3. Moreover, zero sequence circulating current impact on thermal and losses distribution of the power semiconductor devices was discussed. 10 of 11 Energies 2017, 10, 50 Energies 2017, 10, 50 Through simulation results, it can be concluded that while operating in different kinds of conditions associated with circulating current, T1 had a higher temperature than T2 while D2 had a higher temperature than D1. In order to suppress zero sequence circulating current, synchronization issues and zero vector time regulation were considered in this paper. However, such issues increased the temperature and losses of T2, which was higher than T1. Moreover, D1 had a higher temperature than D2. Author Contributions: Chi Zhang developed the basic idea, simulation and wrote the paper. Juan C. Vasquez and Josep M. Guerrero helped to revise the paper. Conﬂicts of Interest: The authors declare no conﬂicts of interest. Conﬂicts of Interest: The authors declare no conﬂicts of interest. References 1. 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https://openalex.org/W3094923452	https://discovery.ucl.ac.uk/id/eprint/10113982/1/herj17020001%20%281%29.pdf	English	null	Identifying aspects of temporal orientation in students’ moral reflections	History education research journal.	2,020	cc-by	12,014	HISTORY EDUCATION RESEARCH JOURNAL ISSN 2631-9713 (Online) Journal homepage: https://www.uclpress.co.uk/pages/history-education- research-journal Identifying aspects of temporal orientation in students’ moral reflections Niklas Ammert , Heather Sharp , Jan Löfström and Silvia Edling Niklas Ammert , Heather Sharp , Jan Löfström and Silvia Edling How to cite this article Ammert, N., Sharp, H., Löfström, J. and Edling, S. (2020) ‘Identifying aspects of temporal orientation in students’ moral reflections’. History Education Research Journal, 17 (2), 132–50. Online. https://doi.org/10.14324/HERJ.17.2.01 Submission date: 8 June 2019 Acceptance date: 10 May 2020 Publication date: 20 October 2020 Submission date: 8 June 2019 Acceptance date: 10 May 2020 Publication date: 20 October 2020 Peer review This article has been peer reviewed through the journal’s standard double-blind peer review, where both the reviewers and authors are anonymized during review. Copyright © 2020 Ammert, Sharp, Löfström and Edling. This is an open-access article distributed under the terms of the Creative Commons Attribution Licence (CC BY) 4.0 https://creativecommons.org/ licenses/by/4.0/, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. HISTORY EDUCATION RESEARCH JOURNAL ISSN 2631-9713 (Online) Journal homepage: https://www.uclpress.co.uk/pages/history-education- research-journal Open access The History Education Research Journal is a peer-reviewed open-access journal. Abstract Using this empirical material, the analysis provides a tentative overarching depiction of students’ expressions of temporal orientation, and reports on findings of how temporal orientations relate to moral reflection. Keywords: temporal orientation; moral reflection; secondary school students; Finland; Sweden Identifying aspects of temporal orientation in students’ moral reflections Niklas Ammert* − Linnaeus University, Sweden Heather Sharp − University of Newcastle, Australia Jan Löfström − University of Turku, Finland Silvia Edling − University of Gävle, Sweden Abstract History education comprises moral issues and moral aspects, often perceived as an important and meaning-making foundation that makes learning relevant and interesting. The interrelationship between time layers fuels historical interpretations and facilitates perceptions of moral issues. This article focuses on a study investigating how secondary school students express inter-temporal relationships in encounters with a morally challenging historical event, which for the participants would have been a moral dilemma. Using historical consciousness as the theoretical framework, a matrix linking two prominent theoretical models – Jörn Rüsen’s (2004) types of narratives and Ann Chinnery’s (2013) strands of historical consciousness – was developed to analyse and categorize secondary school students’ expressions of temporal orientation. To carry out the research, 15-year-old Finnish and Swedish students read an excerpt from Christopher Browning’s (2017) book Ordinary Men: Reserve Police Battalion 101 and the Final Solution in Poland (originally published in 1992). The students answered and discussed open-ended questions regarding the relevance of the text to their lives and others’ lives, and the applicability of this historical situation to Europe now and in the future. Using this empirical material, the analysis provides a tentative overarching depiction of students’ expressions of temporal orientation, and reports on findings of how temporal orientations relate to moral reflection. History education comprises moral issues and moral aspects, often perceived as an important and meaning-making foundation that makes learning relevant and interesting. The interrelationship between time layers fuels historical interpretations and facilitates perceptions of moral issues. This article focuses on a study investigating how secondary school students express inter-temporal relationships in encounters with a morally challenging historical event, which for the participants would have been a moral dilemma. Using historical consciousness as the theoretical framework, a matrix linking two prominent theoretical models g p – Jörn Rüsen’s (2004) types of narratives and Ann Chinnery’s (2013) strands of historical consciousness – was developed to analyse and categorize secondary school students’ expressions of temporal orientation. To carry out the research, 15-year-old Finnish and Swedish students read an excerpt from Christopher Browning’s (2017) book Ordinary Men: Reserve Police Battalion 101 and the Final Solution in Poland (originally published in 1992). The students answered and discussed open-ended questions regarding the relevance of the text to their lives and others’ lives, and the applicability of this historical situation to Europe now and in the future. *Corresponding author − email: niklas.ammert@lnu.se Ammert, N., Sharp, H., Löfström, J. and Edling, S. (2020) ‘Identifying aspects of temporal orientation in students’ moral reflections’. History Education Research Journal, 17 (2), 132–50. Online. https://doi.org/10.14324/HERJ.17.2.01 Introduction History teaching and history education more broadly have an important role in facilitating learning opportunities and staging situations where students encounter history. The situations can be complex and multifaceted, not least because students encounter history, historical narratives, representations of history and uses of history in a multiplicity of ways. Encounters with history also take place outside the formal school classroom, in everyday life such as: through family connections; through visits to public history sites such as museums; incidental encounters such as with public monuments and memorials; through popular culture; and as consumers of the news and other media. History is understood as both personal and as a part of the public sphere, and, as such, encounters with history do not take place in an empty space, devoid of context, but are often based on and framed by the experiences, knowledge and interests of the individual. 133 Identifying aspects of temporal orientation in students’ moral reflections These encounters with history often entail inter-temporal relationships between interpretations of the past, understandings of the present and perspectives on the future, as theoretically framed by, and defined as, a historical consciousness. Questions asked of, and interpretations made of, the past are anchored in the needs and the context of the present (Karlsson, 2016: 117). The concept of historical consciousness has been a strong focus of history didaktik in continental and northern Europe since the 1980s. However, the concept is contested, and it is regarded by some as being too vague or metaphysical (see Straub, 2006: 50–1) for empirical research to be conducted or for its application in the classroom to be viable. One major objection has been that it is not obvious how historical consciousness can be definitively identified, analysed or categorized. The questioning of its veracity underlines the need for further theoretical work and methodological refinement to be undertaken to develop a deeper understanding of the applicability of this concept to educational contexts. Since the influential Youth and History project (Angvik and Von Borries, 1997a, 1997b) was released in the late 1990s, a multitude of studies on historical consciousness have been undertaken (see, for example, Wilschut, 2012; Rosenzweig and Thelen, 1998). An overview of the current state of the field is presented in the recently published anthology Contemplating Historical Consciousness (Clark and Peck, 2019). Introduction The concept refers to how people relate with time, and their ability to construct meaningful relations between the past, the present and the future (temporal orientation, as explained above). In the history education context, how historical consciousness can be identified and analysed has been researched by Andersson Hult (2016) and Alvén (2017), exploring how individuals interpret and discuss the past and its relatedness to the present and the future when they encounter representations of history. Historians routinely work with ethical, moral or value-laden topics. As Mommsen (2000: 48) asserts: ‘The historian deals constantly with values, ideological positions and different normative systems – these are the very fabric of what he studies, and their mutual confrontation constitute in a way, the dynamism of the historical process’ (see also Cotkin, 2008: 298). Moral aspects are central for meaning making (Foster and Yeager, 1998: 1–7; Colby, 2008: 60–2; Löfström, 2014: 517–18). However, moral values do not stop at the basic level of only illuminating or stating something; they are foundational to our relationship to history (Edling and Sharp, 2018). Issues connected to ethical and moral values can thus be assumed to deepen knowledge and stimulate students’ historical consciousness. Milligan et al. (2018: 470) argue that ‘for example, when students assess historical actions, when they seek to understand others’ perspectives, or when they consider how best to move forward from the past, they move into the practice of ethics’. The German historian Jörn Rüsen (2004: 67–8) stresses this reciprocity, and asserts that historical consciousness makes an essential contribution to moral–ethical consciousness by providing a wider context for the moral issues, writing: ‘history clothes values in temporal experience. Historical consciousness transforms moral values into temporal wholes.’ In other words, Rüsen (ibid.) is saying, traditions, rules of conduct, concepts and experiences are mediated and made comprehensible in encounters between temporal dimensions. Factual knowledge about the past also strengthens moral interpretations. At the same time, moral values are important for stimulating a historical consciousness by bridging the distance in time and by facilitating understandings of the past (Ammert, 2010: 25–6). Historical moral dilemmas, or situations when moral values or acceptable conduct have been violated, increase interest in the past and provide opportunities for interconnection between time layers. Introduction Turning points in life and in history seem to act as motivators for people to turn to the past to orient themselves and to interpret what is History Education Research Journal 17 (2) 2020 134 Ammert et al. Ammert et al. 134 happening in the here and now, and what it might mean for the future. Major changes such as wars and terror attacks – but also changes to personal circumstances – can affect thinking in and over time. The theoretical interrelationships between historical consciousness and moral consciousness are complex and interwoven. However, there are two discernible interpretations of the relationships. Rüsen (2004: 78) has argued that historical consciousness could be conceptualized as a ‘synthesis of moral and temporal consciousness’. This interpretation means that moral consciousness is a component of historical consciousness. A partly different view is presented by Carlos Kölbl (2009: 89), who has shown how a ‘touchable past’ – a closeness to the past evoked by ‘plastic, strong and moving images of the past’ – facilitates historical understanding. Our hypothesis, or starting point, is that historical consciousness and moral consciousness are mutually dependent and mutually support each other’s development. Our conclusion is that moral values and moral issues are crucial for developing a historical consciousness (Rüsen, 2001: 253; Gergen, 2005: 101; Bøe, 1999: 19–23; Smith, 2009: 3; Ammert, 2015: 117–19, 121–4), but there is still a lack of empirical evidence supporting this notion. While previous research projects have presented important results of students’ views on moral issues in history (see, for example, Barton and Levstik, 2009; Löfström, 2014), larger studies are required (Endacott and Brooks, 2018: 220–1). The inter-temporal orientation in relation to moral issues is a starting focal point in this study, which aims to identify and analyse 15-year-old students’ expressions of temporal orientation and personal moral reflections by studying their reasoning about interpretations of the past, understandings of the present and perspectives on a possible future. Temporal orientation means if, and how, people connect and interrelate the past, the present and the future. These interrelationships form a web, with reasoning, experiences, knowledge, interpretations and expectations connecting different time layers (Jeismann, 1979). Accordingly, temporal orientation is the central point of a historical consciousness, but a consciousness cannot be observed per se – how temporal orientation is expressed is more tangible. Introduction In this study, the temporal dimension is identified and analysed by exploring secondary school students’ written responses to a task. Students were provided with an excerpt from Christopher Browning’s (2017) book centred on the Second World War and the Holocaust, Ordinary Men: Reserve Police Battalion 101 and the Final Solution in Poland (originally published in 1992). The excerpt describes events leading up to the order of a massacre in the Polish village of Józefów in 1942. In this article, an analysis is presented of how one central element of historical consciousness – temporal orientation – can be conceptualized and operationalized. The analysis and subsequent results are based on a matrix developed for this study and based on the theoretical work of Rüsen (2000, 2001, 2004) and Chinnery (2013) (see Table 1). While there has been other work carried out on student interpretations of Browning’s work and this particular genocidal massacre (see, for example, Nilsen, 2016), this is the first analysis that examines relationships between historical consciousness and moral consciousness. Theoretical framework Developing a theoretical framework with the capacity to categorize and better understand students’ expressions of temporal orientation in relation to moral reflection is a key output of this study. The framework developed combines two theoretical perspectives: Rüsen’s (2004) four types of narrative typology and Ann Chinnery’s (2013) model of how historical consciousness relates to and incorporates ethical and moral History Education Research Journal 17 (2) 2020 Identifying aspects of temporal orientation in students’ moral reflections 135 demands (in encounters with history) on humans. According to Rüsen (2004), historical consciousness can be described as a narrative competence based on, and with the ability to create, meaningful stories about interrelations between the past, the present and the future. His established typology with four types of narratives – traditional, exemplary, critical and genetic – can be applied to identify and analyse how historical consciousness can be expressed (Rüsen, 2004; see also Seixas, 2004: 22–3). The first category, the traditional narrative, describes the past as eternal, permanent and evident. In this way of thinking, nothing changes, and the past may be repeated as sameness over time. The past is not challenged because there is no immediate need for interpretation. Although it is possible to make the past come alive, it happened in, and belongs to, the past. The second category is exemplary narrative, which stresses rules, patterns of life and codes of conduct as guiding principles for society and for people. This is the idea that historia magistra vitae est – people should learn lessons from the past for the present and the future. The third category of historical narrative is described by Rüsen (2004) as the critical type. The critical type takes a clear starting point in the present but does not make direct connections to historical events. The past is no longer understood as absolute and valid from our present point of view. This category indicates a historical consciousness that takes into consideration the fact that time changes, and that is why it is important to formulate a counterpoint or a critical history when considering the past. Alternative narratives offer relief or distance. In this category, the past is considered to be a more or less isolated time dimension. For the purposes of this study, the critical type also includes taking a critical stance to what happened in the past and dissociating from it. Theoretical framework The fourth category, the genetic type, reflects the perception that the past is changing and inherently changeable, and is necessarily embedded in the always-vanishing present. This narrative type expresses the idea that change is natural when regarding the past. Genetic narratives show that every time period reflects what is unique for that era, and that different stories are told at different times. The present is a station of change between the past and the future (Rüsen, 2005: 15). Chinnery (2013) nominates three main strands of historical consciousness that address and connect with ethical and moral demands on humans in the present and their relation to the past – to which we add for this study perspectives on the future. For this analysis, the three strands describe connections between the individual and interrelationships in and between temporal layers within a framing of moral aspects. The first is an existential strand, meaning that an individual considers and reflects on themselves and their life, grounded in the past and the future. There are ethical and moral preconditions for an individual’s perceptions and interpretations. In the existential strand, relationships and interrelationships to the past are fundamental, and they put people in networked contexts where the perception and understanding of the self, relative to wider temporal contexts, are crucial. The second strand is cognitive (knowledge-based). Knowledge about the past can enable a factual interpretation and understanding of – for example – moral problems in the past, as well as in the present and the future. It will also help people analyse the past and make measured decisions about how to act – in the present and in the future. A narrative competence or ability forms the third strand, influenced by the work of Rüsen (2004) and Straub (2006). This competence is the ability to receive, interpret and transform narratives from the past into meaningful contexts (Chinnery, 2013: 254). All three strands – existential, cognitive and narrative competence – are relevant for encounters with moral perspectives. A fourth strand proposed by Chinnery (2013) is an ethics of care. This ethical approach sees people in this context as caring for History Education Research Journal 17 (2) 2020 136 A Ammert et al. 136 past moral actions. Theoretical framework Ethics of care forms an important bridge between historical consciousness and moral consciousness, but for the purposes of the analytical approach selected for this part of the project, it is not possible to isolate an ethics of care from the other strands. A connection expressing caring for the moral actions of the past would necessarily also be based on knowledge-based relations or existential relations. This aspect is more applicable and able to be applied when specifically analysing how a moral consciousness in relation to the past is expressed. The strands provide a richer and more refined way to describe how people relate to moral perspectives or dilemmas in history. Narrative competence is the most advanced strand or type of historical consciousness, because it integrates both existential perceptions and practical knowledge-based abilities into a complex competence of meaning making. It often requires elaborated reasoning. Rüsen’s (2004) and Chinnery’s (2013) typologies were selected to integrate into a matrix to enable an analysis of our data. Rüsen’s (2004) model – our primary typology – has the capacity to grasp inter-temporal relations and also sense-making aspects of temporal dimensions. Historical narratives are charged with moral values and messages from, or manifested in, the past. These messages frame the narratives. Although Rüsen’s (ibid.) typology could be used to analyse the narratives, in this study it is applied to identify and analyse students’ answers to questions about relevance in messages from the past. Chinnery’s (2013) strands enable moral content to be approached in interconnections and interrelations between the past, the present and the future. When merged, Rüsen’s (2004) approach can make explicit Chinnery’s (2013) narrative competence in the form of categories that capture variations in ways of mobilizing historical references in making value judgements, and how these things are interpreted by individuals – in the case of this study, secondary school students. Students’ responses were allocated to categories based on the prescriptive definitions of each item in our matrix (see Table 1). In addition, decisions were made about how to categorize student responses in terms of verbs used to describe what they discussed in their responses, including whether or not participants used verbs associated with higher-order thinking, such as those found in common student learning taxonomies, and comparative verbs. The content of student responses was also used to allocate them to a category when referring to the past, present and/or future. Theoretical framework To ensure integrity of data analysis, categories attributed to the qualitative data received from the students were first coded by two researchers and discussed, before a preliminary determination was made as to where to place the responses in the matrix. This quality assurance process was then checked again by two more researchers, who read the student responses as categorized by the first two researchers, and commented on the validity of their categorization. The final categorization was achieved through consensus by the four researchers. 5. Facts and knowledge help individuals to interpret what is worth bringing on from the past. 8. A factual interpretation could question, dissociate from or criticize (dismiss) what happened in the past (or what happens in the present). 6. Individuals should apply and relate to moral messages from the past as something to learn from or accept. Critical 8. A factual interpretation could question, dissociate from or criticize (dismiss) what happened in the past (or what happens in the present). 9. Individuals could analyse what is not valid in the present and discuss why. An expressed ability to propose counter-narratives. 11. Individuals could interpret and prove what is similar and what is different from the past (and what seems to be important in the future). Individuals could understand moral issues in relation to transformations in society. 12. Individuals could interpret and understand what previous actions really meant in the past and why, but also how the future might be, and how the past and the present will be described in the future. Methodological discussion What happened in the past is connected to the present and what is to come. 11. Individuals could interpret and prove what is similar and what is different from the past (and what seems to be important in the future). Individuals could understand moral issues in relation t t f ti 12. Individuals could interpret and understand what previous actions really meant in the past and why, but also how the future might be, and how the past and the present will be described in th f t Table 1: Combined theoretical typologies for analysing expressions of temporal orientation in relation to moral reflection Table 1: Combined theoretical typologies for analysing expressions of temporal orientation in relation to moral reflection Chinnery’s (2013) strands Rüsens’s (2004) types Methodological discussion This section details the starting points of an empirical study of students’ temporal orientation via reflection in relation to a historical event concerning moral decisions. There are three important points to note. First, historical and moral consciousness involve mental processes that are not immediately observable; thus, the focus of the study is on how the school students who participated in the research project express perceptions of temporal interrelations in relation to moral reflections and motivations. Moral reflections can involve cognitively defining moral issues, but also making moral judgements (see, for example, Rest, 1979), and here, no distinction is made between History Education Research Journal 17 (2) 2020 Identifying aspects of temporal orientation in students’ moral reflections 137 137 Table 1: Combined theoretical typologies for analysing expressions of temporal orientation in relation to moral reflection Chinnery’s (2013) strands Rüsens’s (2004) types Existential strand Relating to oneself, to one’s own opinions, with moral reasoning or arguments, human values. Knowledge- based strand Factual reasoning, not necessarily with moral reflections. Narrative competence Sense making, explicit contextualization, past–present– future is visible. Traditional 1. Reflections of the past and expectations of the future. Relate chronologically to the past and the future. Time does sometimes repeat itself and sometimes times change. Morality as a heritage or an obligation. 2. Individuals could, based on facts, identify and describe historical time and moral issues as continuities. 3. Individuals identify and interpret the past, and moral values from the past, as continuous lines through time. Meaning making. Exemplary 4. Roots and relations are stressed. Individuals could, and should, learn from the past. 5. Facts and knowledge help individuals to interpret what is worth bringing on from the past. 6. Individuals should apply and relate to moral messages from the past as something to learn from or accept. Critical 7. Individuals’ deep-rooted moral perceptions lead them: (1) to say that the narrative is not relevant in the present; (2) to criticize what happened in the past; or (3) to dissociate from the past. 8. A factual interpretation could question, dissociate from or criticize (dismiss) what happened in the past (or what happens in the present). 9. Individuals could analyse what is not valid in the present and discuss why. An expressed ability to propose counter-narratives Genetic 10. Individuals consider existential aspects of narratives from the present, but also in relation to the past and perspectives on the future. 7. Individuals’ deep-rooted moral perceptions lead them: (1) to say that the narrative is not relevant in the present; (2) to criticize what happened in the past; or (3) to dissociate from the past. Genetic 10. Individuals consider existential aspects of narratives from the present, but also in relation to the past and perspectives on the future. What happened in the past is connected to the present and what is to come. Individuals could understand moral issues in relation to transformations in society. History Education Research Journal 17 (2) 2020 138 Ammert et al. these two kinds of approaches to moral issues. Second, secondary school students were not studied continuously in an ethnographical way. Instead, students were asked to respond to stimulus material that connected with issues that are theoretically relevant to historical and moral consciousness. Third, this research is interested in identifying qualitative differences between individual adolescents’ responses, but also in the generally shared features in their responses. Questions and questioning are crucial to learning, to knowing and to explaining something (Ricoeur, 1984: 239; Rüsen, 2004: 19). Explicit questioning is key to being able to analyse students’ responses in accordance with ideas of temporal orientation. One disadvantage to using this method is that the questions could unduly influence the students’ answers. However, as this study is interested in aspects of their reasoning – rather than the content or factual basis of their answers – explicit questions concerning temporal orientation are suitable and even preferred. Rüsen (2001) describes two ways in which questions can arise to stimulate or activate historical consciousness. First, they can arise from something empirically present, for example, artefacts, commemorations, narratives or traditions. Second – and this is the focus in this study – questions can arise from rapid unexpected turning points in society or in private life (ibid.: 253). These kinds of borderline events trigger questions about why the turning points have arisen, about what they mean, about what will or what might happen, and about what could have happened already (Andersson Hult, 2016: 23). In qualitative research, there are many ways that data can be analysed, and various lenses can be applied to attain a deeper understanding of participants’ responses. Because our focus is on the intersections of moral consciousness and historical consciousness, in this research we chose not to take a social psychological or a cognitive approach to data analysis, but to examine how students engaged in their reading of historical texts, with a focus on students’ reactions based on temporality and moral dimensions. Genetic Regarding the design of the study, it could be argued that a limitation is that, given the varying written literacy proficiency of students, some students may have had more difficulty than others in expressing their views. Furthermore, as the research was undertaken in the context of history lessons, this may have sensitized the students in a different way than asking them to do the questionnaire in a non-related class. Conducting the research Students were provided with an excerpt from the book by Christopher Browning, Ordinary Men: Reserve Police Battalion 101 and the Final Solution in Poland (originally published in 1992), a historical study of the operations of a German police battalion in Poland in 1942–3. The extract – adapted from pages 55–7 of the book (Browning, 2017) – describes how the commander of the battalion, Major Trapp, received orders to commit genocidal murder of the Jewish inhabitants of the Polish village of Józefów. The extract tells how the men in the battalion reacted to the task, and it details Major Trapp’s offer that the elderly men of the battalion did not have to take part in direct killing if they did not wish to. The researchers edited the text slightly so that the student participants were not distracted by technical terminology or other information superfluous to the task at hand. The purpose was to present to the students a specific, historical situation, selected due to its ethical complications and morally challenging nature. Browning’s text was chosen based on the aim of our larger study, the objective of which is to investigate students’ expressions of temporal orientation by studying their reasoning about interrelations between dimensions/layers of time, and not their History Education Research Journal 17 (2) 2020 139 Identifying aspects of temporal orientation in students’ moral reflections factual or content knowledge. Accordingly, students were given a written excerpt within a historical context that was familiar to them. Such an infamous historical event was selected as a way to eliminate the risk of students saying that they did not know anything about the situation or that they did not understand the context. By Year 9, Finnish and Swedish students have studied the Second World War and the Holocaust. Also, Nazi officers and soldiers are often described as unambiguously reckless and aggressive. In this excerpt, the expected view of them is suddenly changed, and some of the members of the Reserve Police Battalion are presented counter to the traditional expectation when they chose not to participate in killing innocent people. This counter-narrative provides an opportunity for students to reflect on the situation and their understanding of the messages within the narrative. Following their reading of the text, 15-year-old Finnish and Swedish secondary school students answered a set of eight open-ended questions centring on historical and moral reflections. Conducting the research This article addresses two of the eight questions posed to student participants, Questions 2 and 3. Question 2 read: ‘Does the narrative send a message to you personally? Explain your answers.’ The purpose of this question was to investigate if and how students relate to and interpret a historical text. The analysis is then able to determine whether they find a message in the text and perceive it as relevant, valid and worth bringing into the present, or if they think it should be relegated to the past with no connection to the present or to the future. Question 3 read: ‘Do you think that a similar situation could occur in Europe today or in the future. Why/why not?’ This question was framed in order to: (1) investigate how the students reasoned and argued about the future, in relation to the past from a present-day perspective; and (2) to study whether student participants refer to or relate to moral principles when considering this. The questions were designed to elicit responses that expressed students’ views on continuity and discontinuity between the past, the present and the future, including whether or not the types of moral challenge faced by historical actors were perceived as relevant today or for the future. Thus, the questions addressed students’ abilities to think in terms of diverse temporal interconnections, and to use their knowledge of the past to interpret the world of today and to imagine possible futures. This article reports on responses from 220 participants, made up of 109 females, 104 males and 7 students who did not want to disclose their gender. One student answered only Question 2 and six students answered only Question 3. Two students did not answer either of the two questions. Because some questions were not answered by all student participants, 401 answers have been categorized for analysis. The selection of students is not statistically representative of wider populations. They attend a variety of schools in terms of socio-economic status and geographical location, but they were not selected as being nationally representative of the number of females and males, or the number of students born in Finland or Sweden. Areas included small communities, medium-size towns and large cities. History is a mandatory subject from Year 5 in Finland and from Year 1 in Sweden in compulsory school. Conducting the research Due to the national curricula and syllabuses, all students in Finland and Sweden study the Second World War and the Holocaust, which is explicitly addressed in the syllabus, usually in Year 8 (Finnish National Agency for Education, 2016; Swedish National Agency for Education, 2018). This article reports on responses from 220 participants, made up of 109 females, 104 males and 7 students who did not want to disclose their gender. One student answered only Question 2 and six students answered only Question 3. Two students did not answer either of the two questions. Because some questions were not answered by all student participants, 401 answers have been categorized for analysis. The selection of students is not statistically representative of wider populations. They attend a variety of schools in terms of socio-economic status and geographical location, but they were not selected as being nationally representative of the number of females and males, or the number of students born in Finland or Sweden. Areas included small communities, medium-size towns and large cities. History is a mandatory subject from Year 5 in Finland and from Year 1 in Sweden in compulsory school. Due to the national curricula and syllabuses, all students in Finland and Sweden study the Second World War and the Holocaust, which is explicitly addressed in the syllabus, usually in Year 8 (Finnish National Agency for Education, 2016; Swedish National Agency for Education, 2018). The students completed the activity by accessing a password-protected digital platform during a history lesson, reading the adapted extract from Browning’s (2017) book, and responding to the questions. Students’ responses were then collated and analysed according to the theoretical profiles listed in the matrix categories combining Rüsen’s (2004) and Chinnery’s (2013) typologies (see Table 1). Responses were categorized based on a holistic reading of their responses, rather than on individual The students completed the activity by accessing a password-protected digital platform during a history lesson, reading the adapted extract from Browning’s (2017) book, and responding to the questions. Students’ responses were then collated and analysed according to the theoretical profiles listed in the matrix categories combining Rüsen’s (2004) and Chinnery’s (2013) typologies (see Table 1). Responses were categorized based on a holistic reading of their responses, rather than on individual History Education Research Journal 17 (2) 2020 140 Ammert et al. Conducting the research words, which could potentially be taken out of context, so that an understanding could be gained of the students’ temporal-orientation expressions. After this preliminary analysis, the two answers from each participant were read together, contextually, in order to identify interpretations and aspects that might have been missed in the initial separate readings. In the Findings section, we provide examples of how the coding of answers was carried out. Findings First, we present a quantitative overview of the distribution of the participants’ answers (see Table 2). Second, we discuss participants’ arguments, how they were interpreted and coded, and how the participants express different types of temporal orientation. Third, we analyse the students’ moral reflections, and the relations between temporal orientation and moral reflections. Table 2: Distribution of answers in the combined theoretical typologies of temporal orientation in relation to moral reflection (number of answers, and percentage of the total number of answers) Chinnery’s (2013) strands Rüsens’s (2004) types Existential strand Total: 38% Knowledge- based strand Total: 58% Narrative competence Total: 4% Traditional Total: 31% Question 2: 12 Question 3: 35 Total answers: 47 (12%) Question 2: 32 Question 3: 40 Total answers: 72 (18%) Question 2: 2 Question 3: 3 Total answers: 5 (1%) Exemplary Total: 26% Question 2: 60 Question 3: 1 Total answers: 61 (15%) Question 2: 37 Question 3: 4 Total answers: 41 (10%) Question 2: 4 Question 3: 0 Total answers: 4 (1%) Critical Total: 21% Question 2: 23 Question 3: 6 Total answers: 29 (7%) Question 2: 10 Question 3: 39 Total answers: 49 (13%) Question 2: 3 Question 3: 1 Total answers: 4 (1%) Genetic Total: 22% Question 2: 6 Question 3: 8 Total answers: 14 (3%) Question 2: 3 Question 3: 68 Total answers: 71 (18%) Question 2: 1 Question 3: 4 Total answers: 5 (1%) Note: Total number of answers: 401; number of answers to Question 2: 193; number of answers to Question 3: 208 Table 2: Distribution of answers in the combined theoretical typologies of temporal orientation in relation to moral reflection (number of answers, and percentage of the total number of answers) Alphanumeric codes were used to identify participants: a number was assigned to each student, and a letter represents gender (F for female and M for male; no student identified as anything other than male or female in the demographic questions in the History Education Research Journal 17 (2) 2020 Identifying aspects of temporal orientation in students’ moral reflections 1 sample selected for this article). In this article, we do not analyse potential similarities and differences between gender/sex, nationality, or geographical background. Temporal interrelations: Question 3 – relevance for the present and the future Relating to Question 3 – ‘Do you think that a similar situation could occur in Europe today or in the future. Why/why not?’ – the two most common types of temporal interrelations in the answers were the traditional type and the genetic type, representing approximately 35 per cent of responses each. The traditional type is the base-level way for students to discuss if a specific event in the past could occur in the present or in the future, because it primarily uses a chronological approach. In the traditional type of responses, students related to the past in a non-problematic way, asserting that what had happened probably either will happen or will not happen again. The answers in this category are mainly non-reflective when it comes to discussing transformations of time, and they therefore follow a line of continuity, assuming that similar things might or might not occur again in future. In a response that can be categorized as traditional, Student 157F makes a tentative connection between the situation in the text and the present day by writing in response to Question 3: Yes, of course, we are people and if one thing has happened once, it can surely happen again. I mean if you tie your shoe and the knot goes up, it will surely go up several times before you learn to do double knot. The answer indicates that the past will in all probability repeat itself, as easily as tying up a shoelace; humans remain the same and people just act and react, which does not express a very refined understanding of temporal orientation. It is categorized as traditional because Student 157F identifies the factual context, but does not discuss or problematize the similarities and differences between the past, the present and the future. She seems to regard temporal connections as given and unchangeable. Only five student answers were categorized in the exemplary type of historical consciousness in response to Question 3. The critical type was more frequent, with 25 per cent of the answers within this category. For example, the response from Student 228M reads: We have not had war in Europe in 70 years. According to me there is more and more peace in the world. The student starts his response by stating that (according to his perspective) there has been no war in Europe for 70 years, asserting that the world is more peaceful. Temporal interrelations: Question 3 – relevance for the present and the future The connection to the past is not relevant for him when he considers potential similarities or differences in the present or in the future. The student dissociates from the past by implying that it is not relevant in a future scenario. Concerning the genetic type of historical consciousness, the frequency of students in this category was higher. An explanation for this high result is that Question 3 asked students to reflect explicitly on whether similar situations might occur in the future. Approximately 35 per cent of the participants delivered reflections and arguments in which the past was interwoven in the discussion and the assessment of the situation today and their expectations of the future. The genetic type is a complex, elaborated way of reasoning, and it involves thinking of and between different dimensions of time, History Education Research Journal 17 (2) 2020 142 Ammert et al. Ammert et al. demonstrating temporal orientation, and reflecting on differences and transformations of the issues. The genetic type of responses most frequently occurred in responses from students about whether they believe that similar situations could occur in the present or in the future. To illustrate this category, in a very detailed answer (unlike other participants’ responses), Student 76F wrote: Both yes and no. Given the unrest in Europe today in politics, I would not see it as completely impossible unfortunately. Right-wing extremist parties may have greater influence, e.g. the Swedish Democrats, despite their xenophobic and women-oppressive politics, or the Danish Labour Party, who has now banned the burqa in public places, that is, women’s oppression in the toe tips [a Swedish expression that means, more or less, ‘to the extreme’ or ‘to the edge’, depending on context]. A ban on the burqa does not result in women’s oppression within this culture ceasing, but merely makes the women who actually have the worst conditions be forced to stay home and their opportunities to have their own life go up in smoke. I think it is totally unreasonable that white, rich, highly educated men should decide on these women’s bodies and lives. In this situation, one must listen to the WOMAN behind the burqa. The Nordic Resistance Movement is allowed to stand beside RFSU [the Swedish Assocation for Sexuality Education] at the Almedalsveckan in Visby, and the police are instructed to protect the Nazis, not the vulnerable. Temporal interrelations: Question 3 – relevance for the present and the future [Almedalsveckan is an annual week-long event in July, considered the most important event in Swedish politics. Political parties, organizations and members of the public who are interested in social issues meet in Visby for networking, discussions and seminars.] On the other hand, parties to the left grow too, which means that the two more extreme political types are growing and a ‘gulf’ is growing. I think like this: the more people who settle in either extreme, right or left, the more who disagree in politics. The more disagreeable, the more anxious it becomes and the risk of major conflicts increases. I myself think it is completely unacceptable just in view of this story that Nazism is still living in our society today. We saw how fast Hitler gained influence in Germany. Similarly, the right-wing extremist parties gain more influence in Sweden, which scares me. How can it be legal and accepted by society to be a racist, a Nazi or anti-Semitic? In this extended answer, Student 76F presented reasoning that was advanced and that expressed a deep engagement with, and knowledge of, human rights and democratic issues. It is clear that the excerpt from Browning’s (2017) book resonated with her, and that she was able to communicate clearly her moral reasoning, linking history with present-day politics. Here, her temporal orientation was sophisticated, as her discussions integrated and switched between the past, the present and the future, as she used facts from the past as something people in the present had to bear in mind when engaging with current sociopolitical issues. Existential aspects were central in her reasoning about how human values were oppressed in the past and how they must be protected in the future, while also recognizing present-day threats to freedom and democracy. The genetic type of narrative catches these interrelations in time, and Student 76F presented a discussion on non-democratic tendencies in contemporary society. She used the fact that Nazism is still present in society as a reference for how the situation in Europe today could unfold in the future when she discussed her fear of political polarization and xenophobic perceptions and activities. In this extended answer, Student 76F presented reasoning that was advanced and that expressed a deep engagement with, and knowledge of, human rights and democratic issues. Temporal interrelations: Question 2 – personal message Responding to Question 2 – ‘Does the narrative send a message to you personally? Explain your answers.’ – participants described in different ways, and with different motivations, what message they took from the extract, and their reactions were just as varied. The dominant category of historical consciousness was the exemplary category (52 per cent of responses), followed by the traditional type (24 per cent of responses) and the critical type (18 per cent of responses). Many students interpreted the message in the text as individuals having choice or having the option to follow their own will. The students who related to the exemplary type in their responses often stated that the men who stepped out of the line and did not participate in the executions did the right thing, and that this is a message to apply to current and future contexts. In such exemplary reasoning, students interpreted a positive and hopeful message for the future. Student 63F referred to the soldiers who refused to take part in the execution: In this case, I think that the moral for me today would be the few people who opposed and refused the task of participating in the event. I think it meant to me that you should not lose yourself but stick to what you think is right and what feels good in your stomach. This participant interpreted the story as a historical moral message to learn from the actions of other individuals. It gave her a type of motivation to act ethically in the present day. She related the position of the soldiers to her own moral principles by writing, ‘you should not lose yourself but stick to what you think is right and what feels good in your stomach’. There were also several students who emphasized that what happened during the Nazi regime was terrible, and they expressed a strong dissociation from it. Student 165F illustrates the critical perspective, writing in response to Question 2: No, I personally do not think so, mostly that it is wrong to do this. I think a No, I personally do not think so, mostly that it is wrong to do this. I think a little more that it is like a little story what happened earlier. Temporal interrelations: Question 3 – relevance for the present and the future It is clear that the excerpt from Browning’s (2017) book resonated with her, and that she was able to communicate clearly her moral reasoning, linking history with present-day politics. Here, her temporal orientation was sophisticated, as her discussions integrated and switched between the past, the present and the future, as she used facts from the past as something people in the present had to bear in mind when engaging with current sociopolitical issues. Existential aspects were central in her reasoning about how human values were oppressed in the past and how they must be protected in the future, while also recognizing present-day threats to freedom and democracy. The genetic type of narrative catches these interrelations in time, and Student 76F presented a discussion on non-democratic tendencies in contemporary society. She used the fact that Nazism is still present in society as a reference for how the situation in Europe today could unfold in the future when she discussed her fear of political polarization and xenophobic perceptions and activities. History Education Research Journal 17 (2) 2020 Identifying aspects of temporal orientation in students’ moral reflections 1 143 Moral approach by existential and knowledge- based strands More than 50 per cent of the answers to Question 3 related to factual knowledge (categorized using Chinnery’s (2013) typology) about what happened in the past and the preconditions for society today. The students applied their knowledge when interpreting and discussing perspectives on the future. There could have been moral considerations behind the answers. However, they were not always explicit, nor were they used as main arguments, as was the case with more than 30 per cent of students’ responses. For example, Student 130M’s response to Question 3 provided an example of existential or moral references mentioned in subordinate clauses, not in the main arguments: Yes in Russia. Putin wants to take back what was Russia’s (imperial Russia). The Soviet Union did the same to people but in worse ways, such as letting people starve. I think Russia will at least be the reason why it will be such a disaster in Europe if it now happens because they are the country that wants their ‘land’ back, at any cost. This answer could be defined as relating to existential arguments, as well as to factual and cognitive-based arguments. However, his main argument was the reference to historical interpretation, and accordingly the answer was coded in the cognitive strand. This student described, from within a historical context, what he saw as a possible scenario. The factual arguments dominated, but he implied that it would be a disaster, and he signalled his opinion in how he interprets Russian conduct, today as well as in the past. He was applying his broader knowledge of historical actions taken by Russia and the USSR throughout the twentieth century to the extract, and describing how Russia could ‘be such a disaster in Europe’, drawing mitigated parallels between Nazi Germany and Russia in the present day and in what he saw as a likely future. Existential arguments, in which students discussed questions relating to personal and moral perceptions of right and wrong, were especially frequent in answers to Question 2 (52 per cent of responses) – ‘Does the narrative send a message to you personally? Explain your answers.’ When students reflected on this question, the most common answer was a combination of the exemplary type (Rüsen, 2004) and the existential strand (Chinnery, 2013). Temporal interrelations: Question 2 – personal message This student focused on the fundamental evil activity occurring in the narrative, but she did not personally take on or connect with any message that could come out of the text, and she implied that the narrative of what happened was not very relevant for her current life. The differences could mean that different individuals interpreted different aspects of what they perceive, but it might also be a result of their ability to read and comprehend a difficult and complex text, which illustrates a potential limitation of the study. In this analysis, we are aware that the immediate context was about preparations for the selection and execution of Jewish people living in a rural village. To identify a deeper message about how some of the soldiers reacted, and how Major Trapp (the commander of the battalion) handled the situation, requires a careful and closer reading, which could be a literacy challenge for some students. In some respects, the pattern of a majority of responses to Question 2 falling into the exemplary type is what we might expect. The excerpt that the students read expresses deep moral and human issues, and it would be difficult for a reader’s reactions to be neutral or indifferent. However, there were some students (22 per cent) who reacted by indicating that their reading of the extract was that certain things happened in the past, describing a linear connection over time. They did not communicate if or how the message meant something to them personally, now or in relation to the future. History Education Research Journal 17 (2) 2020 144 Ammert et al. Moral approach by existential and knowledge- based strands The question was personal, and it implicitly encouraged students to go beyond what might be right or wrong, and to relate the case to their experiences, their previous or background knowledge, and their own opinions (see also Sellman and Barr, 2009: 19–20). In several answers, students’ reasoning took its starting point in what seemed to be deep-rooted perceptions of how people should and should not treat each other. Strongly stressed reactions against the Nazi regime and the genocide of the Jewish people made students dissociate from events as described in the extract. Similar reactions, but aiming to learn from the soldiers who stepped out of the line and refused to take part in the killing, characterized the answers in the exemplary type, with participants using the courage of the men as a role model for behaviour. In our research, we also made use of Chinnery’s (2013) narrative competence strand as an elaborated expression of a historical consciousness. This strand is complex and accommodates reasoning that connects dimensions of time to each other, and that explores how relations between them make meaning in people’s lives at different times. Our questionnaire data is open for this kind of interpretation, but the answers are often short, with participants not usually grasping the broader temporal context. History Education Research Journal 17 (2) 2020 145 Identifying aspects of temporal orientation in students’ moral reflections The narrative competence strand is not frequent enough to be used in analysing the specific answers on which this article focuses. However, there are a few examples that show how the students clearly discussed the interrelations between times, how content could be understood differently at different times, and what that meant when they tried to interpret the content in the present day. For example, Student 112F asserted that a similar situation could occur in the future, writing: It could. Not similar to what exactly happened, that they would kill people, but similar in the way that one is manipulated to believe that one has one’s own will, it is difficult to notice, but easy to see afterwards. An exact such situation is probably difficult, because it has happened before, and people decided afterwards that it was wrong. This student described a scenario where people think that they have their own free will, but then expanded on this to state that, in reality, they are being manipulated. Moral approach by existential and knowledge- based strands One way to interpret this response is as suggesting that she is self-identifying fears for a future where people will be misled, as can be seen in her statement, ‘one is manipulated to believe that one has one’s own will, it is difficult to notice, but easy to see afterwards’. This student also discussed how actions in the future will be regarded afterwards – that is, in the future’s future – an elaborated way of reasoning about interrelations between time layers as temporal orientations. This discussion is based on her interpretation of the past. In this way, Student 112F expressed her narrative competence, meaning that she understands that activities and actions could be regarded differently in different times. She expressed a genetic type of narrative, in which the past, the present and the future were interrelated. Discussion and concluding words In the critical type of historical consciousness category, students discussed how the oppression and execution of Jewish people was terrible, but they observed that it was in another time and in a past sociopolitical context and, as such, they dissociated today’s society from that past. In these examples, students discussed their position and their reflections on facts, and they put their trust in people and societies in the future developing by dissociating from what had happened in the past. Many of the students’ answers to Question 2 focused on the battalion commander, Major Trapp, giving his soldiers the opportunity not to participate in the execution of Jewish people. The question elicited personal reflections and arguments, and just over 50 per cent of the answers were characterized in the exemplary type, because students identified the main message as being that it was possible to follow one’s own will and to refuse to do things that one believed were morally or ethically wrong. Students reflected on the message from the past to the present, but they did not explicitly reflect on whether the message was valid in different times. This could indicate: (1) that the primacy of the present was paramount to them in their interpretation; (2) that they posited a linear connection between the past and the present; or (3) that they were not asked to explain that aspect, so they did not do so. The students interwove different temporal layers, but not always in dialogue with the conditions in the past. While 20 per cent of participants did not consider the extract on a personal level, the message received by them was that certain things happened in the past, that is, they understood them through a linear connection over time. However, a convincing finding is that students had the ability to interrelate temporal dimensions when asked. For those sceptical about students’ abilities to undertake such a task, and who might therefore encourage its avoidance in the classroom, this demonstrates that students – when provided with the opportunity to do so – are able to respond in a sophisticated, or complex, manner that demonstrates skills in interlinking temporal dimensions. In the analysis of how a moral approach was expressed by the students, Rüsen’s (2004) types of historical consciousness were combined with the kind of motives and arguments that give fuel to the students’ reasoning, as per Chinnery’s (2013) strands. Discussion and concluding words Students’ reasoning when answering questions after reading the extract provide an empirical foundation to reflect on how their temporal orientation as a form of historical consciousness was expressed. The questions were explicit, but they were open-ended, and they prompted students to reflect on whether a similar situation could happen today or in the future and how (if at all) they perceived a message in the narrative as applicable to their present-day lives. Responses were analysed by categorizing them against the two interconnecting theoretical dimensions of the matrix (see Table 1), one dimension showing how the responses interrelated different layers of time to each other (as per Rüsen’s (2004) typology), and the other one showing how responses approached and reflected on the moral dimension (as per Chinnery’s (2013) three strands). While the empirical study comprised written answers from 220 Finnish and Swedish students, it is not statistically representative for all 15-year-old students in Finland and Sweden. The aim of this study was to identify and analyse students’ expressions of temporal orientation in relation to moral reflection, as part of a heuristic ambition to explore the intersections of historical consciousness and moral consciousness in high-school students on a topic that they have formally learned about in the classroom. Overall, the genetic and the traditional types from Rüsen’s (2004) typology dominated the students’ reasoning about whether similar situations could occur in the future. In the traditional type category, students related to the past in a non- problematizing way, with an assumption that what had happened would probably happen or not happen again. The answers were mainly non-reflective when it came History Education Research Journal 17 (2) 2020 146 Ammert et al. Ammert et al. 146 to discussing transformations of time. The high frequency of students expressing a genetic type response (about 35 per cent of responses) was notable, because the genetic type is complex and involves reflection on continuity and change, similarities and differences and, not least, awareness of the existence of different views at different times. A high percentage of the students provided reflections and arguments about how the past was interwoven in the discussion, and about the assessment of current sociopolitical situations and the expectations that student participants had about the future. Discussion and concluding words More than 50 per cent of the total number of responses related to factual knowledge about what happened in the past and its relevance for society today, possibly indicating the importance that the participants placed on historical knowledge, despite the questions not necessarily requiring any specific historical knowledge. The students used their previous knowledge to identify and interpret what happened in the past, and to discuss factual perspectives on the future. Although there might have been moral considerations behind the answers, they were not always explicit, and they were not the main arguments. There could be several explanations for this. First, students are more familiar with factual reasoning, putting empathy or moral aspects aside. This has been the traditional and established way of teaching, learning and presenting history at school. Second, the question of whether a situation similar to the one in the extract could occur again opens up opportunities for a knowledge-based discussion. These are also indications that personal moral references, as well as changing views of History Education Research Journal 17 (2) 2020 Identifying aspects of temporal orientation in students’ moral reflections 147 moral perceptions and issues, were treated as part of factual considerations by more than 30 per cent of the students. When students reflected on what message the narrative sends to them (Question 2), the most common answer profile was a combination of the exemplary type (Rüsen, 2004) and the existential (here including personal and moral aspects) strand (Chinnery, 2013), with 52 per cent of responses containing existential arguments. The question was personal, and it stimulated students to discuss more than what might be right or wrong, and also to relate to their experiences, their previous knowledge and their own opinions (see also Sellman and Barr, 2009: 19–20). In several answers, the reasoning took its starting point from what seemed to be deep-rooted perceptions of how people should and should not treat each other. Classroom teachers know their students, and know how they learn best. They are at the ‘coalface’, teaching them and interacting with them, usually almost daily. However, this article can assert several general findings: • High-school students can communicate their understanding of the nuances of human behaviour in history. This may help teachers to plan lessons, knowing that their students have the capacity to see historical events beyond the black and white, as they are frequently portrayed in the news and media. Discussion and concluding words • Students can readily connect present-day events (particularly the political) with the past, and this can be of interest to high-school students. Knowing this, teachers could plan lessons to provide an interesting hook to engage students, especially when teaching about traumatic pasts and difficult histories. • Even when students are not asked explicitly to do so, they can draw on historical factual knowledge that they already have – their background knowledge – in their responses. Teachers may use this understanding of their students’ world to construct learning experiences for them that connect with their knowledge and/or experiences. • For teachers, the results suggest that it can be beneficial and valuable, especially when teaching about difficult histories and traumatic pasts, to provide opportunities for students to question ethical implications and moral dimensions in history. Doing so could bring a more in-depth understanding of history and historical thinking. For teachers to do this, professional support will be required, so that teachers are equipped with the pedagogical skills to approach difficult questions in the history classroom. One important finding was that the most common combinations of answers were on existential foundations. Students were able to relate to (and learn from) the past when it came to personal and moral reflection, but they also used knowledge-based explanations of changeable contexts, or dissociated from the past when it came to discussing potential future developments. Participant responses in this study demonstrate that for students to be able to articulate a position of elaborated moral reasoning of historical topics, they must be provided with opportunities to integrate knowledge and content from historical evidence. The students who based their reasoning on existential and moral foundations and experiences neglected neither the factual historical context nor their previous knowledge. At the same time, moral and existential aspects were implicitly present in several answers where students’ main explanations and arguments were based on factual knowledge. This is an important finding because there is a risk that moral reasoning is, by its nature, more or less based on (and biased by) the context of the students’ lives in the present. In this study, the interlinking from both the existential and the knowledge-based positions was present History Education Research Journal 17 (2) 2020 148 Ammert et al. Ammert et al. and palpable. Discussion and concluding words A historical factual foundation is important, but the type of historical consciousness affects (and is affected by) how strong an existential and meaningful thread there is between a person and their relationship to the past. This interlinking strengthens the hypothesis of the importance of investigating the intersections of historical consciousness and moral consciousness in educational settings. Notes on the contributors Niklas Ammert, PhD, is a professor of history with a special focus on educational science and history didactics at Linnaeus University, Sweden. Since 2014, he has been affiliated as an international collaborator at HERMES, University of Newcastle, Australia. His research mainly focuses on history teaching, history education and the uses of history – how individuals and groups encounter, interpret and communicate history at school, in higher education, in politics and in other cultural and societal contexts. Heather Sharp, PhD, is an associate professor at the School of Education, University of Newcastle, Australia, the Convenor of the History Network for Teachers and Researchers (HNTR) and the Special Issues Editor of Historical Encounters. Her current research investigates the teaching of national history, especially through outbound mobility experiences, the influence of public history in teaching, historical representations in school curricula, particularly textbooks, and examines the written and visual texts in picture books that deal with conflict. Jan Löfström, PhD, is an associate professor in social studies education at the University of Turku, Finland. He has researched, among other topics, young people’s historical consciousness in the context of their view of historical responsibility. He is also editor of the multidisciplinary collection of articles, Voiko historiaa hyvittää? (‘Can history be repaired?’, Gaudeamus Helsinki University Press, 2012). Silvia Edling, PhD, is a professor in curriculum studies at the University of Gävle, Sweden, and is a history teacher in upper secondary school. Her research interests include history didactic/didaktik, teacher professionalism, and conditions in and for education. 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https://openalex.org/W4361226212	http://izvestiya.asu.ru/article/download/%282023%291-03/10594	Russian	null	Deterministic Interpretation of the Malus Law and Correlation in Experiments with Entangled Photons	Izvestiâ Altajskogo gosudarstvennogo universiteta	2,023	cc-by	4,303	Deterministic Interpretation of the Malus Law and Correlation in Experiments with Entangled Photons A.I. Goncharov Altai State University (Barnaul, Russia) Рассматривается локальная детерминистическая модель эксперимента с коррелированными фотона- ми. Экспериментальная установка содержит источ- ник, который в каждом акте излучает два линейно поляризованных фотона, два двухканальных анали- затора и детекторы, включенные в схему совпадений. Угол ориентации плоскости поляризации фотонов является случайной величиной с равномерным рас- пределением и одинаков у одновременно излучаемых фотонов. Рассматриваемая модель является обобще- нием «наивного примера теории со скрытыми пара- метрами», описанного в статье А. Аспэ (A. Aspect) Bell's Theorem: The Naive View of an Experimentalist. Исход взаимодействия фотона с анализатором (по- падание в один из двух каналов) однозначно опре- деляется углом между плоскостью поляризации фо- тона и осью анализатора и описывается ступенчатой функцией. Отличие нашей модели состоит в том, что ступенчатая функция содержит большое чис- ло участков разной длины. Расположение и длины участков заданы так, чтобы вероятность попадания фотона в конкретный канал идеального анализатора, определяемая путем усреднения исходов по малым окрестностям углов, при уменьшении максималь- ной длины участка приближалась к закону Малюса. Это делает классическую модель самосогласованной. В случае идеальных анализаторов абсолютная вели- чина коэффициента корреляции показаний детекто- ров в основном не превышает значений из «наивного примера» Аспэ. В случае анализаторов с поглощени- ем рассчитанные корреляции при некоторых распо- ложениях анализаторов превышают квантово-меха- нические значения. This paper examines a local deterministic model of an experiment involving correlated photons. The experimental setup involves a source that emits two linearly polarized photons in each act, two two-channel analyzers, and detectors included in the coincidence scheme. The orientation angle of the photon polarization plane is a random variable with a uniform distribution, and it is the same for simultaneously emitted photons. The model presented here is a generalization of the “naive example of hidden variable theory” described in A. As- pect's article ‘Bell's Theorem: The Naive View of an Expe- rimentalist’. In our model, the outcome of the photon interaction with the analyzer (hit in one of the two channels) is uniquely determined by the angle between the photon polarization plane and the analyzer axis and is described by a step function. The difference in our model is that the step function contains a large number of segments of different lengths. Детерминистическая интерпретация закона Малюса... и корреляции в экспериментах с запутанными фотонами А.И. Гончаров Алтайский государственный университет (Барнаул, Россия) Алтайский государственный университет (Барнаул, Россия) Алтайский государственный университет (Барнаул, Россия) Детерминистическая интерпретация закона Малюса... Детерминистическая интерпретация закона Малюса... УДК 53.01:535.51 УДК 53.01:535.51 Введение Введение В течение последних нескольких десятилетий в экспериментальной и теоретической физике осо- бое место занимают исследования систем частиц в запутанных квантовых состояниях. За исследо- вания фотонов, запутанных по поляризациям, ча- стично описанные в [1–5], Дж.Ф. Клаузеру, А. Ас- пэ и А. Цайлингеру в 2022 г. была присужде- на Нобелевская премия. Как известно, эти ис- следования подтвердили удивительные предска- зания квантовой механики, что обычно интерпре- тируется как нарушение принципа локального ре- ализма (например, [5]). Однако представляются также естественными попытки найти объяснение экспериментальным фактам в рамках классиче- ских локальных моделей [6–9]. Цель настоящей статьи — с помощью расчетов проиллюстриро- вать возможности классического подхода, а также некоторые трудности, которые возникают в ходе поиска классических объяснений результатов экс- периментов с запутанными фотонами. ) (1) ( ) (закон Малюса применительно к отдельным фо- тонам). Таким образом, сейчас полагаем, что ана- лизатор — идеальный, т.е. с единичной вероят- ностью фотон попадает в один из двух каналов. Считаем, что детектор тоже обладает эффектив- ностью в 100%. Если фотоны с определенными, но, вообще го- воря, разными ϕ, равномерно распределенными в [0, 2π), в случайном порядке направлять на анали- затор, то вероятности p±(ϑ) будут определяться путем усреднения вероятностей (1) по ϕ: p± смесь(ϑ) = 1 2π 2π 0 p±(ϕ, ϑ) dϕ = 1 2. (2) (2) 2. Описание двухчастичного экспери- мента. Рассмотрим эксперимент типа [2], в ко- тором источник в каждом акте одновременно из- лучает два фотона, запутанных по состояниям ли- нейной поляризации (см. рис. 2). Каждый из этих двух фотонов попадает в свой анализатор, ориен- тации которых равны соответственно ϑ1, ϑ2. По- казания первого и второго детекторов в одной и той же реализации обозначаем соответственно A и B. Подразумевается, что сигналы A, B поступают на схему совпадений. 1. Взаимодействие линейно поляризо- ванного фотона с анализатором. На рисун- ке 1 анализатор схематически изображен в виде пластинки, расположенной перпендикулярно оси Z. На пластинке имеется выделенное направле- ние — «ось» (на рисунке — пунктир). Ориента- ция пластинки в плоскости XY задается с помо- щью угла ϑ. Пусть на пластинку падают линей- но поляризованные фотоны, до попадания в ана- лизатор движущиеся вдоль оси Z. Ориентация плоскости поляризации фотонов (на рисунке — штрих-пунктир) определяется углом ϕ. Угол меж- ду плоскостью поляризации фотонов и направле- нием оси пластинки равен α = ϕ −ϑ. Рис. 2. Схема эксперимента + - 1 + - 2 A B X Z Рис. 1. Схематическое изображение анализатора X Y Рис. 2. Известия АлтГУ. Физика. 2023. № 1 (129) p−= P{A = −1}. Эти вероятности равны p+(ϕ, ϑ) = cos2 (ϕ −ϑ), p−(ϕ, ϑ) = sin2 (ϕ −ϑ) (1) p−= P{A = −1}. Эти вероятности равны p+(ϕ, ϑ) = cos2 (ϕ −ϑ), p−(ϕ, ϑ) = sin2 (ϕ −ϑ) (1) Deterministic Interpretation of the Malus Law and Correlation in Experiments with Entangled Photons A.I. Goncharov The location and lengths of the segments are set so that the probability of a photon hitting a specific channel of an ideal analyzer, determined by averaging the outcomes over small neighborhoods of angles, approaches the Malus law when the maximum length of a segment decreases. This ensures that the classical model is self-consistent. When ideal analyzers are used, the absolute value of the correlation coefficient of detector readings mainly does not exceed the values from Aspect's ‘naive example’. However, when analyzers with absorption are used, the calculated correlations for some orientations of the analyzers exceed the quantum mechanical values. Key words: linear polarization of photons, Malus law, en- tangled photons, correlations, hidden parameters. Ключевые слова: линейная поляризация фотонов, за- кон Малюса, запутанные фотоны, корреляции, скры- тые параметры. DOI: 10.14258/izvasu(2023)1-03 23 Известия АлтГУ. Физика. 2023. № 1 (129) AB = 1, а при \|θ\| = π/2 — полная антикорреля- ция: AB = −1. ной и той же функцией E(ϕ): A = E(ϕ −ϑ1), B = E(ϕ−ϑ2). Коэффициент корреляции показа- ний детекторов в рассматриваемой модели равен AB = 1 2π 2π 0 E(ϕ−ϑ1)E(ϕ−ϑ2) dϕ. Периодичность подынтегральной функции приводит к тому, что AB зависит только от разности θ = ϑ2 −ϑ1: Во многих работах отмечалась парадоксаль- ность этого экспериментального факта (напри- мер, [11]). Действительно, с классической точ- ки зрения фотоны имеют определенную ориента- цию плоскости поляризации, одинаковую у фо- тонов одной и той же пары, но случайным об- разом меняющуюся от пары к паре. Вероятность попадания отдельных фотонов в тот или иной ка- нал в классическом подходе определяется фор- мулой (2): p± = 1/2. Фотоны — компоненты па- ры взаимодействуют с анализаторами независимо друг от друга. Поэтому можно было бы ожидать, что даже при θ = 0 будут наблюдаться в сред- нем в равных количествах события типа «++», «+−», «−+», «−−». Как было уже отмечено, экс- перименты показывают отсутствие событий типа «+−», «−+» при θ = 0, что находится в пол- ном согласии с предсказанием квантовой меха- ники. При этом парадокса в квантовой механи- ке не возникает, поскольку пара запутанных фо- тонов рассматривается как единая система, а во- прос о механизме, который определяет поведение частиц, в квантовой механике не ставится. AB = 1 2π 2π 0 E(ϕ)E(ϕ −θ) dϕ. (6) (6) Поскольку E2(ϕ) = 1, полная корреляция AB(θ = 0) = 1 имеет место при любой функции E(ϕ), при- нимающей значения ±1. Поскольку E2(ϕ) = 1, полная корреляция AB(θ = 0) = 1 имеет место при любой функции E(ϕ), при- нимающей значения ±1. Чтобы выполнялась полная антикорреляция AB(θ = ±π/2) = −1, функция E(ϕ) должна удо- влетворять условиям E(ϕ ± π/2) = −E(ϕ). (7) (7) Из (7) также следует E(ϕ ± π) = E(ϕ). Если к (7) добавить естественное требование Из (7) также следует E(ϕ ± π) = E(ϕ). Если к (7) добавить естественное требование E(−ϕ) = E(ϕ), (8) (8) Но, как известно, описанный парадокс может быть устранен также и в рамках классическо- го подхода. С классической точки зрения полная корреляция показаний детекторов при θ = 0 (и полная антикорреляция при \|θ\| = π/2) является доказательством того, что при абсолютно точно заданных значениях параметров, определяющих точное (не квантово-механическое) состояние фо- тона и анализатора, исход взаимодействия фото- на с анализатором не является случайным. Набор таких параметров может быть как угодно боль- шим. Введение Схема эксперимента Вероятности попадания отдельных фотонов в каналы «+», «−» согласно квантовой механи- ке и результатам экспериментов равны p± = 1/2. Рассчитываемые с помощью квантовой механики вероятности совпадений и антисовпадений равны Рис. 1. Схематическое изображение анализатора p++(ϑ1, ϑ2) = P{A = 1, B = 1} = = p−−(ϑ1, ϑ2) = 1 2 cos2 θ, p+−(ϑ1, ϑ2) = p−+(ϑ1, ϑ2) = 1 2 sin2 θ, (3) Подразумеваем, что на самом деле анализа- тор представляет собой поляризационную приз- му, при взаимодействии с которой фотон может попасть в один из двух пространственно разде- ленных каналов, которые будем обозначать «+» и «−». Для удобства обозначения факта попада- ния фотона в тот или иной канал введем, следуя Дж. Беллу [10], величину A, которую будем на- зывать показанием детектора: если фотон попал в канал «+», то A = 1, а при попадании в канал «−» A = −1. где θ = ϑ2−ϑ1 (например, [3]). На основе (3) опре- деляется коэффициент корреляции показаний де- текторов AB = p++ + p−−−p+−−p−+ = cos 2θ. (4) (4) Эксперименты [2] подтвердили справедливость формулы (4). В частности, при θ = 0 наблю- дается полная корреляция показаний детекторов: Эксперименты [2] подтвердили справедливость формулы (4). В частности, при θ = 0 наблю- дается полная корреляция показаний детекторов: Вероятности попадания фотона в каналы «+», «−» будем обозначать p+, p−: p+ = P{A = 1}, 24 Детерминистическая интерпретация закона Малюса... Детерминистическая интерпретация закона Малюса... AB = 1, а при \|θ\| = π/2 — полная антикорреля- ция: AB = −1. AB = 1, а при \|θ\| = π/2 — полная антикорреля- ция: AB = −1. AB = 1, а при \|θ\| = π/2 — полная антикорреля- ция: AB = −1. № 1 (129) При N = 4 (λ = π/2) E(ϕ) = sgn(cos 2ϕ) и дли- ны всех участков одинаковы: λ± i = λ/2 = π/4. В работе А. Аспэ [3] этот вариант приведен в каче- стве «наивного примера» модели со скрытыми па- раметрами. Аналогичный пример в терминах из- мерения проекции спина содержится в [10]. В дан- ном случае интеграл (6) может быть вычислен аналитически: При дальнейшем уменьшении λ эти закономерно- сти сохраняются. На рисунке 5 также приведен результат AB(θ) усреднения AB(θ) по окрестностям угла θ ши- риной λ. Из рисунка видно, что функция AB(θ) близка к (cos 2θ)/2. Значение (cos 2θ)/2 коэф- фициента корреляции иногда называют клас- сическим; к нему приводит расчет на основе p++ класс(θ) = 1 2π 2π 0 p+(ϕ)p+(ϕ −θ) dϕ и аналогич- ных формул для p−−, p+−, p−+ (например, [12]), которые обеспечивают лишь частичную корреля- цию при θ = 0 и частичную антикорреляцию при θ = π/2. AB(θ) = 2 −4θ π −1 (θ ∈[0, π]). (11) (11) В классических моделях для описания исхода взаимодействия любых фотонов с анализатором, независимо от наличия или отсутствия квантово- механической запутанности, применяется одна и та же функция E(ϕ). Рассмотрим однофотонный эксперимент с линейно поляризованными фотона- ми при ориентации анализатора ϑ = 0. Необходи- мо согласовать детерминистическое описание экс- перимента A = E(ϕ) с вероятностным описани- ем согласно закону (1). Пусть перед попаданием в анализатор фотоны проходят сквозь реальный поляризатор с ориентацией оси пропускания ¯ϕ. Всем фотонам, выходящим из поляризатора, при- писывается один и тот же угол поляризации ¯ϕ; но истинный угол поляризации отдельных фотонов подвержен неконтролируемым случайным откло- нениям, и поэтому исход взаимодействия каждого фотона с анализатором является случайным. Обо- значим плотность вероятности этих отклонений fσ(ϕ −¯ϕ) (например, нормальное распределение со среднеквадратичным отклонением σ ≪1 рад). Вероятности p±( ¯ϕ) являются результатом усред- нения индикаторов по окрестности угла ¯ϕ: Рис. 4. Осциллирующая кривая — расчет AB(θ) при λ = π/32. Монотонные линии: cos 2θ, (cos 2θ)/2, 1 − 4θ/π Рис. 4. Осциллирующая кривая — расчет AB(θ) при λ = π/32. Монотонные линии: cos 2θ, (cos 2θ)/2, 1 − 4θ/π Рис. 5. Осциллирующая кривая — расчет AB(θ) при λ = π/64. Монотонные линии: cos 2θ, (cos 2θ)/2, 1 − 4θ/π. Кружки — AB(θ) (результат усреднения осцил- ляций AB(θ)) p±( ¯ϕ) = ∞ −∞ I±(ϕ)fσ(ϕ −¯ϕ) dϕ (ϑ = 0). Для самосогласованности модели необходимо, чтобы эти вероятности были близки к закону Ма- люса (1). AB = 1, а при \|θ\| = π/2 — полная антикорреля- ция: AB = −1. Например, должно быть существенным рас- стояние от траектории фотона до каждого атома, расположение электронов в этих атомах в момент пролета фотона и т.д. Но в настоящей статье мы рассмотрим только простейшую феноменологиче- скую модель (будем называть ее «моделью ступе- нек»), в которой состояние анализатора определя- ется одним только углом ϑ, а состояние фотона — углом ϕ. Например, показание A первого детекто- ра при ϑ1 = 0 однозначно определяется некоторой функцией E(ϕ), которая принимает значения ±1. Обозначим L+, L−множества значений ϕ, при ко- торых фотон попадает соответственно в каналы «+», «−». Индикаторы этих множеств обозначим I+ I−: то будут иметь место симметрии то будут иметь место симметрии E(π/2−ϕ) = E(π/2+ϕ), E(π/4−ϕ) = −E(π/4+ϕ), (9) которые приводят, в частности, к AB(θ = ±π/4) = 0. E(π/2−ϕ) = E(π/2+ϕ), E(π/4−ϕ) = −E(π/4+ϕ), (9) которые приводят, в частности, к AB(θ = ±π/4) = 0. E(π/2−ϕ) = E(π/2+ϕ), E(π/4−ϕ) = −E(π/4+ϕ), (9) A (θ E(π/2−ϕ) = E(π/2+ϕ), E(π/4−ϕ) = −E(π/4+ϕ), (9) (9) ( ) которые приводят, в частности, к AB(θ = ±π/4) = 0. Для иллюстрации этого подхода рассмотрим конкретную модель. Весь диапазон ϕ ∈[0, 2π) разобьем на N интервалов одинаковой длины λ, которые будем называть кластерами, с границами ϕi. Каждый кластер содержит один участок типа «+» длиной λ+ i и один участок типа «−» длиной λ− i : 0 < λ+ i , λ− i < λ, λ+ i + λ− i = λ, причем λ+ i = ϕi ϕi−1 cos2 ϕ dϕ, λ− i = ϕi ϕi−1 sin2 ϕ dϕ. (10) (10) Кроме длин участков, необходимо также за- дать расположение участков внутри кластеров. Расположение участков влияет на корреляции по- казаний детекторов, и в этом отношении его роль аналогична роли фазы волновой функции в кван- товой механике. При ϕ ∈[0, π/2) участки типа «+» мы располагаем в левой части кластеров. На рисунке 3 для примера приведен график функции E(ϕ) при N = 16, т.е. при λ = π/8. I±(ϕ) = 1, ϕ ∈L±, 0, ϕ ̸∈L±. (5) (5) Рис. 3. Функция E(ϕ) при λ = π/8 E(φ) π π /2 φ 1 -1 Интересующая нас функция равна E(ϕ) = I+(ϕ) −I−(ϕ). Для идеальных анализаторов I+(ϕ) + I−(ϕ) = 1, E(ϕ) = 2I+(ϕ) −1. Интересующая нас функция равна E(ϕ) = I+(ϕ) −I−(ϕ). Для идеальных анализаторов I+(ϕ) + I−(ϕ) = 1, E(ϕ) = 2I+(ϕ) −1. Так как анализаторы идентичны, результат взаимодействия фотонов с ними описывается од- 25 Известия АлтГУ. Физика. 2023. AB = 1, а при \|θ\| = π/2 — полная антикорреля- ция: AB = −1. Диаграмма функции E(ϕ) с участками E = 0 при λ = π/8, δ = 0,35 в полярных координатах та AB(θ) приведен на рисунке 8. Видно, что име- ются интервалы, на которых \|AB(θ)\| превышает не только \|1 −4θ/π\|, но и квантово-механические корреляции \| cos 2θ\|. На рисунке 9 приведен гра- В двухфотонном эксперименте теряются пары фотонов в случае событий типа «00», «±0», «0±». Чтобы полное число учтенных реализаций оста- лось тем же, число фотонов на остальных участ- ках пропорционально увеличивается. Доля поте- рянных пар, вообще говоря, зависит от θ. Соот- ветственно, от θ будет зависеть и нормировочный множитель. Рис. 8. Жирная кривая — расчет AB(θ) при λ = π/8. Тонкие кривые — cos 2θ, (cos 2θ)/2, 1 −4θ/π При каждом θ вычисляются w±± = 1 2π 2π 0 I±(ϕ)I±(ϕ −θ) dϕ, w±∓= 1 2π 2π 0 I±(ϕ)I∓(ϕ −θ) dϕ, w±∓= 1 2π 2π 0 I±(ϕ)I∓(ϕ −θ) dϕ, Рис. 8. Жирная кривая — расчет AB(θ) при λ = π/8. Тонкие кривые — cos 2θ, (cos 2θ)/2, 1 −4θ/π 0 ненормированный коэффициент корреля- ции ABн(θ) = w++ + w−−−w+−−w−+ и C(θ) = w++ + w−−+ w+−+ w−+. Норми- рованный коэффициент корреляции равен AB(θ) = ABн(θ)/C(θ). 0 ненормированный ненормированный ненормированный коэффициент корреля- ции ABн(θ) = w++ + w−−−w+−−w−+ и C(θ) = w++ + w−−+ w+−+ w−+. Норми- рованный коэффициент корреляции равен AB(θ) = ABн(θ)/C(θ). фик AB(θ) при λ = π/64 (N=128). На многих ин- тервалах \|AB\| > \| cos 2θ\|. Результат AB(θ) усред- нения осцилляций AB(θ) по-прежнему близок к (cos 2θ)/2. Рисунок 10 построен на основе этого же расчета, но на нем оставлены только точки, соответствующие углам θ, кратным π/64. Из ри- сунка 10 видно, что эти точки близки к экспери- ментальной кривой cos 2θ. Отметим, что в моде- лях с двумя скрытыми параметрами для анали- заторов с участками E = 0 возможно получение \|AB(θ)\| ⩾\| cos 2θ\| не только на отдельных интер- валах, но и во всем диапазоне θ ∈[0, 2π). Как и в модели с идеальными анализаторами, разбиваем [0, 2π) на N кластеров одинаковой дли- ны λ. Кластер может содержать несколько участ- ков типа «0»; их суммарная длина в i-м класте- ре равна λ0 i = λ −λ+ i −λ− i . Пусть λ0 i = λδ, где δ = const. Тогда во всех кластерах в среднем бу- дет теряться одинаковая доля фотонов δ. Мини- мальную длину отдельного участка типа «0» в i-м кластере обозначим Di. AB = 1, а при \|θ\| = π/2 — полная антикорреля- ция: AB = −1. (или аналогичной формуле с б´ольшим числом скрытых параметров), удовлетворяет неравенству Белла \|AB(ϑ1, ϑ2) −AB(ϑ1, ϑ2′) + AB(ϑ1′, ϑ2) + AB(ϑ1′, ϑ2′)\| ⩽2. Для точного AB, определяемо- го формулой (4), при некоторых ϑ1, ϑ1′, ϑ2, ϑ2′ это неравенство нарушается (например, [3]). Поэтому (6) в принципе не может привести к (4). Согласно предположению И. Питовского [7], в действитель- ности множества L± неизмеримы, что приводит к невозможности использования формул типа (6). i ̸= 1, ϕ ∈[0, π/4] Di = λ0 i /5. Пусть при ϕ ∈[0, π/4] расположение участков такое, как показано на ри- сунке 6. При ϕ > π/4 E(ϕ) определяется так, что- бы выполнялись симметрии (7)–(9). Ниже при- E( ) Рис. 6. Расположение участков в первых двух кла- стерах. Указаны длины участков, на которых E = 0. Пунктиром показаны границы кластеров 3. Анализаторы с поглощением. В ра- боте [13] на примере частиц со спином 1/2 пока- зано, что можно получить правильные корреля- ции в рамках детерминистической локальной мо- дели со скрытыми параметрами, если допустить, что некоторая доля частиц не регистрируется де- текторами. Аналогичный вывод применительно к двухчастичным экспериментам с фотонами сде- лан в работе [9]. Проиллюстрируем эту идею на примере модели ступенек. 3. Анализаторы с поглощением. В ра- боте [13] на примере частиц со спином 1/2 пока- зано, что можно получить правильные корреля- ции в рамках детерминистической локальной мо- дели со скрытыми параметрами, если допустить, что некоторая доля частиц не регистрируется де- текторами. Аналогичный вывод применительно к двухчастичным экспериментам с фотонами сде- лан в работе [9]. Проиллюстрируем эту идею на примере модели ступенек. ведены результаты расчетов при δ = 0,35. Пусть сначала длина кластера λ = π/8 (N = 16). На рисунке 7 приведена диаграмма функции E(ϕ); направления, в которых E = 1 и E = −1, пока- заны соответственно черным и серым цветом, а направления E = 0 — белым. Результат расче- Рис. 7. Диаграмма функции E(ϕ) с участками E = 0 при λ = π/8, δ = 0,35 в полярных координатах Пусть при некоторых ϕ −ϑ ∈L0 фотон погло- щается в анализаторе; этому соответствует E(ϕ− ϑ) = 0. Покажем, что введение участков типа «0», на которых фотоны теряются, с соответству- ющим увеличением числа фотонов на участках типов «+», «−» для сохранения нормировки, мо- жет привести к функции AB(θ), пересекающейся с cos 2θ в большом числе точек. Уменьшая λ, чис- ло точек пересечения можно сделать как угодно большим. Рис. 7. AB = 1, а при \|θ\| = π/2 — полная антикорреля- ция: AB = −1. Это возможно при достаточно большом числе разбиений N. Каждая окрестность усредне- ния шириной порядка 2σ содержит участки, отно- сящиеся к множествам L+, L−. Если длины λ до- статочно малы, то, согласно (10), λ+( ¯ϕ) ≈λ cos2 ¯ϕ, λ−( ¯ϕ) ≈λ sin2 ¯ϕ. Если при этом λ ≪σ ≪1 рад, то p+( ¯ϕ) ≈λ+( ¯ϕ)/λ ≈cos2 ¯ϕ, p−( ¯ϕ) ≈λ−( ¯ϕ)/λ ≈ sin2 ¯ϕ. Точные равенства достигаются в пределе λ/σ →0, σ →0. Аналогичное понимание веро- ятностей использовалось в работе [6] при анализе мысленных экспериментов с электронами. Рис. 5. Осциллирующая кривая — расчет AB(θ) при λ = π/64. Монотонные линии: cos 2θ, (cos 2θ)/2, 1 − 4θ/π. Кружки — AB(θ) (результат усреднения осцил- ляций AB(θ)) Можно усложнять модель, например, путем разбиения [0, 2π) на кластеры разной длины λi или задавая случайные границы кластеров, зави- сящие от еще одного (вдобавок к ϕ) «скрытого па- раметра», одинакового у фотонов одной и той же пары. Но и в таких моделях в лучшем случае нам удалось получить всего лишь (11) с одновремен- ным точным выполнением закона (1). При E(ϕ) = ±1 любая функция, вычисленная по формуле (6) Вернемся к двухчастичному эксперименту с коррелированными фотонами. На рисунке 4 при- ведены результаты расчета AB(θ) при λ = π/32 (N = 64), а на рисунке 5 — при λ = π/64 (N = 128). На обоих рисунках рассчитанный коэффи- циент корреляции \|AB(θ)\| почти везде, за исклю- чением точек θ = 0, θ = π/2 и небольших об- ластей вблизи θ = π/4, меньше истинного значе- ния \| cos 2θ\| и в основном не превышает \|1−4θ/π\|. 26 Детерминистическая интерпретация закона Малюса... i ̸= 1, ϕ ∈[0, π/4] Di = λ0 i /5. Пусть при ϕ ∈[0, π/4] расположение участков такое, как показано на ри- сунке 6. При ϕ > π/4 E(ϕ) определяется так, что- бы выполнялись симметрии (7)–(9). Ниже при- Рис 6 Расположение участков в первых двух кла D1 3 D1 D D D 3 2 2 2 E( ) 1 -1 (или аналогичной формуле с б´ольшим числом скрытых параметров), удовлетворяет неравенству Белла \|AB(ϑ1, ϑ2) −AB(ϑ1, ϑ2′) + AB(ϑ1′, ϑ2) + AB(ϑ1′, ϑ2′)\| ⩽2. Для точного AB, определяемо- го формулой (4), при некоторых ϑ1, ϑ1′, ϑ2, ϑ2′ это неравенство нарушается (например, [3]). Поэтому (6) в принципе не может привести к (4). Согласно предположению И. Питовского [7], в действитель- ности множества L± неизмеримы, что приводит к невозможности использования формул типа (6). Заключение Предложен самосогласованный вариант «на- ивного примера» А. Аспэ [3] теории со скрытыми параметрами с большим числом интервалов посто- янства индикаторной функции. С помощью рас- четов коэффициента корреляции проиллюстри- рованы возможности классических моделей и их ограниченность. AB = 1, а при \|θ\| = π/2 — полная антикорреля- ция: AB = −1. Пусть D1 = λ0 1/4, а при 27 Известия АлтГУ. Физика. 2023. № 1 (129) Рис. 9. Осциллирующая кривая — расчет AB(θ) при λ = π/64. Монотонные линии: cos 2θ, (cos 2θ)/2. Круж- ки — AB(θ) (усреднение AB(θ) по отрезкам θ шири- ной λ) Рис. 10. Кружки — расчет AB(θ) в точках θ = λ n ∈Z ⩾0; кривая — cos 2θ Рис. 10. Кружки — расчет AB(θ) в точках θ = λn, n ∈Z ⩾0; кривая — cos 2θ Рис. 9. Осциллирующая кривая — расчет AB(θ) при λ = π/64. Монотонные линии: cos 2θ, (cos 2θ)/2. Круж- ки — AB(θ) (усреднение AB(θ) по отрезкам θ шири- ной λ) Рис. 10. Кружки — расчет AB(θ) в точках θ = λn, n ∈Z ⩾0; кривая — cos 2θ Рис. 9. Осциллирующая кривая — расчет AB(θ) при λ = π/64. Монотонные линии: cos 2θ, (cos 2θ)/2. Круж- ки — AB(θ) (усреднение AB(θ) по отрезкам θ шири- ной λ) Соответствует ли предположение о неидеально- сти анализаторов реальной экспериментальной си- туации? Данные на этот счет весьма противоречивы. С одной стороны, например, в работе [14] говорится, что в экспериментах имеют место «колоссальные по- тери фотонов». С другой стороны, согласно [15], по- тери в поляризационных призмах невелики и могут быть снижены до долей процента. Библиографический список 1. Clauser J.F., Horne M.A. Experimental consequences of objective local theories // Phys. Rev. D. 1974. Vol. 10. № 2. 1. Clauser J.F., Horne M.A. Experimental consequences of objective local theories // Phys. Rev. D. 1974. Vol. 10. № 2. 9. Белинский А.В. К поиску разрешения парадокса Бел- ла // Письма в ЖЭТФ. 1996. Т. 64. Вып. 4. 10. Bell J.S. On the Einstein Podolsky Rosen Paradox // Physics. 1964. Vol. 1. № 3. 2. Aspect A., Grangier P., Roger G. Experimental Realization of Einstein–Podolsky–Rosen–Bohm Gedankenexperiment: A New Violation of Bell’s Inequalities // Phys. Rev. Lett. 1982. Vol. 49. № 2.hh 11. Белинский А.В. Теорема Белла для трихотомных наблюдателей // УФН. 1997. Т. 167. № 3. DOI: 10.3367/ UFNr.0167.199703h.0323. 3. Aspect A. Bell’s Th eorem: Th e Naive View of an Expe- rimentalist // Quantum [Un]speakables. Berlin, Heidelberg, 2002. DOI: https://doi.org/10.1007/978-3-662-05032-3_9. 12. Norden B. Quantum entanglement: facts and fi ction — how wrong was Einstein aft er all? // Quarterly Reviews of Biophysics. 2016. Vol. 49. № e17. DOI: 10.1017/ S0033583516000111. 4. Weihs G., Jennewein T., Simon C., Weinfurter H., Zeilinger A. Violation of Bell’s Inequality under Strict Einstein Locality Conditions // Phys. Rev. Lett. 1998. Vol. 81. № 23. 13. Pearle P.M. Hidden-Variable Example Based upon Data Rejection // Phys. Rev. D. 1970. Vol. 2. № 8. 5. Aspect A. To be or not to be local // Nature. 2007. Vol. 446. № 7138. DOI:10.1038/446866a. 14. Хренников А.Ю. Эксперимент ЭПР—Бома и нера- венство Белла: квантовая физика и теория вероятностей // Теоретическая и математическая физика. 2008. Т. 157. № 1. DOI: https://doi.org/10.4213/tmf6266. 6. Pitowsky I. Resolution of the Einstein–Podolsky–Rosen and Bell Paradoxes // Phys. Rev. Lett. 1982. Vol. 48. № 10. 7. Pitowsky I. Deterministic model of spin and statistics // Phys. Rev. D. 1983. Vol. 27. № 10. 15. Белинский А.В. Квантовые измерения. М., 2008. 8. Белинский А.В., Клышко Д.Н. Интерференция све- та и теорема Белла // УФН. 1993. Т. 163. № 8. DOI: 10.3367/ UFNr.0163.199308a.0001. 28
https://openalex.org/W2772735550	https://refubium.fu-berlin.de/bitstream/fub188/21206/1/Dinh-Thanh-2017-Salmonella-0189302.pdf	English	null	Improved sample treatment protocol for accurate detection of live Salmonella spp. in food samples by viability PCR	PloS one	2,017	cc-by	7,504	Mai Dinh Thanh1,2, Gemma Agustı´3, Anneluise Mader2, Bernd Appel2, Francesc Codony3 1 Freie Universita¨t Berlin, Department of Biology, Chemistry and Pharmacy, Berlin, Germany, 2 German Federal Institute for Risk Assessment, Department Biological Safety, Berlin, Germany, 3 GenIUL, Barcelona, Spain Mai Dinh Thanh1,2, Gemma Agustı´3, Anneluise Mader2, Bernd Appel2, Francesc Codony3 1 Freie Universita¨t Berlin, Department of Biology, Chemistry and Pharmacy, Berlin, Germany, 2 German Federal Institute for Risk Assessment, Department Biological Safety, Berlin, Germany, 3 GenIUL, Barcelona, Spain 1 Freie Universita¨t Berlin, Department of Biology, Chemistry and Pharmacy, Berlin, Germany, 2 German Federal Institute for Risk Assessment, Department Biological Safety, Berlin, Germany, 3 GenIUL, Barcelona, Spain * mai.dinhthanh@fu-berlin.de a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * mai.dinhthanh@fu-berlin.de a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 RESEARCH ARTICLE Abstract Culture-based detection is still considered as the standard way for detection of Salmonella in foods, although molecular methods, such as viability PCR (vPCR), have been introduced to overcome some disadvantages of traditional culture methods. Despite the success of the vPCR methodology, the problem of false-positive results is a major drawback, especially when applied to environmental samples, hindering the interpretation of the results. To improve the efficiency of vPCR, many approaches have been introduced by several authors during the last years. In the present work, the combination of PEMAX dye, double tube change, and double photo-activation step was established as a strategy to improve vPCR protocol. By combining these approaches, we developed an improved sample treatment protocol able to neutralize DNA signals of up to 5.0×107 dead cells/sample from both pure culture and artificially contaminated food samples. Our results indicate that vPCR can work reliable and has a potential for high throughput detection of live Salmonella cells in food samples, minimizing false-positive signals. OPEN ACCESS Citation: Dinh Thanh M, Agustı´G, Mader A, Appel B, Codony F (2017) Improved sample treatment protocol for accurate detection of live Salmonella spp. in food samples by viability PCR. PLoS ONE 12(12): e0189302. https://doi.org/10.1371/journal. pone.0189302 Editor: Hideyuki Doi, University of Hyogo, JAPAN Editor: Hideyuki Doi, University of Hyogo, JAPAN Received: May 16, 2017 Accepted: November 23, 2017 Published: December 12, 2017 Copyright: © 2017 Dinh Thanh 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. Detection of live Salmonella in food samples by viability PCR antimicrobial effects, matrices that inhibit detection completely (e.g. cloves), amount of waste due to duplicates and dilutions, plus it is laborious and time-consuming. For instance, 3 to 4 days are required to obtain a negative result and more than 5 days to confirm a positive one (ISO 6579:2002; [3]). Further, viable but nonculturable (VBNC) cells cannot be detected. An alternative approach is polymerase chain reaction (PCR), a powerful method for the detection of microorganisms in various matrices. PCR is rapid, sensitive and allows specific detection of the target microorganism based on the amplification of their DNA. had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. The biggest drawback of the classical PCR is that this method cannot differentiate between live and dead populations or extracellular DNA. Viability PCR (vPCR) has been used to selec- tively detect live microorganisms by molecular procedures. In vPCR, photo-reactive dyes such as ethidium monoazide (EMA), propidium monoazide (PMA) and PEMAXTM (a mix of photo-reactive azide forms of phenanthridium) have been used to exclude DNA from dead cells with compromised cell membrane [4, 5, 6, 7, 8]. By applying a photo-reactive dye to the sample prior to the nucleic acid extraction, the dye can enter compromised cells and can cova- lently link to the nucleic acid through a photo-activation step, with the result that the amplifi- cation of the nucleic acid by molecular tools is inhibited. However, vPCR seems to have specific advantages and shortcomings. The success of vPCR depends on several factors such as dye (type and concentration) incubation conditions (tem- perature and time), light source, microorganism and matrices as well as PCR amplification conditions (e.g. temperature and time) used [9–12]. In addition, membrane integrity of the microorganisms affects the vPCR efficiency. Many efforts were made in recent years to opti- mize the vPCR protocols regarding those critical points. Further, as an approach to solve the problem of “ghost cells”- metabolically inactive and nonculturable cells with intact membrane —Codony et al. [5] combined EMA and PMA to make use of the advantages of both dyes. These authors have shown that the expulsion of EMA (applied in low concentration) from liv- ing Salmonella enteritidis cells is not a passive process. Hence by adding low concentrations of EMA to PMA in conjunction with vPCR buffers, DNA from “ghost cells” can be bound by EMA, whereas live cells would stay unaffected. Despite many efforts, even using well-opti- mized vPCR procedures and in spite of the expected full neutralization of nucleic acids of dead microorganisms, it is not unusual to obtain a partial signal reduction. In this sense, recent investigations demonstrated that, at least for Legionella and Salmonella, a fraction of DNA remains inaccessible to vPCR due to their interaction with the tube wall [6] and in turns gener- ates false positive results. Based on the cumulated knowledge of the recent years regarding optimization of vPCR pro- tocols, we developed a robust vPCR protocol suitable to exclusively detect live Salmonella in several matrices. Our protocol was verified in 33 food samples, which were artificially contami- nated with heat killed Salmonella enterica cells. Further, it was simulated that food samples are contaminated with Salmonella by adding live and dead cells and PCR detection was carried out after enrichment. 1. Introduction Salmonella belongs to one of the most common zoonotic pathogens causing a notable number of foodborne outbreaks and product recalls. In the European Union, 94,625 confirmed sal- monellosis cases were reported in 2015 [1]. For the USA, the Centers for Disease Control and Prevention estimate approximately 1.2 million illnesses and 450 deaths every year [2]. The most important food vehicles for foodborne Salmonella outbreaks were eggs and egg products, pig meat and products thereof, as well as bakery products [1]. Especially bakery products pose a direct risk to the consumers as no further decontamination steps take place before consumption. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: MDT received a grant of the Erasmus Plus program for higher education traineeships from the European Commission that enables us to work abroad at GenIUL research laboratories, Terrassa, Spain. We acknowledge financial support by the German Research Foundation and the OpenAccess Publication Funds of the Freie Universita¨t Berlin, which enables us to publish our manuscript in an open access journal. The funders In order to improve food safety related to Salmonella, it is important to carry out preventive quality controls based on standard detection technologies. According to several governmental regulations or recommendations, Salmonella must not be detectable in 25 g of food. Hitherto, the culture-based detection method is considered as the ideal way for detection of microorgan- isms. However, this method also shows limits and disadvantages such as skill level required, 1 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0189302 December 12, 2017 2.3. PEMAX treatment PEMAXTM dye (GenIUL, Terrassa, Spain), was dissolved in PCR grade water (VWR, Llinas del Valle´s, Spain) to obtain a stock dye solution of 2 mM and was kept at -20˚C until needed. PEMAXTM dye (GenIUL, Terrassa, Spain), was dissolved in PCR grade water (VWR, Llinas del Valle´s, Spain) to obtain a stock dye solution of 2 mM and was kept at -20˚C until needed. All reactions were conducted at a final volume of 200 μl. To create a final concentration of 100 μM PEMAX, 10 μl PEMAX stock solution was added to 200 μl sample. Samples were incu- bated in the dark at 37ºC for 30 min to allow dye penetration into cells with damaged mem- , p ) y p All reactions were conducted at a final volume of 200 μl. To create a final concentration of 100 μM PEMAX, 10 μl PEMAX stock solution was added to 200 μl sample. Samples were incu- bated in the dark at 37ºC for 30 min to allow dye penetration into cells with damaged mem- branes. Thereafter, the suspension was transferred in a new reaction tube (GenIUL). Photo- induced crosslinking of PEMAX was achieved by exposing the samples to 15 min of light, then 10 min of darkness, followed by 15 min of light using the PhAST Blue instrument (GenIUL) at 100% intensity. Then, the samples were transferred to a new reaction tube and were subse- quently centrifuged at 12,100×g for 5 min. 2.5. Amplification efficiency The real-time PCR efficiency was calculated using the slope of the standard curve, which was generated using 10 fold serial dilutions of Salmonella DNA extracted from a pure culture with known concentration. To obtain a standard curve, cycle threshold (Ct) values were plotted against the corresponding log10 cell count. The amplification efficiency (E) was calculated by applying the equation: E = 10−1/slope– 1 [14]. 2.4. DNA extraction and real-time PCR assay Water PCR grade was used as a non-template control. 2.1. Bacterial strain and culture conditions Salmonella enterica subsp. enterica CECT 4594 strain was streaked onto plate count agar (PCA) plates (Liofilchem, Roseto degli Abruzzi, IT) and incubated at 37ºC for 15 to 18 hours. Cells were harvested from the agar plates and suspended in 10 ml phosphate buffered saline (PBS, pH 7.4) to obtain a working bacterial suspension. The cell density was adjusted to an OD600 of 0.35, corresponding to 5.0×108 cfu/ml. 2 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0189302 December 12, 2017 Detection of live Salmonella in food samples by viability PCR 2.2. Dead cells stock production To obtain heat killed cells, 1 ml of the cell suspension was heated at 85˚C for 35 min using a standard laboratory heat block (thermomixer comfort, Eppendorf, Hamburg, Germany) at 900 rpm. The loss of viability of the cells was verified by plating 100 μl of the cell suspension on PCA plates, followed by incubation for 24 hours at 37ºC. 2.4. DNA extraction and real-time PCR assay DNA was extracted using the v-DNA reagent and buffer (GenIUL) according to the manufac- turer’s manual. Briefly, the cell pellets were resuspended in 200 μl of v-DNA reagent and were vortexed at 3,200 rpm (MPS-1 Multi Plate Shaker, bioSan, Riga, Latvia) for 5 min. Then, the cells were incubated at 80ºC for 10 min at 1,200 rpm in the heat block. After that, 600 μl of v- DNA buffer were added and vortexed again at 3,200 rpm for 2 min. Thereafter, the samples were centrifuged at 7,500×g for 2 min and 100 μl of supernatant was transferred in a new tube. DNA was extracted using the v-DNA reagent and buffer (GenIUL) according to the manufac- turer’s manual. Briefly, the cell pellets were resuspended in 200 μl of v-DNA reagent and were vortexed at 3,200 rpm (MPS-1 Multi Plate Shaker, bioSan, Riga, Latvia) for 5 min. Then, the cells were incubated at 80ºC for 10 min at 1,200 rpm in the heat block. After that, 600 μl of v- DNA buffer were added and vortexed again at 3,200 rpm for 2 min. Thereafter, the samples were centrifuged at 7,500×g for 2 min and 100 μl of supernatant was transferred in a new tube. Real-time PCR amplifications were performed using PikoReal™Real-Time PCR System (Thermo Fisher Scientific, Barcelona, Spain) with the following protocol: 15 min at 95˚C, 45 cycles of 15 sec at 95˚C, 40 sec at 60˚C followed by data acquisition. All reactions contained 4 μl of 5X HOT FIREPol Probe qPCR Mix Plus (Solis BioDyne, Tartu, Estonia), 5 μl of DNA template, 0.25 μM of primers and 0.1 μM of specific probe (final volume: 20 μl). The primers and TaqMan probe according to Cheng et al. [13] detected a 262 bp fragment of Salmonella spp. Water PCR grade was used as a non-template control. Real-time PCR amplifications were performed using PikoReal™Real-Time PCR System (Thermo Fisher Scientific, Barcelona, Spain) with the following protocol: 15 min at 95˚C, 45 cycles of 15 sec at 95˚C, 40 sec at 60˚C followed by data acquisition. All reactions contained 4 μl of 5X HOT FIREPol Probe qPCR Mix Plus (Solis BioDyne, Tartu, Estonia), 5 μl of DNA template, 0.25 μM of primers and 0.1 μM of specific probe (final volume: 20 μl). The primers and TaqMan probe according to Cheng et al. [13] detected a 262 bp fragment of Salmonella spp. 2.6. Selectivity of PEMAX on live and dead cells from pure culture Serial dilutions of live Salmonella cells (5.0×107 to 5.0×103 cfu) were mixed with a constant number of 5.0×107 dead Salmonella cells and subjected to 0 and 100 μM PEMAX treatment. As controls, samples containing either only 5.0×107 live cells or dead cells were also tested as previously described. The experiments were conducted by two independent assays, each per- formed in duplicate. 3 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0189302 December 12, 2017 Detection of live Salmonella in food samples by viability PCR 2.8. Detection of live Salmonella in artificially contaminated food samples Ground pork meat and a ready-to-eat salad mix (Cichorium intybus var. foliosum, Cichorium endivia var. latifolium and crispum) obtained from a local supermarket were used for the detec- tion of live Salmonella in the presence of dead Salmonella cells. For each matrix, 25 g were mixed with 225 ml of peptone water (Oxoid, Basingstoke, UK) and RAPID’Salmonella Capsule (Biorad, Hercules, CA, USA) into a filter stomacher bag (IUL S.A., Barcelona, Spain). Then, 1 ml of live cells (5.0×102 cfu/ml) and 2.5 ml of dead cells (5.0×108 cfu/ml) were inoculated to each suspension and homogenized for 30 sec in a stomacher (Maxicator, IUL, Terrassa, Spain). 250 ml of peptone water inoculated with bacteria were used as control. Contaminated samples were incubated at either 37˚C (1 assay with duplicates) or 41.5˚C (3 independent assays with duplicates). At point in time 0 and after 24 hours of incubation, 100 μl of superna- tants were taken for Salmonella detection and were processed as described in 2.7. 2.7. Elimination of dead Salmonella PCR signal in food samples During a 2-month period, a total of 33 food samples and 6 peptone water controls obtained for routine quality controls were subjected to Salmonella detection according to the reference cul- ture method (ISO 6579:2002, [3]) and other routine tests (S1 Table) in a local accredited labo- ratory (Barcelona, Spain). After enrichment, supernatants of the food enrichment broth (FEB) were transported at temperatures between 6-12ºC in a cool box. After arrival at our laboratory, the FEBs were stored at 4˚C until usage, which took place within 24–48 h after arrival. For each sample, 100 μl of the FEB or peptone water control were inoculated with 100 μl of dead Salmonella (5.0×107 cells). The mixtures were centrifuged at 100×g for 2 min, in order to remove rough particles. The supernatants were transferred to a new reaction tube and centri- fuged again at 12,100×g for 5 min. The cell pellets were suspended in 200 μl of PBS and treated either with 0 and 100 μM PEMAX as described previously. In order to examine the back- ground noise, 100 μl of the FEBs or peptone water controls were mixed with 100 μl of PBS without the addition of dead cells. These mixtures were treated with 0 and 100 μM PEMAX. As a negative and a positive control, 100 μl of dead Salmonella cell suspensions mixed with 100 μl of PBS (PBS controls) were subjected to 0 and 100 μM PEMAX as described previously. PLOS ONE \| https://doi.org/10.1371/journal.pone.0189302 December 12, 2017 3.1. Selectivity of PEMAX on live and dead cells in pure culture The selectivity of 100 μM PEMAX was tested on serial dilutions of live Salmonella cells (5.0×107 to 5.0×103 cfu) mixed with 5.0×107 dead cells. A linear regression analysis was performed by plotting the Ct values against the respective log10 live Salmonella cells (Fig 1, left). Samples with- out PEMAX treatment showed mean threshold cycles of 21.17 ± 0.63, which were similar to the controls (Fig 1, right) showing mean Ct values of 22.11 ± 0.22 for live cells without PEMAX, 21.75 ± 0.36 for dead cells without PEMAX and 21.70 ± 0.09 for live cells with PEMAX. Samples of live cells treated with PEMAX showed different threshold cycles. Despite the presence of high dead cell concentration, the linear regression curve of PEMAX treated samples did not lose the linearity, showing a correlation coefficient (R2) of 0.9969. The PCR efficiency of this experiment was 96.11%. Compared to that, the PCR efficiency with serial dilutions of pure DNA was only slightly higher with 98.27% (S1 Fig). These results indicate that PEMAX was able to eliminate PCR signals of 5.0×107 dead Salmonella cells without affecting the PCR efficiency. As depicted in the linear regression data (Fig 1), the quantification limit of the PCR reaction corresponds to 5.0×103 cells/sample (200 μl). Accordingly, with regard to the DNA extraction 4 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0189302 December 12, 2017 Detection of live Salmonella in food samples by viability PCR and DNA template volume, each PCR reaction contained 31 gene copies. At this gene copy level, the mean Ct values were 34.7 ± 0.59. The next tenfold serial dilution step showed no PCR signal. For this reason, Ct values between 35 and 39 should be considered below the practical quantification limit. Occasional Ct values >39 should be considered as non-significant or a possible fluorescence artifact as results of thermal probe degradation. 3.2. Elimination of dead Salmonella PCR signal in food samples FEB of 33 food samples and 6 peptone water negative controls were artificially contaminated with 5.0×107 dead Salmonella cells and were subjected to 0 and 100 μM PEMAX treatment. The goal of this experiment was to examine the applicability of vPCR in food samples. Results are presented in Table 1. The Ct values in spiked samples without PEMAX treat- ment were 23.14 ± 1.16. Once the PEMAX-qPCR procedure has been applied, it was able to completely eliminate the fluorescence signal of 23 samples containing dead Salmonella cells; in 2 samples Ct values were over 37 and in 8 samples Ct values were between 35 and 37. Addition- ally, samples without the addition of dead Salmonella also showed positive PCR signal in 8 cases, although cultural enrichment results showed negative results. As expected, most of the uncontaminated samples with PEMAX treatment showed no PCR signal, however 3 of them showed Ct values around 36. Based on these results, Ct values over 35 might be background noise and cannot be assessed reliable as positive. By setting the limit of detection to Ct 35, all contaminated FEB samples and controls would be assessed as negative. 3.3. Detection of live Salmonella in artificially contaminated food samples Salad and ground pork meat were artificially contaminated with 20 cfu/g live and 5×107 cfu/g dead Salmonella cells, respectively. Enrichment was conducted at 37˚C and 41.5˚C for 24 Fig 1. Effect of PEMAX on serial dilutions of live Salmonella cells in the presence of dead cells. Serial dilutions of live Salmonella cells (5.0×107 to 5.0×103 cfu) in the presence of 5.0×107 dead Salmonella cells were treated with 0 μM (white bars) or 100 μM (grey bars) PEMAX followed by real-time PCR. The control samples contains either 5.0×107 live or dead cells (no mixed population). Means, standard deviations (n = 4) and the linear regression equation and R2 value of PEMAX treated samples are depicted. * In 3 of 4 dead samples treated with PEMAX no PCR signal were detected, therefore only the PCR signal of 1 sample is indicated in the diagram. https://doi.org/10.1371/journal.pone.0189302.g001 Fig 1. Effect of PEMAX on serial dilutions of live Salmonella cells in the presence of dead cells. Serial 3 Fig 1. Effect of PEMAX on serial dilutions of live Salmonella cells in the presence of dead cells. Serial dilutions of live Salmonella cells (5.0×107 to 5.0×103 cfu) in the presence of 5.0×107 dead Salmonella cells were treated with 0 μM (white bars) or 100 μM (grey bars) PEMAX followed by real-time PCR. The control samples contains either 5.0×107 live or dead cells (no mixed population). Means, standard deviations (n = 4) and the linear regression equation and R2 value of PEMAX treated samples are depicted. * In 3 of 4 dead samples treated with PEMAX no PCR signal were detected, therefore only the PCR signal of 1 sample is indicated in the diagram. https://doi.org/10.1371/journal.pone.0189302.g001 https://doi.org/10.1371/journal.pone.0189302.g001 5 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0189302 December 12, 2017 https://doi.org/10.1371/journal.pone.0189302.t001 Artificially contaminated supernatants of the food enrichment broth (FEB) (107 dead Salmonella cells per sample) and uncontaminated FEB were treated with 0 μM and 100 μM PEMAX, followed by real-time PCR. Ct values are indicated. Further, cultural routine test results for the respective food sample are shown..s.: no signal; A: Absence in 25 g RAPID’ Salmonella Medium (Bio-Rad, Hercules, USA) Detection of live Salmonella in food samples by viability PCR Table 1. Effect of PEMAX on 107 dead Salmonella cells in spiked supernatants of the food enrichment broth (FEB). vPCR Ct Availability in 25 g FEB FEB spiked with 107 dead Salmonella cells 25 g food sample Sample number Treatment/ Sample name 0 μM PEMAX 100 μM PEMAX 0 μM PEMAX 100 μM PEMAX RAPID‘ Salmonella 1 Curry vegetables n.s. n.s. 22.67 n.s. A 2 Braised beef n.s. n.s. 21.79 n.s. A 3 Spinach with vegetables n.s. n.s. 22.68 n.s. A 4 Meat croquette n.s. n.s. 24.90 n.s. A 5 Potato salad n.s. 35.77 22.14 n.s. A 6 Braised beef n.s. n.s. 22.36 n.s. A 7 Vegetable cream n.s. n.s. 21.70 n.s A 8 Beans 36.53 n.s. 21.14 n.s. A 9 Broad beans 37.83 36.38 22.91 n.s A 10 Omelet 37.7 n.s. 22.65 n.s. A 11 Green salad, local market 1 n.s. n.s. 24.82 n.s. A 12 Green salad, local market 2 n.s. n.s. 25.55 35.47 A 13 Green salad, local market 3 35.62 n.s. 22.18 35.92 A 14 Green salad, local market 4 n.s. n.s. 22.75 35.52 A 15 Green salad, local market 5 n.s. n.s. 22.21 n.s. A 16 Vegetable soup n.s. 36.23 23.18 n.s. A 17 Grilled fish n.s. n.s 23.04 n.s. A 18 Curry vegetables n.s. n.s. 22.12 38.54 A 19 Grilled sausage 37.51 n.s. 24.41 n.s A 20 Seafood salad n.s. n.s. 24.76 35.62 A 21 Sausage with vegetables n.s. n.s. 24.22 n.s. A 22 Stewed lentils n.s. n.s. 22.93 n.s. A 23 French fries n.s. n.s. 24.60 36.93 A 24 Macaroni 31.01 n.s 22.32 35.75 A 25 Fish soup n.s. n.s. 22.86 n.s. A 26 Fried fish n.s. n.s. 23.16 n.s. A 27 Lasagne n.s. n.s. 21.54 n.s. A 28 Vegetable soup 34.47 n.s. 21.73 n.s. A 29 Burger n.s. n.s. 23.13 n.s. A 30 Curry vegetables n.s. n.s. 24.30 38.36 A 31 Chicken in sauce n.s n.s. 24.44 n.s A 32 Mashed potatoes n.s. n.s. 24.08 35.25 A 33 Grilled chicken n.s. hours in peptone water. The aim of this study was to demonstrate that PEMAX treatment can fully suppress PCR signals of dead cells in food samples without showing any negative effect on the detection on live cells after enrichment. 4.1. vPCR protocol improvement In theory, vPCR is a rapid, sensitive, and reliable method to simply detect live cells in a sample by molecular methods. However, the practical experience from many researchers raised doubts about their effectiveness [12, 15, 16]. Despite their huge potential, incomplete suppres- sion of PCR signals resulting in false positive results, in particular with high background of dead cells [17–19], has been one of the major limitations in the spread of this technique and their application in routine quality control. To address this issue, researchers around the world have made efforts for improving the effectiveness of vPCR methodology in order to detect only live cells in the sample. Factors such as the ratio between viable and dead bacterial cells, organic material composition and concentration have been highlighted as potential inhibitors for the vPCR procedure, DNA extraction and qPCR [11, 15]. With the aim to overcome false-positive results, we developed an improved vPCR protocol. In the present work, the improved vPCR protocol was tested in various experiments to demon- strate the applicability of this in real food samples. In these studies, we combined several improvements suggested in the literature in conjunction with our previous vPCR works regarding Salmonella in pure cultures [6]. Our focus was to keep the protocol as simple as pos- sible and suitable for adaption in the routine quality control. In most of the vPCR experiments, heat treatment was used to obtain dead bacteria cells [4, 17–19], therefore we also chose this method to inactivate the cells. Nevertheless, thermally- treated bacteria might differ in their performance compared to bacteria treated with other inactivation processes. Our killing condition could successfully kill the cells without releasing DNA as the signal of live and dead cells were similar (Fig 1). In the case of heat treatment, cell membrane becomes compromised and viability dyes can easily penetrate into the cell. In our case, we chose the newly appeared reagent PEMAX, which is a blend of viability dyes with different molecular weights. One of them has a certain level of permeability in intact cellular membranes. At least for Salmonella, previous investigations have demonstrated that metabolically active cells can be able to extrude certain levels of viability dyes (EMA) that crossed the cellular membrane. By keeping the less selective dye in a smaller concentration, e.g. 10 μM EMA and 50 μM PMA, live cells were not affected [5]. n.s 24.35 35.17 A A ifi i ll i d f h f d i h b h (FEB) (107 d d S l ll ll l ) d i d FEB d Artificially contaminated supernatants of the food enrichment broth (FEB) (107 dead Salmonella cells per sample) and uncontaminated FEB were treated with 0 μM and 100 μM PEMAX, followed by real-time PCR. Ct values are indicated. Further, cultural routine test results for the respective food sample are shown..s.: no signal; A: Absence in 25 g RAPID’ Salmonella Medium (Bio-Rad, Hercules, USA) hours in peptone water. The aim of this study was to demonstrate that PEMAX treatment can fully suppress PCR signals of dead cells in food samples without showing any negative effect on the detection on live cells after enrichment. The results are summarized in Table 2. At point in time 0 hours, the mean Ct values of all samples without PEMAX treatment were 30.34 ± 1.29, whereas PEMAX treated samples showed negative PCR results. After 24 hours of enrichment at 37˚C, untreated and PEMAX 6 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0189302 December 12, 2017 Detection of live Salmonella in food samples by viability PCR treated samples showed comparable results with mean Ct of 29.05 ± 2.13 and 29.64 ± 2.26, respectively, showing the amplification signal of live cells growing in the samples. However, enrichment at 41.5˚C could yield mean Ct of 24.05 ± 3.53 and 24.78 ± 3.77 for untreated and PEMAX treated samples, respectively, showing that at 41.5ºC, as expected, the cells had grown more efficiently and faster than at 37ºC. In addition, the Ct values for the particular food sam- ples varied heavily, resulting in the high standard deviation of the given Ct values. For both enrichment temperatures, salad showed higher Ct values than meat or the control. PLOS ONE \| https://doi.org/10.1371/journal.pone.0189302 December 12, 2017 4.1. vPCR protocol improvement Indeed, in case of our Salmonella vPCR experiment, no differ- ence occurred between PEMAX treated and untreated live cells. By using PEMAX, we ex- tended the possibility to avoid detection of “ghost cells”, which have an intact membrane but are metabolically inactive. Since, the Salmonella detection is done after the enrichment to ensure the absence of Salmonella in 25 g, the use of the viability dye PEMAX ensures the detec- tion of cells with intact cell membrane and active metabolism. With our improved vPCR protocol, PCR signal of dead cells with concentrations of up to 5×107 cells/sample could be completely suppressed. Complete signal reduction for Salmonella spp. has been obtained before by Xiao et al. [19] by using PMA, but only for concentration up to 106 cell/ml. In their experiments, PMA concentration of up to 200 μM was applied, yet PLOS ONE \| https://doi.org/10.1371/journal.pone.0189302 December 12, 2017 7 / 12 Detection of live Salmonella in food samples by viability PCR DNA of dead cells with concentrations beyond 106 cells/ml could not be neutralized. On the other hand, Martin et al. [17] were able to neutralize huge amounts of approximately 108 dead cells/g by applying 100 μM PMA and using large amplicons as a PCR target. Nonetheless, in this case the method for preparing dead cells was highly destructive (boiling for 10 min) for the membranes. Treatment with high temperature around 100˚C could lead to a release of DNA or make the membrane highly accessible to viability dyes [20–22]. For improving the effectiveness of vPCR, we made use of the findings of Li and Chen [23] as well as Martin et al. [17]. In their studies, the authors tested different primers resulting in PCR amplicon lengths varying between 65 and 260 or 75 and 417 bp, respectively. Their results showed that amplifications of PMA treated samples using primers for long amplicons could yield higher PCR signal suppression than short amplicons. Further, we adopted the tube change approach published by Agustı´et al. [6] to our protocol and extended it for a further tube change step after the photo-activation. By changing the tubes after the dark incubation these authors could yield a further signal suppression of 5 Ct compared to the control group. Further they showed that by changing the tube, residual DNA attached to the reaction tube could be avoided. 4.1. vPCR protocol improvement Moreover, the samples were treated with a double photo-activation step of dye, however without additional reagent addition. To our knowledge, this procedure has not been previously reported. By applying double photo-activation with a short dark period in between, we could extent the signal suppression for dead cells by at least 2 Ct (S2 Fig). Thus, this simple approach provides a new way to improve the effectiveness of vPCR. Some authors reported the use of double dye treatment, which increased the ΔCt of dead cell samples around 1–2.8 Ct compared to a single dye treatment for Listeria monocytogenes and Mycobacterium avium subsp. paratu- berculosis [24, 25]. Nevertheless, complete suppression of the fluorescent signals could not be obtained. In our study, a comparable trend was obtained by applying only one dye dosage cou- pled with double photo-activation instead of double dye treatment. Our results showed that the ratio of live to dead cells did not affect the efficiency of the method (Fig 1) as some reports indicate [21, 26]. These reports showed that the quantification of live cells by vPCR in the presence of high numbers of dead cells was difficult when the con- centration of live cells was lower than 105 cells per sample. Not least, dye incubation time and temperature is also a critical point affecting the effective- ness of vPCR. Nkuipou-Kenfack et al. [18] reported a combined effect of these two factors; for Salmonella an increased in ΔCt of about 2 for incubation at 40˚C instead of 20˚C could be shown. A further increase of about 2 Ct could be yielded when dye incubation time was 30 min instead of 5 min. Based on this finding, we set the PEMAX incubation condition to 30 min at 37˚C to improve the vPCR protocol. PLOS ONE \| https://doi.org/10.1371/journal.pone.0189302 December 12, 2017 Detection of live Salmonella in food samples by viability PCR Table 2. Detection of live Salmonella in artificially contaminated food samples (with live and dead cells) by vPCR method. Time 0h 24h Treatment 0 μM PEMAX 100 μM PEMAX 0 μM PEMAX 100 μM PEMAX Temperature Sample 37˚C (n = 2) Control 30.39 ± 0.64 >40* 26.61 ± 0.29 27.23 ± 0.25 Meat 30.29 ± 0.11 >40* 29.21 ± 0.04 29.44 ± 0.17 Salad 32.38 ± 0.37 >40* 31.35 ± 0.2 32.26 ± 0.29 41.5˚C (n = 6) Control 30.44 ± 1.55 >40 22.97 ± 0.55 23.52 ± 0.53 Meat 29.99 ± 1.14 >40 22.01 ± 2.78 22.49 ± 3.09 Salad 29.93 ± 1.35 n.s. 27.19 ± 4.03 28.35 ± 3.88 Threshold cycle (Ct) value of meat and salad artificially contaminated with Salmonella before and after enrichment. Enrichment was conducted at 37˚C and 41 5˚C Samples were either let untreated (0 μM PEMAX) or were treated with 100 μM PEMAX n s no PCR signal in 6 samples nella in artificially contaminated food samples (with live and dead cells) by vPCR method. Table 2. Detection of live Salmonella in artificially contaminated food samples (wi Table 2. Detection of live Salmonella in artificially con Threshold cycle (Ct) value of meat and salad artificially contaminated with Salmonella before and after enrichment. Enrichment was conducted at 37˚C and 41.5˚C. Samples were either let untreated (0 μM PEMAX) or were treated with 100 μM PEMAX. n.s. no PCR signal in 6 samples * 1 sample out of 2 without PCR signal ** at least 4 samples out of 6 without PCR signal https://doi.org/10.1371/journal.pone.0189302.t002 the results for cultural detection were negative for the 10 mentioned FEBs, the positive PCR signals over Ct 35 did now mean the presence of Salmonella. On the other hand, no signal would not mean negative result. In case that vPCR should become a routine test for Salmo- nella, there is a need to find a common sense for setting the practical quantification limit. Additionally, it should be discussed about further procedures to approve the results beyond the real-time PCR quantification limit. In our opinion, it is not necessary to choose between vPCR and cultural detection method. To the contrary, a combination of both methods would be an ideal way to detect live and viable but nonculturable cells. 4.2. Detection of Salmonella in food samples Usually, real samples are complex matrices, which may interfere with the efficacy of the vPCR treatment [10], therefore the new protocol was evaluated for dead Salmonella cells in various food samples. With our vPCR protocol using PEMAX, we could yield complete PCR signal suppression for 5.0×107 dead Salmonella cells per reaction in most of FEB samples (23 out of 33 samples). Nevertheless, we still have positive signals with Ct beyond our quantification limit in 10 samples. Within our practical quantification limit of 35 Ct, PCR signals below the limit could reliably be assessed as positive. The theoretical detection limit was about 38 Ct, but sig- nals higher than 35 Ct might be caused by residual unspecific amplification or due to thermal degradation of the Taqman probe. In addition, Ct values beyond 35 could not be assessed as reliable, because corresponding gene copy number per PCR reaction was low and would also cause false negative results. As PLOS ONE \| https://doi.org/10.1371/journal.pone.0189302 December 12, 2017 8 / 12 Threshold cycle (Ct) value of meat and salad artificially contaminated with Salmonella before and after enrichment. Enrichment was conducted at 37˚C and 41.5˚C. Samples were either let untreated (0 μM PEMAX) or were treated with 100 μM PEMAX. n.s. no PCR signal in 6 samples * 1 sample out of 2 without PCR signal ** at least 4 samples out of 6 without PCR signal PLOS ONE \| https://doi.org/10.1371/journal.pone.0189302 December 12, 2017 S1 Table. Microbiological status of 33 food samples tested in a laboratory accredited according to ISO 9001:2008. (DOCX) S1 Table. Microbiological status of 33 food samples tested in a laboratory accredited according to ISO 9001:2008. (DOCX) S1 Table. Microbiological status of 33 food samples tested in a laboratory accredited according to ISO 9001:2008. S1 Fig. Salmonella standard curve. Cycle threshold (Ct) values were plotted against the corre- sponding log10 cell count (3.7–8.7). (TIF) Since food matrices can be very complex, maybe some positive signals beyond or around Ct 35 could also be explained by incomplete DNA neutralization as result of matrix interference. In order to overcome this problem, the use of detergents during the dye incubation [11] might contribute to an improvement. However, we did not explore this approach because under our point of view, Salmonella detection in routine analysis is usually done after 24 hours of enrich- ment, as regulations claim the absence of live cells in 25g. In case that the samples contain live Salmonella cells, Ct values would reach the reliable range for positive signals after enrichment due to growth of cells. This criterion is well supported by the results shown in Table 2. In this sense, practical quantification limit of 35 Ct (corresponds to 5.0×103 cells/sample) does not meet the requirement for quantification of low concentration, but is sufficient for qualitative detection. In the last experiment, we demonstrated the applicability of the improved vPCR protocol to a more realistic scenario. Since our aim was to mimic a real world scenario, we decided for lower concentration of dead cells (5.0×107 cells/g = 5.0×105 cells/sample) than tested in the experiment before (107 cells/sample). The start inoculum of live cells was 20 cfu/g (accord- ingly, 0.2 cfu/sample). This concentration was considered to be sufficient to demonstrate the ability to detect low concentrations after enrichment and still ensure the presence of live cells in the contaminated food sample. The results indicate that the improved vPCR protocol was able to selectively detect live cells without being affected by dead cells. After enrichment, a clear positive signal could be observed, however Ct values varied. The reason might be the presence of other microorganisms in meat and salad. The autochthonous flora of salad might cause a competitive situation in the sample during enrichment, which affect the growth of Sal- monella. In addition, meat is a further source of nutrition and might affect the growth of Sal- monella positively. PLOS ONE \| https://doi.org/10.1371/journal.pone.0189302 December 12, 2017 9 / 12 Detection of live Salmonella in food samples by viability PCR Author Contributions Conceptualization: Mai Dinh Thanh, Gemma Agustı´, Francesc Codony. Investigation: Mai Dinh Thanh. Methodology: Mai Dinh Thanh, Francesc Codony. Project administration: Mai Dinh Thanh. Supervision: Gemma Agustı´, Anneluise Mader, Francesc Codony. Writing – original draft: Mai Dinh Thanh. Writing – review & editing: Gemma Agustı´, Anneluise Mader, Bernd Appel, Francesc Codony. Conceptualization: Mai Dinh Thanh, Gemma Agustı´, Francesc Codony. Project administration: Mai Dinh Thanh. Supervision: Gemma Agustı´, Anneluise Mader, Francesc Codony. Writing – original draft: Mai Dinh Thanh. Writing – original draft: Mai Dinh Thanh. Writing – review & editing: Gemma Agustı´, Anneluise Mader, Bernd Appel, Francesc Codony. 4.3. Summary and conclusion The present report showed that with the use of combined procedures the results obtained with vPCR are improved and the critical weaknesses of this technique might be solved. These mini- mal changes in the classical vPCR procedure provides hope to work with complex samples get- ting complete PCR signal reduction in dead cells avoiding thus, the false positive results. In the present work, a serial dilution of live cells could be detected properly without losing the linear- ity despite the presence of high dead cell concentration. Our improved vPCR protocol could demonstrate that the problem of false-positive results can be overcome and the same approach can probably be effective for detection of other food pathogens as well. In conclusion, our proposed vPCR protocol using PEMAX might be suitable for usage in routine analysis, because it is simple and showed reliable suppression of dead cells. In addition, this protocol harbors a potential for high throughput detection because it is quick and easy to conduct. Acknowledgments We acknowledge support by the German Research Foundation and the OpenAccess Publica- tion Funds of the Freie Universita¨t Berlin. 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https://openalex.org/W2753909683	https://jased.net/index.php/jased/article/download/615/1974	Norwegian	null	Hva vet vi (ikke) om elevers opplevelser med friluftsliv i norsk skole – en gjennomgang av empiriske studier 1974–2014	Journal for Research in Arts and Sports Education	2,017	cc-by	8,483	Peer-reviewed article Peer-reviewed article Peer-reviewed article Journal for Research in Arts and Sports Education Hva vet vi (ikke) om elevers opplevelser med friluftsliv i norsk skole en gjennomgang av empiriske studier 19742014 Kristian Abelsen1, og Petter Erik Leirhaug2 1Norges idrettshøgskole, seksjon for kroppsøving og pedagogikk, Norge; 2Høgskulen pa˚ Vestlandet, campus Sogndal, Norge Sammendrag Artikkelen presenterer en kunnskapsoversikt basert pa˚ en systematisk gjennomgang av empiriske studier om friluftsliv i norsk skole der elevers opplevelser eller stemmer kommer til uttrykk. En slik oversikt er ikke tidligere sammenfattet, selv om det er over 40 a˚r siden ordet friluftsliv kom inn i fagplanformuleringene med Mønsterplanen av 1974. 1974 ble derfor valgt som utgangspunkt for studiens søkeperiode, selv om det kan hevdes at opplæring og undervisning som omfatter aktivitet, læring og opphold i natur allerede fra tidlig 1900-tall har vært en del av norsk skoles oppdrag og virke. For søkeperioden fram til 2014 avdekkes 24 studier som møter inklusjonskriteriene, hvorav 18 er master- og hovedfagsoppgaver. Resultatet forteller at foreliggende empirisk basert kunnskap om elevers opplevelser med friluftsliv i skolen er begrenset og ikke tillater a˚ trekke syntetiserte slutninger. Med den reservasjonen som hovedkonklusjon drøfter artikkelen hvilken kunnskap de inkluderte studier kan gi om friluftsliv i skolen ut fra elevers perspektiv. purpose, even commercially, provided the original work is properly cited and states its license. Citation: K. Abelsen og P. E. Leirhaug. ‘‘Hva vet vi (ikke) om elevers opplevelser med friluftsliv i norsk skole en gjennomgang av empiriske studier 19742014.’’ Journal for Research in Arts and Sports Education, Vol. 1, 2017, pp. 1831. http://dx.doi. org/10.23865/jased.v1.615 #2017 K. Abelsen og P. E. Leirhaug. This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license. Citation: K. Abelsen og P. E. Leirhaug. ‘‘Hva vet vi (ikke) om elevers opplevelser med friluftsliv i norsk skole en gjennomgang av empiriske studier 19742014’’ Journal for Research in Arts and Sports Education Vol 1 2017 pp 1831 http://dx doi copy and redistribute the material in any medium or format and to remix, transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license. Citation: K. Abelsen og P. E. Leirhaug. ‘‘Hva vet vi (ikke) om elevers opplevelser med friluftsliv i norsk skole en gjennomgang av empiriske studier 19742014.’’ Journal for Research in Arts and Sports Education, Vol. 1, 2017, pp. 1831. http://dx.doi. org/10.23865/jased.v1.615 Keywords: School-based friluftsliv; outdoor education; student perspective; physical education; Norway Received: November, 2016; Accepted: April, 2017; Published: September, 2017 Keywords: School-based friluftsliv; outdoor education; student perspective; physical education; Norway Received: November, 2016; Accepted: April, 2017; Published: September, 2017 Forma˚let med artikkelen er a˚ gjennomga˚ og analysere empiriske studier om elevers opplevelser med friluftsliv i skolen, og drøfte hva disse studiene kan fortelle om friluftsliv som del av opplæringen i norsk skole. Friluftsliv som begrep kom inn i fagdelene av norske læreplaner først med Mønsterplanen fra 1974 (M-74; KUF, 1974), men bruk av natur, og a˚ bygge det som kan kalles friluftslivsverdier, har vært betraktet som en del av skolens oppgave gjennom hele 1900-tallet (Augestad, 2003; Haslestad, 2000). Etter en gjennomgang av M-74 og utkast fra arbeidet mot ny Mønsterplan i 1987 (M-87; KUF, 1987) uttalte Mytting (1991) at han ikke var i tvil om at (opplæring i) friluftsliv har kvaliteter som er sentrale i ma˚lsetningene for skolen og derfor potensielt et viktig satsingsfelt. Tilsvarende konklusjoner trekker Repp (1993) i sin grundige analyse av M-87, men presiserer samtidig at «fordi friluftslivsbegrepet er sa˚pass lite avklart i planen, sa˚ vil likevel bruken av natur kunne bli svært ulik fra klasse til klasse, fra skole til skole» (s. 98). Backman (2011) har i nyere studier fra Sverige, hvor læreplanene ligner de norske (Annerstedt, 2008), demonstrert at vektlegging av friluftsliv vil kunne bidra til opplæringens ma˚l om a˚ utvikle likeverd og demokratiske holdninger. Ba˚de M-74 og M-87 var rammeplaner hvor fagplandelen beskrev innhold som skoler og lærere skulle gjøre valg ut fra. Reformene pa˚ 1990-tallet representerte en ny og mer forpliktende læreplanforsta˚else, hvor friluftsliv med egne ma˚lformuleringer ble noe alle elever skulle møte i opplæringen. Dette ble fulgt opp ved innføring av Kunnskapsløftet i 2006 (LK06). Med økt fokus pa˚ grunnleggende ferdigheter og ma˚lbart læringsbytte, samt reduksjon i antall ma˚lformuleringer for friluftsliv, har det blitt hevdet at reformen innebar en svekking av friluftslivets plass i læreplanen (Gurholt, 2005; Leirhaug & Klepsvik, 2010). Leirhaug og Arnesen (2016) argumen- terer for at den tilsynelatende svekkingen skyldes føringer som ble gjort gjeldende for alle fag og emner, og konkluderer derfor med at friluftsliv ma˚ sies a˚ ha en sterk posisjon i LK06. Gjennom tia˚rig grunnskole og inntil tre a˚r i viderega˚ende opplæring skal elevene ha fa˚tt varierte opplevelser i friluftsliv, og opparbeidet seg kompetanse til a˚ kunne gjennomføre og verdsette enkle turer i norsk natur. Abstract The article aims to provide a systematic review of empirical studies on students’ experiences with school-based ‘friluftsliv’ (word used to describe a Nordic tradition of outdoor life). Even though 40 years have passed since the word ‘friluftsliv’ first entered the national curriculum in Norway and became part of the expected student experience, no such reviews of empirical research from schools have been conducted. The authors reviewed literature published between 1974the year ‘friluftsliv’ became a formulated goal in the new curriculumand 2014. For this period only 24 studies, of which 18 were master’s theses, met the inclusion criteria. The result shows that current research-based knowledge about students’experienceswith‘friluftsliv’andnature-basedoutdooreducationinschoolsisextremelylimited, and does not allow us to draw synthesized conclusions. With this as a backdrop, the article presents and discusses how the included studies can contribute to the knowledge of school-based ‘friluftsliv’. Korrespondanse: Kristian Abelsen, Norges idrettshøgskole, Postboks 4014 Ulleva˚l Stadion, 0806 Oslo, Noway. E-mail: kristian.abelsen@nih.no 2017 K. Abelsen og P. E. Leirhaug. This is an Open Access article distributed under the terms of the Creative mmons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), allowing third parties to py and redistribute the material in any medium or format and to remix, transform, and build upon the material for any rpose, even commercially, provided the original work is properly cited and states its license. #2017 K. Abelsen og P. E. Leirhaug. This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license. Citation: K. Abelsen og P. E. Leirhaug. ‘‘Hva vet vi (ikke) om elevers opplevelser med friluftsliv i norsk skole en gjennomgang av empiriske studier 19742014.’’ Journal for Research in Arts and Sports Education, Vol. 1, 2017, pp. 1831. http://dx.doi. 18 Hva vet vi (ikke) om elevers opplevelser med friluftsliv i norsk skole Hva vet vi (ikke) om elevers opplevelser med friluftsliv i norsk skole Keywords: School-based friluftsliv; outdoor education; student perspective; physical education; Norway Received: November, 2016; Accepted: April, 2017; Published: September, 2017 Før presentasjon og diskusjon av va˚re funn relatert til problemstillingen, vil neste avsnitt gjøre rede for studiens metodiske overveielser og valg. «skal kunne planlegge og gjennomføre turer til ulike a˚rstider, ogsa˚ med overnatting» (Utdanningsdirektoratet, 2015b). Tilsvarende finner vi i læreplanen for aktivitetslære 2 pa˚ idrettsfag dette kompetansema˚let: «Elevene skal kunne planlegge, gjennomføre og vurdere tur med overnatting ute i nytt naturmiljø» (Utdanningsdirektoratet, 2006). Gjennom programfag friluftsliv som en valgmulighet i viderega˚ende opplæring og innføring av valgfaget natur, miljø og friluftsliv for trinn 810 gis det mulighet for et utvidet tilbud til friluftslivsinteresserte elever. Samtidig som disse to nyvinningene indikerer skolepolitisk vilje for mer friluftsliv i skolen, viser Leirhaug og Arnesens (2016) studie at omfanget friluftslivsundervisning i skolehverdagen for de fleste elever ikke samsvarer med opplæringens intensjoner og læreplanens ma˚lsetninger. Med Goodlad og hans samarbeidspartneres (Goodlad, Klein og Tye, 1979) læreplanniva˚er som teoretisk rammeverk sammenligner de friluftslivets posisjon i den formelle læreplan med hvor mye tid som brukes til friluftslivsundervisning i kroppsøvingsfagets operasjonaliserte læreplan. Leirhaug og Arnesen (2016) avslutter sin studie av friluftsliv i kroppsøvingsfaget med understreking av at det er først med kunnskap om det Goodlad, Klein og Tye (1979) kaller den erfarte læreplan, hva elevene opplever og fa˚r ut av undervisningen, vi kan vurdere om undervisningen fungerer og om friluftslivets potensielle bidrag til læringsutbytte realiseres. Denne artikkelen søker a˚ bidra med en empirisk basert kunnskapsoversikt for den erfarte læreplan knyttet til friluftsliv. Utgangspunktet er en systematisk gjennomgang av norske empiriske studier pa˚ friluftsliv i skolen fra perioden 19742014. Med en a˚pen inngang har vi ikke gjort noen avgrensning av friluftslivsbegrepet, men overlatt til studiene selv a˚ definere friluftsliv i skolen som sitt undersøkelsesomra˚de. Vi har formulert følgende problemstilling: Hvilken empirisk kunnskap har vi om elevenes opplevelser med og tanker om friluftsliv i skolen? Før presentasjon og diskusjon av va˚re funn relatert til problemstillingen, vil neste avsnitt gjøre rede for studiens metodiske overveielser og valg. Keywords: School-based friluftsliv; outdoor education; student perspective; physical education; Norway Received: November, 2016; Accepted: April, 2017; Published: September, 2017 Under den generelle del av læreplanen heter det at undervisningen bør «nøre hugen til a˚ ferdes utanfor oppstukne veger, til a˚ bruke kropp og sansar til a˚ oppdage nye stader og til a˚ utforske omverda» (Utdanningsdirektoratet, 2015a). Undervisning og opplæring i friluftsliv skal bidra til felles dannelse og identitetsskaping i samfunnet (Utdanningsdirektoratet, 2015a, Utdanningsdirektoratet, 2015b). Stortingsmeldinga Friluftsliv Natur som kilde til helse og livskvalitet (Meld. St. 18 (20152016), 2016) bekrefter at satsing pa˚ friluftsliv i skolen skal innga˚ som et ledd i a˚ møte framtidige utfordringer. Ser vi pa˚ kroppsøving som felles fag, legger LK06 opp til et skolefriluftsliv hvor progresjon og elevenes selvstendiggjøring sta˚r sentralt. Et av kompetansema˚lene etter 10. trinn sier at elevene 19 K. Abelsen og P. E. Leirhaug «skal kunne planlegge og gjennomføre turer til ulike a˚rstider, ogsa˚ med overnatting» (Utdanningsdirektoratet, 2015b). Tilsvarende finner vi i læreplanen for aktivitetslære 2 pa˚ idrettsfag dette kompetansema˚let: «Elevene skal kunne planlegge, gjennomføre og vurdere tur med overnatting ute i nytt naturmiljø» (Utdanningsdirektoratet, 2006). Gjennom programfag friluftsliv som en valgmulighet i viderega˚ende opplæring og innføring av valgfaget natur, miljø og friluftsliv for trinn 810 gis det mulighet for et utvidet tilbud til friluftslivsinteresserte elever. Samtidig som disse to nyvinningene indikerer skolepolitisk vilje for mer friluftsliv i skolen, viser Leirhaug og Arnesens (2016) studie at omfanget friluftslivsundervisning i skolehverdagen for de fleste elever ikke samsvarer med opplæringens intensjoner og læreplanens ma˚lsetninger. Med Goodlad og hans samarbeidspartneres (Goodlad, Klein og Tye, 1979) læreplanniva˚er som teoretisk rammeverk sammenligner de friluftslivets posisjon i den formelle læreplan med hvor mye tid som brukes til friluftslivsundervisning i kroppsøvingsfagets operasjonaliserte læreplan. Leirhaug og Arnesen (2016) avslutter sin studie av friluftsliv i kroppsøvingsfaget med understreking av at det er først med kunnskap om det Goodlad, Klein og Tye (1979) kaller den erfarte læreplan, hva elevene opplever og fa˚r ut av undervisningen, vi kan vurdere om undervisningen fungerer og om friluftslivets potensielle bidrag til læringsutbytte realiseres. Denne artikkelen søker a˚ bidra med en empirisk basert kunnskapsoversikt for den erfarte læreplan knyttet til friluftsliv. Utgangspunktet er en systematisk gjennomgang av norske empiriske studier pa˚ friluftsliv i skolen fra perioden 19742014. Med en a˚pen inngang har vi ikke gjort noen avgrensning av friluftslivsbegrepet, men overlatt til studiene selv a˚ definere friluftsliv i skolen som sitt undersøkelsesomra˚de. Vi har formulert følgende problemstilling: Hvilken empirisk kunnskap har vi om elevenes opplevelser med og tanker om friluftsliv i skolen? Datagrunnlag og metode Selv om disse varierer i kvalitet, vil vi slik Borgen og Engelsrud (2015) argumenterer for kroppsøvingsfaget, hevde at «masterstudentene ma˚ betraktes som kunnskapsprodusenter for feltet» (s. 66). Oppgavene utgjør en vesentlig del av foreliggende empirisk dokumentert kunnskap om friluftsliv i skolen, samtidig som det ma˚ understrekes at dette er materiale produsert i forskerutdanning og at konklusjoner ma˚ leses i lys av dette. 54 studier møtte kriteriene. 24 av disse hadde empiri med elevperspektiv eller elev- og lærerperspektiv, og det er disse som utgjør datagrunnlaget i denne artikkelen. Kun en (Jordet, 2007) av de inkluderte studiene er publisert etter kriterier som tilfredsstiller universitets- og høgskolesektorens krav til fagfellevurdering. Resterende studier er enten forskningsrapporter publisert i regi av institusjoner eller forskningskonferanser eller studentarbeider. Master- og hovedfagsoppgaver teller 18 av de inkluderte studiene. Selv om disse varierer i kvalitet, vil vi slik Borgen og Engelsrud (2015) argumenterer for kroppsøvingsfaget, hevde at «masterstudentene ma˚ betraktes som kunnskapsprodusenter for feltet» (s. 66). Oppgavene utgjør en vesentlig del av foreliggende empirisk dokumentert kunnskap om friluftsliv i skolen, samtidig som det ma˚ understrekes at dette er materiale produsert i forskerutdanning og at konklusjoner ma˚ leses i lys av dette. Utarbeidelse av en tabelloversikt med hensiktsmessig indeksering (Timmins & McCabe, 2005) for hele materialet dannet grunnlaget for analyse av de 24 studiene med elevperspektiv. I tillegg til forskningsperspektiv sorterte indekseringen studiene i henhold til publikasjonsa˚r, skoleslag og a˚rstrinn, metodisk tilnærming og type publikasjon. Vi hentet fram problemstillingene og skrev et resyme av hovedfunn i hver enkelt studie. Arbeidet med oversikt og indeksering avdekte en rekke problemstillinger knyttet til friluftsliv i skolen, men ved gjennomgang av studiene med elevperspektiv, hvor ma˚let var a˚ gjøre en tematisk syntetisering (Thomas & Harden, 2008), ble det klart at det er noen tema som fa˚r mer oppmerksomhet i studiene, ba˚de fra elevenes og forskernes side. Sammen med betraktninger over materialet som helhet vil vi i de pa˚følgende delene av artikkelen løfte fram disse temaene og drøfte hvordan de bidrar med kunnskap om va˚r problemstilling. Begge artikkelforfatterne er vokst opp med friluftsliv og arbeider med lærerutdan- ning og friluftsliv, noe som kan ha betydning for tolkning og analyse. I va˚r analyse av materialet og den videre diskusjonen har vi tatt pa˚ alvor Baumeisters (2013) ra˚d om a˚ prøve a˚ opptre som dommer og jury heller enn advokat ved gjennomføring av litteraturreviewer. Datagrunnlag og metode Vi tilstreber med andre ord a˚ presentere resultatene nøytralt og samtidig synliggjøre va˚re overveielser. Datagrunnlag og metode Materialet, som vi baserer denne artikkelen pa˚, er samlet i sammenheng med utarbeidelse av en systematisk oversikt over norske empiriske studier pa˚ friluftsliv i skolen fra 1974 til 2014. Starta˚r ble valgt fordi friluftsliv med M-74 for første gang kom eksplisitt til uttrykk i fagplanene. Litteratursøk ble gjort i 2015 og vi valgte a˚ inkludere studier publisert ut 2014. Med utgangspunkt i ulike kombinasjoner av søkeord som «friluftsliv», «skole», «uteskole», «undervisning» og «pedagogikk» ble det søkt via bibliotekenes søkesystem Oria, i Cristin og databasene ERIC og Idunn. Det ble ogsa˚ gjort søk i rapportserier utgitt i re´gi av høgskoler med idretts- og friluftslivsstudier. Videre ble referanselister i identifiserte arbeider gjennomga˚tt pa˚ jakt etter enna˚ ikke registrerte publikasjoner. Etter en første sortering ut fra utgivelsesa˚r, titler og beskrivelser sto vi igjen med 93 titler. Disse ble skaffet til veie og gjennomga˚tt ut fra følgende inkluderingskriterier: Studien skal 20 Hva vet vi (ikke) om elevers opplevelser med friluftsliv i norsk skole . ha empiri om friluftsliv i grunnskole eller viderega˚ende skole (dette betyr at barnehage og høyere utdanning ekskluderes, og at studien skal presentere resultat fra forskning i skolehverdag eller blant lærere og/eller elever) . ha empiri om friluftsliv i grunnskole eller viderega˚ende skole (dette betyr at barnehage og høyere utdanning ekskluderes, og at studien skal presentere resultat fra forskning i skolehverdag eller blant lærere og/eller elever) . ha empiri om friluftsliv i grunnskole eller viderega˚ende skole (dette betyr at barnehage og høyere utdanning ekskluderes, og at studien skal presentere resultat fra forskning i skolehverdag eller blant lærere og/eller elever) . inneholde redegjørelse for metodisk tilnærming . være publisert som forskningsresultat (i form av artikkel, rapport, avhandling eller oppgave pa˚ minimum masterniva˚) i tidsrommet fra 1974 til og med 2014 . være publisert som forskningsresultat (i form av artikkel, rapport, avhandling eller oppgave pa˚ minimum masterniva˚) i tidsrommet fra 1974 til og med 2014 54 studier møtte kriteriene. 24 av disse hadde empiri med elevperspektiv eller elev- og lærerperspektiv, og det er disse som utgjør datagrunnlaget i denne artikkelen. Kun en (Jordet, 2007) av de inkluderte studiene er publisert etter kriterier som tilfredsstiller universitets- og høgskolesektorens krav til fagfellevurdering. Resterende studier er enten forskningsrapporter publisert i regi av institusjoner eller forskningskonferanser eller studentarbeider. Master- og hovedfagsoppgaver teller 18 av de inkluderte studiene. Ingen empiriske studier før 1993 Vi har ikke funnet empiriske arbeider fra de første 18 a˚rene av søkeperioden. Repp (1993) er første studie som tilfredsstiller inklusjonskriteriene. Derimot er det godt 21 21 K. Abelsen og P. E. Leirhaug med eksempler pa˚ mer teoretisk og pedagogisk baserte artikler om hva friluftsliv i skolen kan og bør være. Haugsja˚ (1975), Ka˚rhus (1975) og Mytting (1991) er eksempler pa˚ dette. Det ble ogsa˚, i løpet av disse fra forskningens empiriske «stumme a˚r», gjort utviklingsarbeid med friluftsliv i skolen flere steder. Dokumentasjon og spor av dette finnes, men da uten en klar metode for innsamling av empiri. Den erfarte læreplan omtales sjelden, og elevperspektivet berøres stort sett bare indirekte. Skogvang (1993) gjør en kartlegging i ettertid av friluftslivsvaner hos voksne som var med pa˚ et friluftslivsopplegg i gymnasregi i 1970-a˚ra, og i va˚r sammenheng kan vi notere at 25 % uttrykker at undervisningsopplegg i friluftsliv har hatt positiv innvirkning pa˚ deres friluftslivsinteresse. En annen konsekvens av manglende studier pa˚ 1970- og 80-tallet, er at det ikke foreligger empirisk basert kunnskap om elevers opplevelser med for eksempel friluftsliv valgfag i ungdomsskolen, et valgfag som vi vet var relativt utbredt og kunne være omfattende ba˚de i tid og innhold (Aamelfot, 1994). Men tilbake til va˚rt første funn, Repp (1993). Vi drister oss til a˚ pa˚sta˚ at studien er materialets mest ambisiøse pa˚ friluftslivets vegne. Den tenderer mot det ideologiske, og tyngdepunktet ligger i friluftsliv- og læreplananalyser og diskusjoner om a) hvilke potensiale friluftslivet har for skolen, og b) hva synes a˚ begrense en mer helhetlig og omfattende bruk av friluftsliv i skolen. Repp (1993) finner at friluftsliv har en begrenset plass i de praksisene han observerer, og selv om han har observert undervisnings- dynamikk i to skoler fa˚r elevenes stemme aldri eksplisitt plass i det empiriske materialet. 1Programfag friluftsliv er et valgfritt 5 timers fag for elever pa˚ Vg1 og Vg2, a˚pent for alle elever men plassert under utdanningsprogram for idrettsfag. Oversikt over inkluderte studier Grønningseter, Halla˚s & Kristiansen, 2005; Jordet, 2007; Jørgensen, 1999; Rødby, 2006). Jordet (2010) definerer uteskole som en «ma˚te a˚ arbeide med skolens innhold pa˚ hvor elever og lærere bruker nærmiljø og lokalsamfunn som ressurs i opplæringen for a˚ supplere og utfylle klasseromsundervisningen» (s. 34). En slik definisjon som inkluderer alle former for læringsprosjekter utenfor klasserommet har bred oppslutning, og forteller at uteskole ikke er ensbetydende med friluftsliv i skolen. De inkluderte uteskolestudiene beskriver alle en praksis hvor skolen nytter lokale naturomgivelser og friluftsliv kan sies a˚ ha en sentral plass. Det betyr at materialet ut over uteskolestudiene besta˚r av kun sju studier (Eriksen, 1997; Jacobsen mfl., 2002; Jacobsen mfl., 2006; Jakobsen, 2007; Kongsrud, 2004; Repp, 1993; Sølvik, 2003), hvorav to er basert pa˚ samme empiri, som fanger opp elevperspektiver mer direkte knyttet til ma˚lformuleringer for friluftsliv i grunnopplæringa. Analysen og oversikten over studiene fører oss til a˚ spørre om det er idrettsfaget som pa˚ høyere trinn bidrar til a˚ opprettholde vitaliteten og legitimiteten til friluftsliv i opplæringa? Friluftsliv har siden opprettelse av idrettslinjer hatt en sentral plass i faget aktivitetslære. I tillegg ble det i 2006 a˚pnet for a˚ opprette programfag friluftsliv1. Arbeid med friluftsliv har i disse fagene helt andre betingelser og tidsrammer enn hva lærere uttrykker at skolehverdagen ellers tillater (Jacobsen mfl., 2007; Leirhaug & Arnesen, 2016). Av de 10 studiene fra viderega˚ende opplæring er fire gjennomført pa˚ idrettsfag (Bamle, 2009; Gøtz, 2014; Naustdal, 2012; A˚ vangen, 2008) og fire pa˚ programfag friluftsliv (Foss, 2011; Hvattum, 2012; Nyga˚rd, 2012; Valdal, 2013). Dermed er det kun to studier fra viderega˚ende skole som tar for seg friluftsliv i kroppsøvingsfaget (Mikkelsen, 2013; Wiken, 2011). Vi skal i det følgende se nærmere pa˚ empirien i de inkluderte studiene, hvilke sider ved friluftsliv i skolen de tematiserer og hva de kan fortelle om elevers opplevelser. Oversikt over inkluderte studier 24 studier møtte inklusjonskriteriene. 14 fra grunnskolen og 10 fra viderega˚ende skole. Studiene i grunnskolen er fra perioden 19932007, mens de 10 studiene i viderega˚ende skole er alle fra perioden 20082014. Bare tre studier er publisert før 2002. De resterende 21 fordeler seg relativt jevnt fra 2002 til 2014. Dette kan være et uttrykk for en generell interessedreining ba˚de politisk og pedagogisk i retning den erfarte læreplan, fra undervisning og innhold til hva elever lærer og opplever. For friluftsliv kan interessen i studier med elevperspektiv i tillegg synes a˚ ha flyttet seg fra grunnskole til viderega˚ende skole, en dreining som kan ha sammenheng med innføringen av LK06 i 2006. Skolereformen medførte nye læreplaner for hele opplæringsløpet. Motivene var flere: bedre elevenes evne og lyst til a˚ lære, styrke de grunnleggende ferdighetene og gjøre grunnopplæringen bedre i stand til a˚ møte kunnskapssamfunnets utfordringer (St.meld. nr. 30 (20032004), 2004). Det kan hevdes at friluftsliv som tverrfaglig emne og naturen som læringsarena fikk da˚rligere betingelser etter denne reformen i hvert fall i de første a˚rene av grunnskolen. I «Har skulen snudd ryggen til naturen?» problematiserer Leirhaug og Klepsvik (2010) dette ut fra økt fokus pa˚ fag, regning, lesing og skriving i grunnopplæringa, noe som igjen koples til resultater blant annet i PISA-undersøkelsene. En annen observasjon na˚r vi ser pa˚ studiene fra grunnskolen, er at sju av 14 studier er relatert til uteskole (Abelsen, 2002; Fjørtoft & Larsen, 2005; Garborg, 2003; 22 Hva vet vi (ikke) om elevers opplevelser med friluftsliv i norsk skole Grønningseter, Halla˚s & Kristiansen, 2005; Jordet, 2007; Jørgensen, 1999; Rødby, 2006). Jordet (2010) definerer uteskole som en «ma˚te a˚ arbeide med skolens innhold pa˚ hvor elever og lærere bruker nærmiljø og lokalsamfunn som ressurs i opplæringen for a˚ supplere og utfylle klasseromsundervisningen» (s. 34). En slik definisjon som inkluderer alle former for læringsprosjekter utenfor klasserommet har bred oppslutning, og forteller at uteskole ikke er ensbetydende med friluftsliv i skolen. De inkluderte uteskolestudiene beskriver alle en praksis hvor skolen nytter lokale naturomgivelser og friluftsliv kan sies a˚ ha en sentral plass. Det betyr at materialet ut over uteskolestudiene besta˚r av kun sju studier (Eriksen, 1997; Jacobsen mfl., 2002; Jacobsen mfl., 2006; Jakobsen, 2007; Kongsrud, 2004; Repp, 1993; Sølvik, 2003), hvorav to er basert pa˚ samme empiri, som fanger opp elevperspektiver mer direkte knyttet til ma˚lformuleringer for friluftsliv i grunnopplæringa. Metode og forskningsspørsma˚l i studiene Inkluderte studier er i all hovedsak kvalitative. Kun tre studier er basert pa˚ kvantitative data, og to av disse faller litt pa˚ siden av va˚r problemstilling ved at de undersøker elevenes aktivitetsniva˚ ved uteundervisning og uteskole (Fjørtoft & Larsen, 2005; Grønningseter, Halla˚s & Kristiansen, 2005). Begge studiene er sma˚skala og har et lite utvalg, men pa˚viser at elevene ofte kommer opp i en intensitet som bidrar til helsefremmende effekter. Grønningseter, Halla˚s og Kristiansen (2005) sammenlignet inne- og uteundervisning og fant at intensitetsverdiene pa˚ dager med lavest intensitet i uteskolen fortsatt var høyere enn de høyeste verdiene pa˚ en tradisjonell dag inne pa˚ skolen. Tredje studie med kvantitativ tilnærming er Wiken (2011), som har spurt 229 elever fra tre viderega˚ende skoler om erfaringer med friluftsliv i kroppsøvingsfaget. 3040 % oppgir i liten grad a˚ kjenne fagets kompetansema˚l. Det er i friluftsliv, orientering og dans de oppgir a˚ ha lært minst. Nesten halvparten rapporterer en 23 K. Abelsen og P. E. Leirhaug holdning som tilsier at det er unødvendig a˚ lære om friluftsliv. Elevrapporteringene i studien støttes av andre masterstudier (Dalebø, 1996; Lund, 2014) som peker pa˚ at lærere har store utfordringer med a˚ tilrettelegge for et friluftsliv i tra˚d med læreplanens intensjoner og forventninger. De tre studiene med kvantitativ tilnærming viser at hva det forskes pa˚ og hvilke spørsma˚l som stilles, berører mer grunnleggende spørsma˚l om hva friluftsliv i skolen er, hva det kan være og hva det skal bidra med. Kartlegging av aktivitetsniva˚ knytter bruken av friluftsliv og natur til et folkehelseperspektiv. Det er i tra˚d med ma˚lsettinger i stortingsmeldinga om friluftsliv (Meld. St. 18 (20152016), 2016), men inviterer til a˚ diskutere mening fra et friluftsfaglig sta˚sted; er det aktivitetsniva˚et i friluftslivs- undervisningen som er vesentlig med tanke pa˚ skolens oppdrag om a˚ bygge elevenes friluftslivskompetanse, og a˚ opprettholde og levendegjøre friluftsliv som en del av kulturarven. Resultatene fra Wiken (2011) kan tolkes som at ba˚de elevenes kjennskap til hva som forventes relatert til friluftsliv, og hva de synes og tenker om friluftsliv i skolen kan være ganske forskjellig. Va˚r gjennomgang og analyse av de kvalitative studiene representerer kun sma˚ glimt fra noen enkelte elever, gjerne ved en enkelt skole eller med utgangspunkt i et bestemt friluftslivsopplegg. Hva vet vi om elevenes opplevelser og læring i friluftsliv? De forskjellige studiene og informantene benytter ulike ord og begreper, men de opplevelser og læringsutbytter som hyppigst løftes fram i studiene er relatert til sosial tilhørighet og gevinsten av samarbeid og samhandling (Foss, 2011; Garborg, 2003; Gøtz, 2014; Hvattum, 2012; Jakobsen, 2007; Rødby, 2006; Sølvik, 2003; A˚ vangen, 2008). Observasjonsstudier av uteskole viser en praksis hvor elevers medvirkning blir synlig, og hvordan elevene ser og anerkjenner hverandre i fri lek (Garborg, 2003; Jørgensen, 1999; Rødby, 2006). Selv om elevenes tanker og opplevelser bare unntaksvis kommer direkte til uttrykk i studiene fra trinn 17, beskrives det hvordan natur og romlighet inviterer til nye elevroller. God plass til utfoldelse synes a˚ virke positivt pa˚ dynamikken elevene imellom, pa˚ konfliktniva˚ og mulighet for tilpasset opplæring. Sølvik (2003), som har studert adferdsvansker mer spesifikt, skriver at friluftsliv tilbyr sammenheng mellom oppgaver, løsninger og konsekvenser som oppleves meningsfull og forsta˚elig for ungdommen. Atferdsproblematikken trer i bakgrunnen og elevene opplever sosial mestring. Særlig turer med lengre varighet gir anledning til a˚ vise flere sider ved personligheten og utfordrer faste samhandlings- og adferds- mønster (Sølvik, 2003). Avbrekk fra det teoretiske skolearbeidet, det a˚ se hverandre i nye roller og hvordan fellesskapsopplevelser utvikler og styrker samholdet, er sentrale kvaliteter na˚r elever i viderega˚ende skole selv skal forklare turenes verdi (Hvattum, 2012; A˚ vangen, 2008). Et annet moment vi ser i flere av studiene, er hvordan elever verdsetter under- visningsformer i friluftsliv hvor de blir delaktige og ansvarliggjort. Mikkelsen (2013) har gjennomført et undervisningsopplegg inspirert av selvbestemmelsesteorien til Deci og Ryan (2002). Elevene uttrykker at de har liten erfaring med medbestemmelse og valgfrihet i skolesammenheng. A˚ vangen (2008) løfter opp selvbestemmelse som en særlig kvalitet i elevenes erfaringer, noe de kan tenke seg ytterligere forsterket. I en kvalitativ studie av et femdagers tverrfaglig friluftslivsopplegg, løfter Kongsrud (2004) fram det positive forholdet mellom selvbestemmelse og engasjement som et hovedfunn. Det kan selvsagt diskuteres hvor mye motivasjonsteoriens begreper farger resultatene i de tre studiene, men vi merker oss gjennomga˚ende a) at elever uttrykker lite erfaring med medbestemmelse og involverende arbeidsformer, og b) at de kan tenke seg mer av denne type undervisning. Medvirkning og valgmulighet virker a˚ generere positive opplevelser av likeverdighet pa˚ tur og større motivasjon for ba˚de forberedelser og det som skjer underveis. Ser en forbi opplevelsene av medvirkning og friluftslivets sosiale og rekreative kvaliteter, forteller elevstemmene lite om hva som læres i skolens friluftslivsunder- visning. Metode og forskningsspørsma˚l i studiene Der Haslestad (2000) og Repp (1993) viser hvordan friluftslivets mer ideologiske talsmenn har vært opptatt av skolen som bidragsyter for et tryggere friluftsliv og ansvar for natur og miljø som friluftslivspedagogisk utfordring, er dette bare perifert berørt i studiene med elevperspektiv. Ut over uteskole, som er et felles tema i fem av de kvalitative studiene, gir studiene sma˚ beretninger om friluftsliv i skolen som sosialiseringsarena, om betydning for motivasjon og læringsmiljø, noe om vurdering og om elevers opplevelser og læring i friluftsliv er i tra˚d med læreplanenes ma˚l med friluftsliv. Selv om tre oppgaver har sett spesielt pa˚ opplevelser for elever med innvandrerbakgrunn, framtrer ikke perspektiver av hvordan mening og forsta˚else i friluftsliv konstrueres i skolen som sosial og kulturell kontekst. Spørsma˚l om hvordan elever er introdusert for, og fa˚tt hjelp til a˚ verdsette, natur og friluftslivslivets egenverdi, tematiseres ikke. Det er, tørr vi si, overraskende lite om hva elevene kan og tenker om friluftsliv, om elevenes naturopplevelser, om deres refleksive prosesser og læring underveis. Det er, for a˚ skjele til læreplanens generelle del, lite om hvordan elevene bruker «kropp og sansar til a˚ oppdage nye stader og til a˚ utforske omverda» (Utdanningsdirektoratet, 2015a). Det er mulig a˚ se en svak utvikling i at elevenes opplevelser fa˚r komme mer direkte til uttrykk i studiene utover 2000-tallet, men det er vanskelig a˚ si om dette skyldes en dreining i fokus eller om det har med at elevene i disse studiene er eldre og mer nærliggende a˚ intervjue for a˚ fa˚ del i deres refleksjoner og opplevelser. Med forbehold om at de fleste studiene bare representerer sma˚ glimt fra utvalgte elevers opplevelser med friluftsliv i skolen har vi allerede antydet hvilke tema som er løftet opp. Det er disse vi na˚ skal se nærmere pa˚. 24 Hva vet vi (ikke) om elevers opplevelser med friluftsliv i norsk skole K. Abelsen og P. E. Leirhaug K. Abelsen og P. E. Leirhaug Goodlad, Klein og Tye (1979) skrev at det er vanskelig a˚ innhente gode data for den operasjonaliserte læreplan, men at samme prosjekt for den erfarte læreplan er «even more slippery» (s. 63). Det at elevene opplever a˚ ikke lære noe i friluftsliv kan like gjerne være et uttrykk for hvordan elevene har en snever forsta˚else av begrepet læring. Studiene beskriver vesentlige læringsutbytter relatert til sosial læring og kompetanse, men det oppfattes ikke av elevene som læring. Na˚r elevene hos Foss (2011) i programfag friluftsliv sier de i liten grad lærer noe nytt og stort sett repeterer kunnskap de allerede har, kan det ogsa˚ leses som oppfyllelse av læreplanens intensjoner om ansvarlighet og selvstendiggjøring (Gurholt, 2008; Leirhaug & Arnesen, 2016). Hvis mange av ungdommene allerede har kompetanse til selv a˚ utøve enkelt friluftsliv uten en aktiv nærværende lærer, gir det ny forsta˚else til at halvparten av elevene i Wiken (2011) oppga det som unødvendig a˚ lære om friluftsliv. Vi ser et behov for studier om hvordan lærere og elever kommuniserer rundt friluftsliv og forventet læring, men ogsa˚ teoretiske studier om hvordan progresjon skal forsta˚s hvis den er ment a˚ invitere til verdifulle naturopplevelser, til nysgjerrig oppdaging og utforsking av natur og seg selv i møtet med natur. Friluftsliv er ogsa˚ en arena for konkret refleksjon om klodens bærekraft og hvordan menneskets handlinger alltid samspiller med naturen som kosmos. I lys av skolepolitiske intensjoner representerer utvikling av slike perspektiver, som altsa˚ er relativt fraværende i hva vi vet om elevenes opplevelser, en vesentlig utfordring, ikke bare for lærerkompetansen, men ogsa˚ til forskningen. Her er det et behov for a˚ utarbeide friluftslivsdidaktisk teori og praksis, parallelt med en kontinuerlig drøfting av hva friluftsliv i skolen skal innebære. Hvis selvstendiggjøring og opplevelse av egenverdi i møte med natur og friluftsliv er sentrale ma˚l skolefriluftslivet, er ekspertopplegg som krever ekstern spisskompetanse, særlig tilrettelagte anlegg eller avansert utstyr neppe veien a˚ ga˚. Vi skal ikke forfølge disse refleksjonene, men vende tilbake til analysen. Vi vil ta for oss vurdering og friluftslivshabitus, to tema hvor elevenes beretninger og opplevelser synes a˚ sprike i de studiene vi har sett pa˚. Hva vet vi om elevenes opplevelser og læring i friluftsliv? Sett under ett indikerer de 24 inkluderte studiene at læringsutbyttet na˚r det gjelder friluftsliv i den erfarte læreplan er relativt tilfeldig. Relatert til grunnopplærin- gen drøfter ba˚de Abelsen (2002) og Jordet (2007) utfordringer med manglende bevissthet rundt elevenes læring i uteskole som praktisert arbeidsform. I andre enden av opplæringen, viderega˚ende skole, uttrykker elever at det er uklart hva de skal lære i friluftsliv (Bamle, 2009; Wiken, 2011). 25 Utfordringer med vurdering Vi har sett at det som skal læres i friluftsliv framsta˚r uklart for elevene. Da er det ikke underlig at vurderingskriterier ogsa˚ oppfattes uklare og mangelfulle, og at usikkerhet forbundet med karaktergrunnlag kan føre til frustrasjon for elever (Naustdal, 2012). Likevel trenger ikke dette være tilfelle. Elevene i Hvattum (2012) opplever programfag friluftsliv som et avbrekk fra det teoretiske skolearbeidet og en ellers hektisk og vurderingsopptatt skolehverdag. Elevene i Bamles (2009) studie pa˚ idrettsfag uttrykte uklarhet omkring forventet læringsutbyttet i friluftsliv, men er eksplisitte pa˚ at de ønsker gode karakterer og opplever at innsats, samarbeid og gode holdninger vektlegges i vurderingen. I lys av læringsutbyttet som hyppigst beskrives, virker dette meningsfullt. To studier som inkluderer elever med innvandrerbakgrunn demon- strerer at sa˚ enkelt er det ikke. Gøtz (2014) har undersøkt hvordan elever med ulik kulturell bakgrunn opplever undervisningen i friluftsliv pa˚ idrettsfag, og skriver at de «flerkulturelle elevene i denne 26 Hva vet vi (ikke) om elevers opplevelser med friluftsliv i norsk skole oppgaven er enige om at hvis de hadde øvd mer pa˚ a˚ sta˚ pa˚ ski pa˚ forha˚nd, ville de fa˚tt bedre karakter» (s. 72). Manglende vinterlige forutsetninger viser seg avgjørende for vurdering og virker negativt pa˚ trivsel og motivasjon. Naustdal (2012) beskriver hvordan elever mister fokus pa˚ det sosiale og naturopplevelse pa˚ grunn av manglende kompetanse; perspektivet blir instrumentelt og opplevelsene preget av karakterjag. De tidligere beskrevne elevopplevelser av medbestemmelse og ansvar i friluftslivsunder- visningen, ser ikke ut til a˚ inkludere arbeid med kriterier og vurdering. De fire studiene (Bamle, 2009; Hvattum, 2012; Naustdal, 2012 og Gøtz, 2014) som belyser elevers opplevelser med vurdering er alle fra etter innføringen av LK06. Denne voksende interessen for vurdering i studienes spørsma˚lsstillinger kan tolkes som uttrykk for at reformen medførte et generelt økt fokus pa˚ vurdering og vurderingens rolle i opplæringen. Ingen av studiene kommer med eksempler pa˚ hvordan vurdering kan brukes produktivt inn mot læringsma˚l. De illustrerer derimot at et økt fokus pa˚ vurdering kan komme til a˚ overskygge andre verdier som elevene uttrykker sentrale i skolefriluftslivet. «. . .til ønsker om a˚ kunne videreføre friluftslivsgleden til venner og familie med telt- og skiturer. De har begge størst erfaring med friluftsliv fra venner, barnehage Utfordringer med manglende friluftslivshabitus Friluftsliv og innvandrere er et annet tema som i likhet med vurdering har fa˚tt økt fokus i senere tid. De tre studiene som inkluderer informanter med innvandrerbakgrunn og deres opplevelser, gir et tvetydig bilde av friluftsliv i skolen. Samtidig som de diskuterer hvordan fravær av primærsosialisering (Eriksen, 1997) og manglende forutsetninger eller habitus (Gøtz, 2014; Naustdal, 2012) bidrar til lav mestringsforventning og negative opplevelser, er det utsagn i disse studiene som tilsier at elevene opplever friluftsliv som en positiv avveksling, noe nytt og spennende en aktivitetsform egnet for nye utfordringer og sosiale opplevelser. Informantene uttrykker seg positivt om prosesser som krever samarbeid, ansvarstaking og kommunikasjon kompetanser som vi ikke bare finner igjen i opplæringens ma˚l, men ogsa˚ vesentlig bidrag for a˚ bli bedre kjent og utvikle vennskap. Eriksen (1997) spør om friluftsliv er en egnet arena for integrering, noe som samfunnsmessig og politisk er interessant fordi forskning viser lavere deltakelse i friluftsliv blant innbyggere med minoritetsbakgrunn (NINA, 2014). Stortingsmel- dinga om friluftsliv understreker at god informasjon til denne gruppen er spesielt viktig (St. Meld. 18 (20152016), 2016, s. 81). En utfordring er at informantene hos Eriksen (1997) ikke virker a˚ se overføringsverdien av opplevelser fra friluftslivsun- dervisning til dagliglivet. Denne studien er omtrent 20 a˚r gammel og det flerkulturelle Norge har utviklet seg mye i mellomtiden. Selv om va˚rt materiale fram til 2014 ikke framviser en økt interesse for det flerkulturelle, kan vi forvente dette i kommende a˚r. Det er allerede kommet flere studier etter va˚r undersøkelsesperiode. En av disse er Tune (2016), der to jenter med innvandrerbakgrunn eksemplifiserer andre historier enn Eriksen (1997). Pa˚ spørsma˚l om hvorfor de har valgt programfag friluftsliv, viser jentene: «. . .til ønsker om a˚ kunne videreføre friluftslivsgleden til venner og familie med telt- og skiturer. De har begge størst erfaring med friluftsliv fra venner, barnehage 27 K. Abelsen og P. E. Leirhaug eller skole. Programfag friluftsliv gir dem kompetansen de føler de trenger til a˚ kunne gi en form for reversert sosialisering til friluftsliv med deres egen familie» (Tune, 2016, s. 7071). eller skole. Programfag friluftsliv gir dem kompetansen de føler de trenger til a˚ kunne gi en form for reversert sosialisering til friluftsliv med deres egen familie» (Tune, 2016, s. 7071). Med manglende primærsosialisering til friluftsliv, har jentene gjennom sekundær- sosialiseringen tilegnet seg erfaringer med a˚ være ute i naturen og lært a˚ glede seg over dette. Utfordringer med manglende friluftslivshabitus Begge har ga˚tt i en «skole der de vektlegger a˚ legge deler av undervisningen ut i naturen» (s. 58), og Tune (2016) kommenterer at skolens effekt og rolle som sosialiseringsarena har vært sentral i elevenes dannelse og utdannelse til friluftsliv. Selv om dette er et enkelteksempel, skrevet fram i en masteroppgave, ser vi her at friluftsliv gjennom skolen kan fungere inkluderende for elever som ikke møter norsk natur og friluftsliv via familie og oppvekst ellers. Men bak denne type gode eksempler lurer mange uavklarte problemstillinger. Det kan gjelde innhold i friluftslivsunder- visningen, hvordan friluftslivets egenverdi rekonstrueres i en skolesetting, timer til ra˚dighet, skolens avstand til naturomra˚der, muligheter for medbestemmelse og valg, lærernes kompetanse og samarbeid med lokale ressurspersoner og organisasjoner. Biography Kristian Abelsen er ansatt ved Norges Idrettshøgskole, Seksjonen for Kroppsøving og Pedagogikk. Forskning og undervisning er relatert til faglærerutdanningen og studieprogrammene i friluftsliv ved seksjonen. Han har bakgrunn som uteskolelærer i ungdomsskolen og lærer i folkehøgskolen. Forskningsinteressen sentrerer omkring didaktikk for friluftsliv i skolen. Petter Erik Leirhaug er førsteamanuensis ved Høgskulen pa˚ Vestlandet. Forskning og undervisning er knyttet til lærerutdanningen og bachelor- og masterstudiene innen friluftsliv, idrett og kroppsøving. Han har bakgrunn som lærer i grunnskolen og i viderega˚ende skole, og har skrevet doktorgrad om vurdering for læring i kroppsøving ved Norges idrettshøgskole. Hva vet vi (ikke) om elevers opplevelser med friluftsliv i norsk skole Hva vet vi (ikke) om elevers opplevelser med friluftsliv i norsk skole For a˚ oppsummere er det fristende a˚ si at friluftsliv i elevperspektivets prisme framtrer som et mangfoldig fenomen. Med 24 inkluderte studier, hvor tema og elevgruppe varierer, er det ikke mulig a˚ avgjøre om det er studienes perspektiver som styrer hva vi fa˚r vite noe om, mer enn hva elevene faktisk lærer og opplever i skolefrilutslivet. I forlengelsen av dette er det vi spør hva vi ikke vet om elevenes opplevelser og læringsutbytte. Det kan like gjerne være ma˚ten studiene er gjennomført pa˚, spørsma˚lsstillingene og teoriene det bygges pa˚, som fører til at enkelte perspektiver løftes frem og andre tilskygges. Vi kan derfor gjenta Leirhaug og Arnesens (2016) pa˚pekning av et akutt behov for a˚ utforske og dokumentere hva elever fa˚r ut av eksisterende friluftslivsundervisning. Vi vil ogsa˚ understreke skolen og lærernes ansvar overfor det intensjonelle og formelle læreplanniva˚et og pa˚ behov for større og mer langsiktige utviklingsprosjekter, og da spesielt med et fokus rundt didaktisk praksis, naturopplevelser, elevenes læring og kroppsliggjøring av kunnskap i friluftsliv. Det hevdes stadig, senest i nevnte Stortingsmelding (Meld. St. 18 (20152016), 2016), at friluftsliv kan inkluderes i mange av skolens fag, at nærmiljøet representerer en viktig kunnskapskilde og læringsarena. Meldingen relaterer til spennende prosjekter og undervisningspraksis hvor friluftsliv synes a˚ fa˚ særlig oppmerksomhet. I fortsettelsen blir det viktig at slike eksempler blir gjenstand for forskningsmessig tilnærming. Oppsummering og konklusjon I denne studien har vi gjennomga˚tt og analysert empiriske studier 19742014 med elevperspektiv pa˚ friluftsliv i skolen. Materialet teller totalt 24 studier. Disse er i hovedsak kvalitative (21) og undersøkelsene fordeler seg pa˚ uteskole (7), kroppsøving i grunnskolen (7) og viderega˚ende skole (2), idrettsfag (4) og programfag friluftsliv (4). Na˚r 18 av de 24 inkluderte studiene er utført pa˚ master- og hovedfagsniva˚ vil vi pa˚peke et bemerkelsesverdig fravær av bidrag fra etablerte forskere. Dette fører oss til a˚ konkludere med at det knapt finnes empirisk forskningsbasert kunnskap om den erfarte læreplan av norsk skoles friluftslivsopplæring. Materialets omfang tillater ikke en tematisk syntetisering av resultatene (Thomas & Harden, 2008). Vi kan derfor ikke trekke konklusjoner om hva som er typiske elevopplevelser fra friluftsliv i skolen, eller hva som preger effektiv friluftslivsundervising. Men innenfor rammen av de 24 inkluderte studiene uttrykker elever pa˚ tvers av skoleslag og alder at friluftsliv i skolen har kvaliteter som fremmer samhold, samarbeid og andre sider ved sosial kompe- tanse. Samtidig illustrerer analysen at friluftsliv og dets mulige kvaliteter konstitueres gjennom didaktiske praksiser der elever og lærere samhandler. Friluftsliv i skolen er ikke et ferdig forsta˚tt fenomen. Friluftslivet kan ikke leses rett ut av læreplanene, det ma˚ fylles med mening og verdi. Va˚r gjennomgang av studier med elevperspektiv, selv om de er fa˚, viser nettopp dette. Det er studier der elevene i hovedsak beskriver friluftsliv som avbrekk fra en ellers teoripreget skolehverdag, samtidig som vi ogsa˚ finner studier der elever opplever karakterjag i friluftsliv. I materialet er det to studier hvor elever med innvandrerbakgrunn opplever at det forventes bakgrunnskunnskap og ferdigheter som de ikke har grunnlag for a˚ mestre, mens friluftslivsundervisningen i andre studier framtrer positivt og motiverende ved at elevene opplever reelt ansvar og medvirkning. 28 Litteraturliste Aamelfot, A.M. (1994). Valgfag friluftsliv i ungdomsskolen: en kartlegging av utbredelse og omfang av valgfag friluftsliv pa˚ ungdomstrinnet i Norge i 1989. Hovedfagsoppgave. Oslo: Norges idrettshøgskole. Abelsen, K. (2002). Uteskole og lærerprofesjonalitet. Visjoner og virkelighet. Hovedfagsoppgave. Oslo: Norges idrettshøgskole. dt, C. (2008). 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https://openalex.org/W4293105639	https://research-information.bris.ac.uk/files/334692615/s41598_022_18505_0.pdf	English	null	Basin-scale multi-decadal analysis of hydraulic fracturing and seismicity in western Canada shows non-recurrence of induced runaway fault rupture	Scientific reports	2,022	cc-by	11,983	Rodriguez, G., Eaton, D. W., & Verdon, J. P. (2022). Basin-scale multi-decadal analysis of hydraulic fracturing and seismicity in western Canada shows non-recurrence of induced runaway fault rupture. Scientific Reports, 12(1), Article 14463. https://doi.org/10.1038/s41598-022-18505-0 Publisher's PDF, also known as Version of record License (if available): CC BY Link to published version (if available): 10.1038/s41598-022-18505-0 This is the final published version of the article (version of record). It first appeared online via Nature Publications at https://doi.org/10.1038/s41598-022-18505-0 . Please refer to any applicable terms of use of the publisher. Rodriguez, G., Eaton, D. W., & Verdon, J. P. (2022). Basin-scale multi-decadal analysis of hydraulic fracturing and seismicity in western Canada shows non-recurrence of induced runaway fault rupture. Scientific Reports, 12(1), Article 14463. https://doi.org/10.1038/s41598-022-18505-0 Rodriguez, G., Eaton, D. W., & Verdon, J. P. (2022). Basin-scale multi-decadal analysis of hydraulic fracturing and seismicity in western Canada shows non-recurrence of induced runaway fault rupture. Scientific Reports, 12(1), Article 14463. https://doi.org/10.1038/s41598-022-18505-0 University of Bristol – Bristol Research Portal General rights This document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/red/research-policy/pure/user-guides/brp-terms/ www.nature.com/scientificreports Basin‑scale multi‑decadal analysis of hydraulic fracturing and seismicity in western Canada shows non‑recurrence of induced runaway fault rupture , David W. Eaton 1 & James P. Verdon 2* , David W. Eaton 1* Hydraulic fracturing (HF) is a reservoir stimulation technique that has been widely deployed in recent years to increase the productivity of light oil and/or natural gas from organic-rich, low-permeability formations. Although the process of fracturing a rock typically results in microseismic events of magnitude < 0, many cases of felt seismic events (typically magnitude 3.0 or larger) have also been reported. In the Western Canada Sedimentary Basin (WCSB), where more than 40,000 wells have been drilled and hydraulically fractured in the past two decades, the occurrence of HF-induced events has surged in some areas. Yet, many other areas of the WCSB have not experienced a significant increase in induced seismicity, despite a sharp increase in both the number of HF wells and the volumes of injected fluid. The relationship between injected volume and induced magnitudes can be quantified using the seismic efficiency ratio (SEFF), which describes the ratio between the net seismic moment release and the injected fluid volume. Runaway rupture, in which the fault rupture is dominated by the release of accumulated tectonic stresses, is inferred to be marked by an abrupt increase in SEFF to a value > 0.5. Most previous studies of induced earthquakes have been limited to a single operation and/or seismicity sequence. To better understand the observed variability of the seismic response to HF stimulations at a basin scale, we compiled HF data for all unconventional wells hydraulic fractured in the WCSB between 2000 and 2020, together with the seismicity reported during the same period. We grouped these observations into bins measuring 0.2° in longitude and 0.1° in latitude, or approximately 13 by 11 km. We identified 14 areas where large magnitude events resulted in high SEFF values, implying runaway rupture had taken place. However, we find that in these areas, sustained fluid injection did not lead to persistent high SEFF values. Instead, as injection continued, SEFF values returned to values less than 0.5. This suggests that there is a limited budget of tectonic strain energy available to generate runaway rupture events: once this is released, event magnitudes decrease even if high volume injection persists. Hydraulic fracturing (HF) is a reservoir stimulation technique that has been extensively used to enhance the production of hydrocarbons from organic-rich, low-permeability shale formations. Basin‑scale multi‑decadal analysis of hydraulic fracturing and seismicity in western Canada shows non‑recurrence of induced runaway fault rupture A causal association between hydraulic fracturing and induced (anthropogenic) earthquakes has been documented around the world1, includ- ing in the Bowland Shale in the UK2,3, the Sichuan Basin in China4, the Utica Shale in Ohio5, the Woodford Shale in Oklahoma6, and the Montney7 and Duvernay8 Formations in the Western Canada Sedimentary Basin (WCSB). In the Western Canada Sedimentary Basin (WCSB) more than 40 000 wells have been drilled and hydrauli Hydraulic fracturing (HF) is a reservoir stimulation technique that has been extensively used to enhance the production of hydrocarbons from organic-rich, low-permeability shale formations. A causal association between hydraulic fracturing and induced (anthropogenic) earthquakes has been documented around the world1, includ- ing in the Bowland Shale in the UK2,3, the Sichuan Basin in China4, the Utica Shale in Ohio5, the Woodford Shale in Oklahoma6, and the Montney7 and Duvernay8 Formations in the Western Canada Sedimentary Basin (WCSB). In the Western Canada Sedimentary Basin (WCSB), more than 40,000 wells have been drilled and hydrauli- cally fractured between the years 2000 and 2020 (Fig. 1)9. Over this time, there has been a progressive increase in well depth, as deeper formations are explored, and an increase in the length of the lateral sections of horizontal wells. Taken together, this has led to higher volumes of fluid pumped into each HF well (Fig. 2). Many areas of the WCSB have not experienced any significant increase in seismicity, despite a large increase in both the number of hydraulically fractured wells and the volumes of fluid pumped10. However, in certain areas of western In the Western Canada Sedimentary Basin (WCSB), more than 40,000 wells have been drilled and hydrauli- cally fractured between the years 2000 and 2020 (Fig. 1)9. Over this time, there has been a progressive increase in well depth, as deeper formations are explored, and an increase in the length of the lateral sections of horizontal wells. Taken together, this has led to higher volumes of fluid pumped into each HF well (Fig. 2). Many areas of the WCSB have not experienced any significant increase in seismicity, despite a large increase in both the number of hydraulically fractured wells and the volumes of fluid pumped10. However, in certain areas of western 1Department of Geoscience, University of Calgary, Calgary, AB, Canada. 2Present address: School of Earth Sciences, University of Bristol, Bristol, UK. Basin‑scale multi‑decadal analysis of hydraulic fracturing and seismicity in western Canada shows non‑recurrence of induced runaway fault rupture The moment magnitude of earthquakes and suspected blasts from the compiled public seismic catalogs45,49 are shown in (c), together with the date of the implementation of magnitude-based traffic-light protocols for induced seismicity (IS-TLP) in North Peace and the Kiskatinaw Seismic Monitoring and Mitigation Area (KSMMA) areas for the Montney Formation, and Fox Creek and Red Deer areas for the Duvernay formation (red vertical lines). The strong correlation between the number of hydraulic fractured wells and the number of seismic events, shown in (d), is the main motivation of this study. (e) Reference map of North America, with the studied area shown in (a) highlighted in red. The number of suspected blasts has also increase in western Canada since 2014; these were removed from the seismicity catalog prior to calculating response paths. The HF wells and earthquakes shown in (a) were grouped into bins measuring 0.2° in longitude and 0.1° in latitude (approximately 11 × 13 km) to calculate the total fluid pumped, total seismic moment and maximum earthquake magnitude inside each bin, as shown in Fig. 2. The temporal variation of the seismic and hydraulic fracturing activity illustrated in this figure is also shown in Video S1 in the supplementary materials. All geoLOGIC systems ltd. data and software is copyright 2022. Canada, particularly near Fox Creek11 and Red Deer12 in central Alberta, and in the Horn River Basin13 and Fort St. John area in NE British Columbia14, the rate of seismicity has grown in conjunction with increasing intensity of hydraulic-fracturing operations. This spatial correlation has prompted provincial agencies to introduce new regulations, most notably Traffic Light Protocols (TLPs) to manage the risk of induced seismicity15,16. Canada, particularly near Fox Creek11 and Red Deer12 in central Alberta, and in the Horn River Basin13 and Fort St. John area in NE British Columbia14, the rate of seismicity has grown in conjunction with increasing intensity of hydraulic-fracturing operations. This spatial correlation has prompted provincial agencies to introduce new regulations, most notably Traffic Light Protocols (TLPs) to manage the risk of induced seismicity15,16. g yfi g g y Given the impacts of induced seismicity, both on hydrocarbon producers and on the public who live near to their operations17, much recent research has focussed on the factors that control the magnitudes of induced earthquakes2,18,19. Broadly speaking, two end-member scenarios have been proposed. Basin‑scale multi‑decadal analysis of hydraulic fracturing and seismicity in western Canada shows non‑recurrence of induced runaway fault rupture *email: german.rodriguez@bristol.ac.uk; eatond@ucalgary.ca; james.verdon@bristol.ac.uk Scientific Reports \| (2022) 12:14463 \| https://doi.org/10.1038/s41598-022-18505-0 Scientific Reports \| (2022) 12:14463 www.nature.com/scientificreports/ nature.com/scientificreports/ Figure 1. Hydraulically fractured (HF) wells in the Western Canada Sedimentary Basin (WCSB) between January 1, 2000 and January 1, 2020, and the reported seismicity during the same period. The HF wells, colour- coded by the stimulated formation, together with the boundary of the Duvernay and Montney formations, are shown in (a), and the number of stimulated wells per formation is shown in (b). The moment magnitude of earthquakes and suspected blasts from the compiled public seismic catalogs45,49 are shown in (c), together with the date of the implementation of magnitude-based traffic-light protocols for induced seismicity (IS-TLP) in North Peace and the Kiskatinaw Seismic Monitoring and Mitigation Area (KSMMA) areas for the Montney Formation, and Fox Creek and Red Deer areas for the Duvernay formation (red vertical lines). The strong correlation between the number of hydraulic fractured wells and the number of seismic events, shown in (d), is the main motivation of this study. (e) Reference map of North America, with the studied area shown in (a) highlighted in red. The number of suspected blasts has also increase in western Canada since 2014; these were removed from the seismicity catalog prior to calculating response paths. The HF wells and earthquakes shown in (a) were grouped into bins measuring 0.2° in longitude and 0.1° in latitude (approximately 11 × 13 km) to calculate the total fluid pumped, total seismic moment and maximum earthquake magnitude inside each bin, as shown in Fig. 2. The temporal variation of the seismic and hydraulic fracturing activity illustrated in this figure is also shown in Video S1 in the supplementary materials. All geoLOGIC systems ltd. data and software is copyright 2022. Figure 1. Hydraulically fractured (HF) wells in the Western Canada Sedimentary Basin (WCSB) between January 1, 2000 and January 1, 2020, and the reported seismicity during the same period. The HF wells, colour- coded by the stimulated formation, together with the boundary of the Duvernay and Montney formations, are shown in (a), and the number of stimulated wells per formation is shown in (b). Basin‑scale multi‑decadal analysis of hydraulic fracturing and seismicity in western Canada shows non‑recurrence of induced runaway fault rupture In the arrested rupture sce- nario, events primarily release strain that has been introduced by the industrial activity, and hence the maximum magnitude is limited by the injection volume20–22. In the runaway rupture scenario, the fault rupture is initiated by the industrial activity, but it extends beyond the fluid-perturbed region and releases tectonically accumulated strain energy. In this scenario, the maximum magnitude is limited only by tectonic factors23. Scientific Reports \| (2022) 12:14463 \| https://doi.org/10.1038/s41598-022-18505-0 www.nature.com/scientificreports/ Figure 2. (a) Total fluid pumped (TFP) in every unit area of 0.2° longitude by 0.1° latitude (the unit areas are shown in Fig. 1a), and the moment magnitude of the seismic events (shown in Fig. 1c). The black dots show the maximum magnitude and total volume as of January 1, 2020 reported inside each unit area that contains hydraulically fractured (HF) wells, for seismic events that occurred after the start of HF operations and up to 90 days after completion. The grey lines show the response path, which tracks the magnitude-volume relationship over time. Although almost every black dot falls approximately at or below the McGarr moment cap25 marked by the red line, 14 response paths significantly surpass this relationship. In 93% of such cases (13 out of the 14 cases), continued fluid injection within the same unit area as the induced earthquake did not trigger another large seismic event. (b) Total well length (TWL) or measured depth (MD) of the HF wells, with the true vertical depth and total fluid pumped (TFP) during the HF stimulation of each well until January 1st, 2020 (c). The fluid intensity of the hydraulic stimulation (i.e., TFP/TWL, in m3/m), is also shown in (b). The temporal variation of the total fluid pumped per HF well and maximum moment magnitudes illustrated in this figure is also shown in Video S1 in the supplementary materials. All geoLOGIC systems ltd. data and software is copyright 2022. Figure 2. (a) Total fluid pumped (TFP) in every unit area of 0.2° longitude by 0.1° latitude (the unit areas are shown in Fig. 1a), and the moment magnitude of the seismic events (shown in Fig. 1c). The black dots show the maximum magnitude and total volume as of January 1, 2020 reported inside each unit area that contains hydraulically fractured (HF) wells, for seismic events that occurred after the start of HF operations and up to 90 days after completion. www.nature.com/scientificreports/ induced earthquakes from water disposal wells in the same basin did not exceed McGarr’s maximum magnitude cap. Similarly, seismicity at the Pohang enhanced geothermal site in South Korea appears to have significantly exceeded the McGarr volume-based maximum magnitude28. In this “runaway rupture” scenario28, the injection creates a perturbation that initialises earthquake rupture within the stimulated rock volume, but this rupture extends along larger faults into rock volumes that are unaf- fected by injection-induced pressure changes. In this scenario, the upper limit to induced earthquake magnitudes is controlled by the structural, geomechanical and tectonic characteristics of the formation in question, rather than by the scale of the injection operations. We note that even in a runaway rupture scenario, assuming a rate and state nucleation model, earthquake rate is still expected to scale with stressing rate, which in turn might be expected to scale with injection volume. Therefore, a scaling or correlation between injection volume and seismicity should still be expected in a runaway rupture scenario, but with no upper bound to the value of any scaling coefficient. We also note in passing that this scenario does not necessarily imply that the maximum mag- nitude, MMAX, for induced seismicity will be the same as tectonic MMAX estimates, since the formations targeted for hydraulic fracturing typically have lower stresses, and smaller faults, than mid-crustal rocks where larger earthquakes are typically generated29, which are the primary control on tectonic MMAX estimates.h The phenomenon of runaway rupture may impose its own limits on the amount of seismic moment released over the longer term. Tectonic strain energy is accumulated over geological timescales, meaning that there is no opportunity for this energy to be recharged over the timescales in which hydraulic fracturing takes place (years and/or decades). In principle, once this energy has been released, further injection in the vicinity of a reactivated fault will only be able to release the strain directly imparted by injection. Given the above, we might expect to observe the following with respect to earthquake magnitudes. During initial operations, tectonic strain energy can be released, leading (in some areas and some formations, where critically-stressed faults are present) to rapid escalation of event magnitudes. However, as injection continues within an area and the tectonic strain energy budget has been consumed, the cumulative seismic moment would be expected to revert to the bounds imposed by a volumetric cap. Runaway rupture and volume‑based magnitude limits 24 Runaway rupture and volume‑based magnitude limits McGarr24 proposed a relationship between the cumulative seismic moment (ΣM0) of induced seismic events observed in oilfields and copper mines in the 1960s and 1970s, based on the volume changes (\|ΔV\|) created either by injection or extraction of fluids, or removal of rock volumes by mining. McGarr25 refined this relationship for cases of seismicity induced by the injection of large volumes of fluid—mostly saltwater disposal, enhanced geothermal systems (EGS) and unconventional hydrocarbon wells. In addition to the relationship between ΔV and ΣM0 (1) M0 = 2µ\|V\| (1) McGarr25 further derived the expected moment of the largest induced event, M0(max), by assuming an earth- quake stress drop equal to half of the stress buildup during a tectonic cycle and a b-value of 1 for the Gutenberg- Righter distribution30 of the magnitudes of the induced events: (2) M0(max) = µV (2) The maximum moment magnitude of induced events, MMAX, can be calculated with the moment-magnitude cale from Hanks and Kanamori MMAX = (log10M0(max) −9.05)/1.531. g10 In addition to the above-referenced assumptions, the McGarr relationship represents an upper bound, in that it assumes that the strain generated by subsurface injection is released as seismic energy. In reality, for many sites, much of the deformation may be released as aseismic deformation (for example, by creep on fractures and/or poroelastic expansion of the reservoir rocks). As a result, for most injection sites, ΣM0 falls well below the McGarr limit, and M0(max) ≪µV . To account for this, Hallo et al.21 proposed a modification to the McGarr relation- ship, introducing a Seismic Efficiency Ratio (SEFF) parameter as a calibration factor to McGarr’s relationship, (3) M0 = SEFFµ\|V\| (3) SEFF has been observed to range between 10–6, to a ratio of unity (or higher)21. Noting that Eq. (2) assumes a stress drop corresponding to 50% of the maximum stress drop over a seismic cycle, Li et al.32 derived a physical interpretation for SEFF, (4) SEFF = 1 2(1 −c) (4) where c is the fraction of the full co-seismic stress drop during a tectonic loading cycle. This formulation implies that SEFF ≥ 0.5 and has no upper limit. A value of SEFF that is less than 0.5 does not require the release of any tec- tonic strain energy; rather, it indicates the prevalence of deformation processes associated with fluid injection21. www.nature.com/scientificreports/ Alternatively, if the accumulated tectonic strain budget is sufficiently high, runaway rupture may persist for extended periods of time. In this study, we examine and compare the temporal evolution of induced earthquake magnitudes and injec- tion volumes in hydraulic fracturing wells in the WCSB over the past two decades. The availability of hydraulic fracturing and induced seismicity data from the WCSB, a region which covers over 1000 km from north to south, provides an opportunity to evaluate the hypotheses described above at a regional (basin) scale. Basin‑scale multi‑decadal analysis of hydraulic fracturing and seismicity in western Canada shows non‑recurrence of induced runaway fault rupture The grey lines show the response path, which tracks the magnitude-volume relationship over time. Although almost every black dot falls approximately at or below the McGarr moment cap25 marked by the red line, 14 response paths significantly surpass this relationship. In 93% of such cases (13 out of the 14 cases), continued fluid injection within the same unit area as the induced earthquake did not trigger another large seismic event. (b) Total well length (TWL) or measured depth (MD) of the HF wells, with the true vertical depth and total fluid pumped (TFP) during the HF stimulation of each well until January 1st, 2020 (c). The fluid intensity of the hydraulic stimulation (i.e., TFP/TWL, in m3/m), is also shown in (b). The temporal variation of the total fluid pumped per HF well and maximum moment magnitudes illustrated in this figure is also shown in Video S1 in the supplementary materials. All geoLOGIC systems ltd. data and software is copyright 2022. In 1976, McGarr24 developed a formulation to relate the volume of rock and/or fluid extracted during mining to the maximum seismic moment released as induced seismicity. In 2014, McGarr25 adapted this formulation to estimate the maximum seismic moment release generated by subsurface fluid injection. The McGarr equation, and the resulting upper bound for seismic moment release, is based on several assumptions, including that all of the strain released by the seismicity is directly generated by the subsurface deformation induced by industrial activities. However, detailed microseismic observations of induced seismicity sequences indicate that hydraulic fracturing induced seismicity occurs on pre-existing tectonic faults, and therefore may be releasing stored tec- tonic strain energy, in addition to any strain imparted by the injection operations26,27. Atkinson et al.10 presented a selection of case studies from the WCSB where induced earthquake magnitudes appear to exceed the McGarr maximum magnitude based on volumes in HF wells, indicating that runaway rupture had occurred, whereas the https://doi.org/10.1038/s41598-022-18505-0 Scientific Reports \| (2022) 12:14463 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ and hence earthquake magnitudes, are limited by the injection volume. We note that, in practice, a sequence of induced seismicity may release both tectonically stored energy and the injection-induced strain at a rate that produces an overall SEFF < 0.5. In such situations, discriminating between scenarios will be difficult in practice, unless high-resolution microseismicity observations are available from which deformation processes can be imaged in detail. aged deta . Galis et al.22 proposed an alternative model for arrested rupture cases where g Galis et al.22 proposed an alternative model for arrested rupture cases where (5) M0(max) = γ V3/2, M0(max) = γ V3/2, (5) in which γ is determined by the reservoir thickness, bulk modulus and coefficient of dynamic friction. However, the Galis et al. model is based on an assumption that the injection-induced perturbation can be represented as an expanding cylinder within the reservoir22, a situation that is unlikely to be representative of hydraulic fracturing in shale reservoirs, where the evolution and distribution of pressures may be strongly controlled by, for example, the presence of permeable structures such as fracture networks within the reservoir27.h The alternative endmember to the arrested rupture scenario is that induced earthquakes, while initiated by injection, generate ruptures that release significant quantities of stored tectonic strain energy. We refer to this as the runaway rupture scenario. Within the Hallo et al. SEFF framework, a runaway rupture scenario would be represented by values of SEFF that exceed 0.5, implying that the seismic moment released exceeds the amount of deformation created by the injection, and therefore that a significant portion of the seismic moment is gener- ated by the release of tectonic strain energy. The behaviour of such cases over an extended period of injection then becomes of particular significance. If, for a given area, we initially observe runaway rupture and SEFF > 0.5, but over time SEFF trends back to 0.5 or lower, then we can assume that initial seismicity was dominated by the release of tectonic energy (i.e., runaway rupture), but that the tectonic strain energy budget was limited, and that subsequent injection is dominated by arrested ruptures, and is not able to generate seismicity at a rate that exceeds the McGarr cap. p Figure 3 shows schematic examples of conceptual response paths of seismic activity according to the scenarios described in the preceding paragraphs. www.nature.com/scientificreports/ Scenario 1 shows a situation with a relatively stable SEFF around 0.5, at the upper limit for propagation of arrested ruptures22, following the relationship described by Eqs. (3 and 4). Scenarios 2 and 3 show sharp increases in the seismic activity during injection operations that exceed the McGarr cap, implying the occurrence of runaway rupture and the release of significant amounts of stored tectonic strain energy. In Scenario 2, the contribution from the tectonic strain energy is then used up, and the evolution of magnitudes returns to that described by the McGarr cap. In Scenario 3, ΣM0 continues to exceed the McGarr cap over an extended period of time, implying a continued contribution to the overall seismic strain release from tectonic strain. Figure 3b shows the evolution of these scenarios with respect to SEFF: in the arrested rupture case (Scenario 1), SEFF is below 0.5 throughout; in Scenario 2, SEFF is initially greater than 0.5, but then drops below 0.5 as injection continues, and in Scenario 3 SEFF persists at values greater than 0.5 over an extended period.hi j EFF p g p The extent to which these scenarios occur is of great significance with respect to long-term assessments of seismic hazard from hydraulic fracturing-induced seismicity. It is already established that in certain set- tings, runaway rupture has occurred, and as such we cannot use the McGarr cap as an upper bound for event magnitudes10,28. The extent to which runaway rupture can persist is also of significance. Hydraulic fracturing- induced seismicity hazard assessments are often performed using observed rates of seismicity per well33, with these rates extrapolated into the future without any consideration of whether the rates of seismicity observed during initial fault reactivation might be representative of longer-term behaviour. If the budget of tectonic strain energy is high relative to the amount released by hydraulic fracturing, then we might expect the rates of induced seismicity to persist at a high level, whereas if the budget of tectonic strain energy is significantly depleted by the induced seismicity, then we might expect rates of seismicity to decrease over time. Runaway rupture and volume‑based magnitude limits 24 Hence, hereafter, we refer to the situation whereby induced earthquake magnitudes are limited by the injected volume, as per McGarr25, as the arrested-rupture scenario, since the implication is that rupture dimensions, https://doi.org/10.1038/s41598-022-18505-0 Scientific Reports \| (2022) 12:14463 \| www.nature.com/scientificreports/ Scientific Reports \| (2022) 12:14463 \| Data and analysis (3), with SEFF = 0.5. Scenario 2 (orange) shows a runaway rupture case, where ΣM0 exceeds this cap given by Eq. (3). However, over time, once the tectonic strain energy budget has been released the cumulative seismic moment relationship reverts to that described by Eq. 3. Scenario 3 (red) shows a runaway rupture case where the tectonic strain energy continues to contribute to the induced seismicity over an extended period, with ΣM0 continuing to exceed the Eq. (3) cap. In (b) we show the same conceptual scenario paths viewed with respect to SEFF. Figure 3. Schematic illustrations of conceptual induced seismicity response paths. In (a) we show the potential evolution of ΣM0 relative to ΔV for three conceptual scenarios. Scenario 1 (in green) shows an arrested-rupture case, where magnitudes are always limited by the injection volume according to Eq. (3), with SEFF = 0.5. Scenario 2 (orange) shows a runaway rupture case, where ΣM0 exceeds this cap given by Eq. (3). However, over time, once the tectonic strain energy budget has been released the cumulative seismic moment relationship reverts to that described by Eq. 3. Scenario 3 (red) shows a runaway rupture case where the tectonic strain energy continues to contribute to the induced seismicity over an extended period, with ΣM0 continuing to exceed the Eq. (3) cap. In (b) we show the same conceptual scenario paths viewed with respect to SEFF. susceptible to induced seismicity—for example, there are no reliably-documented cases of hydraulic fracturing induced seismicity from wells targeting shallow, Cretaceous-age formations in the WCSB. In our analysis, we only include wells that target formations below the base-Mannville unconformity, which is a major basin-wide stratigraphic feature of Lower Cretaceous age that marks the onset of clastic deposition in a foreland basin setting. By contrast, Palaeozoic and lower Mesozoic deposition in the WCSB took place on an extensional/transtensional passive margin37. Notwithstanding these steps, the assessment of whether an earthquake (or sequence of seismic- ity) is induced (and if induced, by what particular activity) is not trivial38, and so the inclusion of any particular event in our analysis does not mean that we explicitly assign causation to a particular activity. We computed the temporal evolution of SEFF within each block (Fig. 5), in order to assess the extent to which each of the endmember scenarios described in Fig. 3 might apply to the induced seismicity within the WCSB. Data and analysis In this study, we compiled hydraulic-fracturing data available from provincial regulators (see “Methods and datasets” section) for wells in western Canada. We grouped wells into “blocks” of 0.2° in longitude by 0.1° in latitude (approximately 13 × 11 km), aggregating the total volume of fluid injected inside each HF well located inside each block. This area size was chosen to implement a similar spatiotemporal association filter proposed for HF-induced seismicity in western Canada in recent studies8, where only seismic events located less that 5 km from any well pad were associated with the HF stimulation of any unconventional well. This 5 km radius (or 10 × 10 km for gridded areas) was then rounded to the nearest 0.1 degree in geographical coordinates for sim- plicity. This spatial discretisation enables analysis of fluid volumes injected at the scale of well pads (i.e., multiple horizontal wells drilled at close distances from each other), instead of individual wells8, thereby recognising that wells from multiple different pads could influence the same fault structures. The block dimensions are also larger than typical location uncertainties of the seismic events reported in regional catalogs34. A sensitivity analysis of the block size is presented in our supplementary materials. y To calculate SEFF for each block, we compared injection volumes to the cumulative seismic moments of earth- quakes recorded within the block (Fig. 4). To ensure that we only consider events that may have been induced by hydraulic fracturing, we only use events that occurred within 3 months after any hydraulic fracturing activity within a block. This temporal filter for HF-induced earthquakes has also been suggested by Schultz et al.8, and reflects maximum observed delay-times between injection and induced seismicity identified by Verdon and Bommer30 in their worldwide compilation of hydraulic fracturing induced seismicity case studies. Sequences of wastewater disposal induced seismicity have also been identified in the WCSB—such sequences are often easy to identify as persistent, long-standing clusters of seismicity35. Events within such sequences were removed from our analysis. Verdon and Bommer36 have demonstrated that only certain formations within the WCSB are Scientific Reports \| (2022) 12:14463 \| https://doi.org/10.1038/s41598-022-18505-0 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 3. Schematic illustrations of conceptual induced seismicity response paths. In (a) we show the potential evolution of ΣM0 relative to ΔV for three conceptual scenarios. Scenario 1 (in green) shows an arrested-rupture case, where magnitudes are always limited by the injection volume according to Eq. Data and analysis We also computed the b-value and magnitude of completeness of the Gutenberg-Righter distribution of the seismic events for each block with at least 20 seismic events (shown in Figs. 4, 6 and Video S2, and listed in Table S1 in the supplementary material). To calculate b values for each block, we first measured the catalog completeness (Mcomp in Fig. 4c) as the magnitude with the largest numbers of events, and then calculated the a and b values of the Gutenberg-Righter distribution (log10 N = a − bM, where N is the number of events with magnitudes greater than or equal to M) using the Maximum Likelihood Estimate method39 (see example in Fig. 4c). Results Figure 5a,b show the evolution of SEFF for all the blocks in our study area in which SEFF is close to 0.5 at any point during the two decades covered by our study. We omit grid blocks where SEFF ≪ 0.5, since these cases will not contribute to any assessment of the extent to which runaway rupture can occur and persist. We observe that https://doi.org/10.1038/s41598-022-18505-0 Scientific Reports \| (2022) 12:14463 \| www.nature.com/scientificreports/ nature.com/scientificreports/ Figure 4. Example of the Seismic Efficiency Ratio (SEFF) calculated for one unit area of 0.2° in Longitude by 0.1° in Latitude (or approximately 13 × 11 km) near the town of Fox Creek in central Alberta. Most of the wells hydraulic fractured in this area were from the Duvernay and Montney formations (b), whereas the entire seismic activity in the same area (d) occurred only during the hydraulic stimulations of the Duvernay wells (e) despite all wells being located only a few kilometers apart from each other (shown in f). The shallow depth distribution of most of the same seismic events (shown in g), of less than 5 km, also suggests that these events where induced by the hydraulic fracturing stimulations of wells from the Duvernay formation (as natural events tend to have deeper locations). The seismic activity (d) has no clear correlation with the injection in the two water disposal (WD) wells (also shown in e) located inside the same unit area. The Gutenberg-Richter distribution of the seismic events from this area (shown in c), and calculated using a Maximum Likelihood Estimate method39), has a b-value close to 1 [as assumed in Eq. (2)], and a magnitude of completeness (Mcomp) of 2.3. The SEFF calculated for all unit areas in western Canada, shown in the background map in a), is also h h h h l l d b l d b d d d l fi Figure 4. Example of the Seismic Efficiency Ratio (SEFF) calculated for one unit area of 0.2° in Longitude by 0 1° in Latitude (or approximately 13×11 km) near the town of Fox Creek in central Alberta Most of the Figure 4. Example of the Seismic Efficiency Ratio (SEFF) calculated for one unit area of 0.2° in Longitude by 0.1° in Latitude (or approximately 13 × 11 km) near the town of Fox Creek in central Alberta. Results Most of the wells hydraulic fractured in this area were from the Duvernay and Montney formations (b), whereas the entire seismic activity in the same area (d) occurred only during the hydraulic stimulations of the Duvernay wells (e) despite all wells being located only a few kilometers apart from each other (shown in f). The shallow depth distribution of most of the same seismic events (shown in g), of less than 5 km, also suggests that these events where induced by the hydraulic fracturing stimulations of wells from the Duvernay formation (as natural events tend to have deeper locations). The seismic activity (d) has no clear correlation with the injection in the two water disposal (WD) wells (also shown in e) located inside the same unit area. The Gutenberg-Richter distribution of the seismic events from this area (shown in c), and calculated using a Maximum Likelihood Estimate method39), has a b-value close to 1 [as assumed in Eq. (2)], and a magnitude of completeness (Mcomp) of 2.3. The SEFF calculated for all unit areas in western Canada, shown in the background map in a), is also shown in Fig. 6 together with the calculated b-value and observed maximum moment magnitude, and also in Video S2 and listed in Table S1 in the supplementary materials. All geoLOGIC systems ltd. data and software is copyright 2022. values of SEFF higher than 0.5 are evident, indicating that runaway rupture has likely occurred. We identified 14 such cases, which are shown in detail in the Supplementary Material. However, the general trend for these cases is for SEFF to decrease to < 0.5 as injection proceeds, in accordance with Scenario 2 of Fig. 3. Indeed, there are no cases for which SEFF is greater than 0.5 after the cumulative injection of more than 105 m3. Given that typical injection volumes for Montney, Horn River and Duvernay wells are 20,000 m3 or more, this would indicate that runaway rupture does not persist after stimulation of more than 4 or 5 wells within a given block. values of SEFF higher than 0.5 are evident, indicating that runaway rupture has likely occurred. We identified 14 such cases, which are shown in detail in the Supplementary Material. However, the general trend for these cases is for SEFF to decrease to < 0.5 as injection proceeds, in accordance with Scenario 2 of Fig. 3. Results Indeed, there are no cases for which SEFF is greater than 0.5 after the cumulative injection of more than 105 m3. Given that typical injection volumes for Montney, Horn River and Duvernay wells are 20,000 m3 or more, this would indicate that runaway rupture does not persist after stimulation of more than 4 or 5 wells within a given block. y p pt g In Fig. 6, we provide cross-plots of relevant variables for each grid block, including ΣM0, SEFF, the cumulative injection volume ΔV, the largest observed magnitude MMAX, and b-values. Figure 6 also shows the gradient, m, of the least-squares fit between each pair of variables (for ΣM0, SEFF, and ΔV, fitted in log space), the Pearson’s correlation coefficient, R, and the P values for assessing the null hypothesis that there is no relationship between the variables. Some of the observed correlations are trivial and expected, such as between SEFF and ΣM0 (as per Eq. 3), and between MMAX and ΣM0.i We note the statistically significant negative correlation between SEFF and ΔV: this supports the situation dis- ussed in the previous paragraph where high SEFF values do not persist as larger volumes are injected, leading to https://doi.org/10.1038/s41598-022-18505-0 Scientific Reports \| (2022) 12:14463 \| www.nature.com/scientificreports/ Figure 5. (a) Response path of the cumulative fluid pumped unit areas of 0.2° in Longitude by 0.1° in Latitude shown in F the unit area near Fox Creek obtained from the Total Fluid P and cumulative seismic moment of all seismic events inside and their magnitudes shown in Fig. 4d). The response path c also shown in Video S2 in the supplementary materials. The Fox Creek, shown in (b), shows two clear runaway rupture s Fig. 3b), attributable to the reactivation of two different (but A constant SEFF of 0.5, which corresponds to a seismic cycle in (a) and (b) for reference. The True Vertical Depth (TVD) (c), together with the TVD of the Precambrian basement (re Alberta48). All geoLOGIC systems ltd. data and software is c Figure 5. (a) Response path of the cumulative fluid pumped in all hydraulic-fractured wells located inside the unit areas of 0.2° in Longitude by 0.1° in Latitude shown in Fig. 4a (in grey), highlighting the response path of the unit area near Fox Creek obtained from the Total Fluid Pumped per well from this area (shown in Fig. Results 4e), and cumulative seismic moment of all seismic events inside the same area (shown in Fig. 4f near the HF wells, and their magnitudes shown in Fig. 4d). The response path calculated for all unit areas in western Canada, are also shown in Video S2 in the supplementary materials. The variability of the SEFF for this example unit area near Fox Creek, shown in (b), shows two clear runaway rupture sequences (similar than the scenario 2 illustrated in Fig. 3b), attributable to the reactivation of two different (but relatively close) faults located inside this unit area. A constant SEFF of 0.5, which corresponds to a seismic cycle with zero stress drop [from Eq. (4)] is also shown in (a) and (b) for reference. The True Vertical Depth (TVD) of the same hydraulic fractured wells are shown in (c), together with the TVD of the Precambrian basement (retrieved from the 3D provincial Geological Model of Alberta48). All geoLOGIC systems ltd. data and software is copyright 2022. Figure 5. (a) Response path of the cumulative fluid pumped in all hydraulic-fractured wells located inside the unit areas of 0.2° in Longitude by 0.1° in Latitude shown in Fig. 4a (in grey), highlighting the response path of the unit area near Fox Creek obtained from the Total Fluid Pumped per well from this area (shown in Fig. 4e), and cumulative seismic moment of all seismic events inside the same area (shown in Fig. 4f near the HF wells, and their magnitudes shown in Fig. 4d). The response path calculated for all unit areas in western Canada, are also shown in Video S2 in the supplementary materials. The variability of the SEFF for this example unit area near Fox Creek, shown in (b), shows two clear runaway rupture sequences (similar than the scenario 2 illustrated in Fig. 3b), attributable to the reactivation of two different (but relatively close) faults located inside this unit area. A constant SEFF of 0.5, which corresponds to a seismic cycle with zero stress drop [from Eq. (4)] is also shown in (a) and (b) for reference. The True Vertical Depth (TVD) of the same hydraulic fractured wells are shown in (c), together with the TVD of the Precambrian basement (retrieved from the 3D provincial Geological Model of Alberta48). All geoLOGIC systems ltd. data and software is copyright 2022. a negative correlation. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 6. Cross-plots of variables for each grid block, including SEFF, MMAX, ΣM0, ΔV, and b-values. The dashed lines show the least-squares fit between each pair of variables. The gradient of this line, M, the Pearson’s correlation coefficient, R, and the statistical significance of this value, P, are reported in each sub-plot. Figure 6. Cross-plots of variables for each grid block, including SEFF, MMAX, ΣM0, ΔV, and b-values. The dashed lines show the least-squares fit between each pair of variables. The gradient of this line, M, the Pearson’s correlation coefficient, R, and the statistical significance of this value, P, are reported in each sub-plot. fluid injection rather than the release of tectonic stress. Overall, the strong correlations between the rates and magnitudes of the induced seismicity and the SEFF value shows that, for improved seismic hazard assessment, it is vital that we understand the controls on seismic efficiency, and how it might vary over both space and time for a given target formation and type of industrial activity. Understanding the controls on SEFF are more important the b values, which tend to revert to tectonic values once large earthquakes, driven by release of tectonic stresses, begin to occur. Results We note the absence of correlation between ΔV and MMAX: this is because the occurrence of large events is primarily controlled by the presence of high SEFF values, rather than high injection volumes. As a result, we observed strongly significant correlation between MMAX and SEFF. We observe negative correlation between b-values and SEFF: high SEFF values (SEFF > − 1) are universally associated with b-values of approximately 1.0 or less, equivalent to values commonly observed for tectonic earthquakes40. In contrast, many of the blocks with lower SEFF values are associated with higher b-values. This is consistent with the hypothesis that cases with high SEFF represent situations where tectonic stress is released, and hence the b-values are similar to those observed for tectonic earthquakes. In contrast, higher b-values are often argued to indicate seismicity driven by fluid-movement within fault and fracture networks41, and hence indicative of seismicity driven directly by https://doi.org/10.1038/s41598-022-18505-0 Scientific Reports \| (2022) 12:14463 \| Discussion Figures 4 and 5 show an example of the temporal evolution of SEFF calculated for one unit block near the town of Fox Creek in central Alberta. A similar plot of the SEFF for each unit block is shown in Video S2 in the Sup- plementary Material. The response path of this area, as delineated by the evolution of SEFF, shows evidence for runaway rupture with an initial sharp increase in the cumulative seismic moment. Over time, however, while seismic activity continues, it does so at a more gradual rate, resulting in a decrease in the SEFF, implying that seismicity becomes dominated by arrested ruptures that release strain energy imparted by the injection process. Figures S5 to S18 in the Supplementary Material highlights the unit blocks from Video S2 that had runaway ruptures (i.e., areas with SEFF higher than 0.5 at any moment). p EFF g y Kao et al.42 calculated the rate of tectonic moment accumulation across the WCSB, based on geodetic obser- vations of tectonic strain. They found that injection induced seismicity typically occurred in areas where the tectonic moment rate was MO TS = 1–2 × 106 Nm/km2/yr. Adjusting this rate for our blocks with areas of 11 × 13 km gives a tectonic moment rate of MO TS = 1.43–2.86 × 108 Nm/block/yr. On this basis, the occurrence of a single M 3.0 induced earthquake within a grid block, if it primarily releases accumulated tectonic strain energy, repre- sents the tectonic strain accumulated over 100,000 years. This implies that the tectonic strain rate in the WCSB is insufficient to reload a fault and thus allow continued occurrence of runaway rupture within a given area that is subject to repeated injection activities. This observation is supported by our compilation of SEFF values within the basin: runaway rupture, as indicated by larger magnitude events occurring after small injection volumes, giving SEFF > 0.5, can (and does) occur. However, SEFF > 0.5 does not appear to persist as injection continues in a https://doi.org/10.1038/s41598-022-18505-0 Scientific Reports \| (2022) 12:14463 \| www.nature.com/scientificreports/ Figure 7. (a) Number of earthquakes of magnitude ≥ 3.0 per year reported inside the Fox Creek area designated in the Alberta Energy Regulator’s Subsurface Order No. 2 (SSO2)16 (shown in Figs. 1a and 4a), and total volume injected in HF wells inside the same area. Discussion (b) Average injected volume per year per HF well inside the Fox Creek area. Note the gradual decrease in the number of earthquakes per year of magnitude ≥ 3.0 since 2015, despite the constant increase in the total injected volume per year in HF wells, and in the average injected volume per HF well. Figure 7. (a) Number of earthquakes of magnitude ≥ 3.0 per year reported inside the Fox Creek area designated in the Alberta Energy Regulator’s Subsurface Order No. 2 (SSO2)16 (shown in Figs. 1a and 4a), and total volume injected in HF wells inside the same area. (b) Average injected volume per year per HF well inside the Fox Creek area. Note the gradual decrease in the number of earthquakes per year of magnitude ≥ 3.0 since 2015, despite the constant increase in the total injected volume per year in HF wells, and in the average injected volume per HF well. given location, which is consistent with the fact that a handful of induced events of moderate magnitude may be sufficient to release the tectonic strain accumulated over many thousands, or indeed millions of years. given location, which is consistent with the fact that a handful of induced events of moderate magnitude may be sufficient to release the tectonic strain accumulated over many thousands, or indeed millions of years. In the Fox Creek area in central Alberta, Reyes-Canales et al.43 performed an assessment of event rates and Gutenberg-Richter b-values. This area is the subject of the Alberta Energy Regulator’s Subsurface Order No. 2 (SSO2)16, imposing a Traffic Light Protocol with a red light of M 4.0. The rate of occurrence of M > 3.0 events in this area shows a gradual decrease from 2015 through to 2020, while injection volumes into the Duvernay within this area have continued to rise (Fig. 7a). Reyes-Canales et al.43 suggested that some of this reduction in seismicity could be attributed to the targeting areas less susceptible to induced seismicity. However, it is also clear that there has been a reduction in the seismic activity of the eastern region inside the Fox Creek area that previously exhibited higher seismic activity. This reduction in the seismic activity occurred despite the per-well injection volumes in this area continuing to rise during this time (Fig. 7b). Methods and datasets To study the recent seismicity associated with hydraulic fracturing operations in the WCSB, we first compiled public seismic catalogs from the Composite Alberta Seismicity Catalog (CASC, retrieved from https://www.induc edseismicity.ca/catalogues/, last accessed on August 2021) that includes all seismic events reported in Alberta and NE British Columbia until January 2020, in the seismic catalog from the Geological Survey of Canada (GSC), Alberta Geological Survey (AGS), US Geological Survey (USGS), and the TransAlta/Nanometrics seismic network installed in 2013 around the Brazeau Dam in west-central Alberta45. The CASC catalog first eliminates duplicate events that appear in different catalogs, and classifies each event as an earthquake or a suspected blast (discriminated from seismic events from its time of occurrence -as every mine blast is schedule in afternoon hours only to ensure plenty of daylight during each blast– and its epicenter proximity to open pit coal mines or quarries). The AGS also provides in their seismic catalog (publicly available from https://ags-aer.shinyapps.io/ Seismicity_waveform_app/) a similar discrimination for each reported seismic event, as Suspected Earthquake (SE), Suspected Induced (SI) or Known Induced (KI), although for KI events it does not specify induced by what (i.e., hydrocarbon production, enhanced oil recovery, salt water disposal, or hydraulic fracturing, all of them previously reported in western Canada46).h The CASC catalog also assigns a moment magnitude (MwAsg) to each event to normalize the magnitudes reported in different catalogs and using different scales (mostly local magnitude)45. The seismic events in the catalogs from the Horn River Basin47 and the GSC for the provinces of Saskatchewan and Manitoba were also added to the CASC catalog to cover the seismicity reported in the entire basin between 2000 and 2020.hl g y p The stimulation data from the wells hydraulic fractured in the same period (as injected fluid volumes, target formation, and well depth, length, and orientation) was retrieved with the software geoSCOUT (see Data avail- ability). We then clustered the earthquake and well data in unit areas of 0.2° in Longitude by 0.1° in Latitude (or approximately 13 × 11 km, somewhat larger than a standard township of 6 × 6 miles) to calibrate McGarr’s cap function (Eq. Discussion Reyes-Canales et al.40 suggested that the reduction could have been driven by the implementation of the SSO2 TLP, which encourages the operators to exercise additional precautions and mitigation strategies to avoid induced seismicity. fi In the Fox Creek area in central Alberta, Reyes-Canales et al.43 performed an assessment of event rates and Gutenberg-Richter b-values. This area is the subject of the Alberta Energy Regulator’s Subsurface Order No. 2 (SSO2)16, imposing a Traffic Light Protocol with a red light of M 4.0. The rate of occurrence of M > 3.0 events in this area shows a gradual decrease from 2015 through to 2020, while injection volumes into the Duvernay within this area have continued to rise (Fig. 7a). Reyes-Canales et al.43 suggested that some of this reduction in seismicity could be attributed to the targeting areas less susceptible to induced seismicity. However, it is also clear that there has been a reduction in the seismic activity of the eastern region inside the Fox Creek area that previously exhibited higher seismic activity. This reduction in the seismic activity occurred despite the per-well injection volumes in this area continuing to rise during this time (Fig. 7b). Reyes-Canales et al.40 suggested that the reduction could have been driven by the implementation of the SSO2 TLP, which encourages the operators to exercise additional precautions and mitigation strategies to avoid induced seismicity. Scientific Reports \| (2022) 12:14463 \| https://doi.org/10.1038/s41598-022-18505-0 www.nature.com/scientificreports/ Our results presented above suggest an alternative hypothesis to explain the reduction in seismicity seen in the Fox Creek region since 2015. Rather than being primarily driven by the introduction of the TLP, it instead may represent a situation in which runaway rupture has become less prevalent as the tectonic strain accumulated over millennia is released by initial hydraulic fracturing in the area, with later wells subsequently limited by the the moment cap indicated by Eq. (3) with respect to the maximum available moment that could be generated. The relative significance of these factors could be further addressed by examining in more detail the extent to which operators have successfully taken pro-active steps to mitigate induced seismicity in practice—no actual examples of such were presented by Reyes-Canales et al.40, although detailed information of operators’ mitigation steps is seldom publicly available. Even in cases where seismogenic faults have been identified, mitigation steps taken by operators have often not been effective28,44. Conclusionsh The WCSB has a wide variety of geological and tectonic features that influence the seismic response of unconven- tional wells to hydraulic fracturing stimulations. Responses range from virtually zero seismicity near shallower Bakken and Viking wells located in the east side of the basin, to the seismically active areas near some longer and deeper Duvernay and Montney wells on the west side of the basin. In some cases, the maximum magnitude has surpassed the expected levels estimated with McGarr’s cap function, which is commonly used to estimate the maximum seismicity level associated with fluid-injection operations. We fit a seismic efficiency ratio (SEFF), the ratio of the net seismic moment release and the forecasted maximum moment, and find that the obtained SEFF exhibits a complex evolution in such areas, with anomalously high seismic activity arising from inferred runaway rupture processes on pre-existing faults. In 93% of cases where exceedance of SEFF = 0.5 occurred (i.e., in 13 out of 14 cases), representing the presumed onset of stored tectonic stress release, continued injections within the same 0.2° × 0.1° area (in Latitude x Longitude, of approximately 13 × 11 km) did not lead to further seismicity with characteristics of runaway rupture. Discussion The fact that per-well injection volumes have continued to increase indicates that the TLP has not led to any major changes in operators’ completion strategies. Moreover, the reduction in seismicity in the Fox Creek area is observed at all magnitudes, whereas the SSO2 TLP, with a red light at M 4.0, is only designed to mitigate larger-magnitude events. Methods and datasets 2) to estimate the maximum magnitude of an induced seismic event by calculating for each unit area a Seismic Efficiency Ratio (SEFF) from the total fluid pumped in every hydraulic fractured well, and the cumulative seismic moment from every seismic event reported inside each unit area. In the case of Alberta, we were also able to compare the wells’ proximity to the basement, as shown in Figs. 4g and 5c (another key geo- logical parameter in relation to seismicity induced by water injection operations including hydraulic fracturing) from the depth of the Precambrian basement included in the province’s 3D geological model48. This was not possible for other unit areas within the same WCSB, particularly in NE British Columbia (as shown in Video S2 in the supplementary material), where no detailed map of the Precambrian basement has been released to date. Finally, the location of the water disposal wells, as the ones shown in Fig. 4f, were retrieved from the public databases of the provincial regulators of Alberta and British Columbia, and their monthly injection data is also publicly available in https://petroninja.com/ (see more details in Data availability). The authors thank geoLOGIC systems ltd. for their contribution of data and software used in this study. All geoLOGIC systems ltd. data and software is copyright 2022. Additional information. The supplementary material contains two animations (Videos S1 and S2) and one table (Table S1) based on Figs. 1 and 4, showing the temporal and spatial variation of hydraulic fracturing and seismic activity in the WCSB between the years 2000 and 2020. A sensitivity analysis of grid areas of differ- https://doi.org/10.1038/s41598-022-18505-0 Scientific Reports \| (2022) 12:14463 \| www.nature.com/scientificreports/ ent sizes and locations used for the estimation of the Seismic Efficiency Ratio (SEFF) inside each area, and their response path and variability of SEFF overtime (based on Fig. 5), are shown in Figs. S1 to S4, and the 14 cases of runaway rupture observed in our study are shown in detail in Figs. S5 to S18. ent sizes and locations used for the estimation of the Seismic Efficiency Ratio (SEFF) inside each area, and their response path and variability of SEFF overtime (based on Fig. 5), are shown in Figs. S1 to S4, and the 14 cases of runaway rupture observed in our study are shown in detail in Figs. S5 to S18. Data availability Th l The regional seismic catalogs from western Canada used in this study are publicly available (see “Methods and datasets”). For convenience, the well information from the hydraulic fractured wells in the WCSB was retrieved using geoSCOUT software from geoLOGIC Systems Ltd., licensed to the Microseismic Industry Consortium, University of Calgary. Information from the HF stimulation from the same wells is also publicly available in http://fracfocus.ca/en and in the public databases of the provincial regulators of Alberta and British Columbia (https://www1.aer.ca/ProductCatalogue/WELL.html; https://reports.bcogc.ca/ogc/app001/r/ams_reports/1), that also include the location of the water disposal (WD) wells in both provinces. The monthly injection data of the WD wells reproduced in this manuscript and in its supplementary material is also publicly available from https://petroninja.com/. Received: 6 January 2022; Accepted: 4 May 2022 References 1. Schultz, R. et al. Hydraulic fracturing-induced seismicity. Rev. Geophys. 58, 1–43 (2020). 1. Schultz, R. et al. Hydraulic fracturing-induced seismicity. Rev. 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Solid Earth 119, 1008–1019 (201 Maximum magnitude earthquakes induced by fluid injection. J. G g q yl j p y 26. Eyre, T. S., Eaton, D. W., Zecevic, M., D’Amico, D. & Kolos, D. Author contributionsi G.R.P. first compiled the hydraulic fracturing and seismicity datasets from the WCSB to illustrate in this manu- script the spatial variability of the seismic response of large-scale HF stimulations in a full basin, multi-year timescale, based on the methodology of previous studies of HF-induced seismicity from D.W.E. and J.P.V. All authors discussed the methods and results and jointly produced the interpretations and conclusions. G.R.P. and J.P.V. co-wrote and edited the article. www.nature.com/scientificreports/ h l Th l lb b f h k d k ( ) ( ) ( ) 48. Alberta Geological Survey. 3D provincial geological framework model of Alberta, version 2 (Alberta Energy Regulator/Alberta Geological Survey, 2019).h 48. Alberta Geological Survey. 3D provincial geological framework model of Alberta, version 2 (Alberta Energy Regulator/Alberta Geological Survey, 2019).h g y 49. Schultz, R. & Stern, V. The regional Alberta observatory for earthquake studies network (RAVEN). CSEG Rec. 40(8), 34–37 (2 Acknowledgements g We would like to thank all the sponsors of the Bristol University Microseismicity ProjectS group and the Micro- seismic Industry Consortium for their support, and geoLOGIC Systems Ltd. for providing access to the well data analyzed in this study through their geoSCOUT platform. We are also very thankful for the discussions with Melanie Popp and Alemayehu Aklilu we had during the preparation of this manuscript. We thank geoLOGIC Systems Ltd. for their contribution of data and software used in this study. All geoLOGIC Systems Ltd. data and software is copyright 2022. This research was supported by the Natural Science and Engineering Research Council of Canada (NSERC, Grant Number IRCPJ/485692-2014), and by the UK Natural Environment Research Council (NERC, Grant Number NE/R018162/1). Competing interests g The BUMPS and MIC consortia are both sponsored by a range of hydrocarbon operating companies, some of whom have conducted hydraulic fracturing activities in the WCSB. JPV has acted as an independent consultant for a variety of organisations including hydrocarbon operating companies and governmental organisations on issues pertaining to induced seismicity. The results and conclusions are those of the authors—representatives of our sponsors have not influenced the analyses presented here. References Microseismicity reveals fault activation before Mw 4.1 hydraulic- fracturing induced earthquake. Geophys. J. Int. 218(1), 534–546. https://doi.org/10.1093/gji/ggz168,July (2019). 26. Eyre, T. S., Eaton, D. W., Zecevic, M., D’Amico, D. & Kolos, D. 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https://openalex.org/W4286203526	https://www.researchsquare.com/article/rs-1855705/latest.pdf	English	null	Factors associated with personal recovery in an outpatient sample of people with Schizophrenia in Brazil	Research Square (Research Square)	2,022	cc-by	6,310	Factors associated with personal recovery in an outpatient sample of people with Schizophrenia in Brazil Tiago Ribeiro Silva (  tiago.rs10@gmail.com ) Federal University of São Paulo (UNIFESP) Mário César Rezende Federal University of São João Del-Rei (UFSJ) Zlatka Russinova Boston University Marianne Farkas Boston University Ary Gadelha Federal University of São Paulo (UNIFESP) Rodrigo A. Bressan Federal University of São Paulo (UNIFESP) Tiago Ribeiro Silva (  tiago.rs10@gmail.com ) Federal University of São Paulo (UNIFESP) Conclusion This study replicated findings from the international literature about the domains of personal recovery and its associations with important clinical constructs. It also identified specific cultural aspects such as the importance of affective and social relationships (friends, family) for life and as support for processes recovery. Purpose This study aimed to assess the level of personal recovery and its associated factors in a Brazilian sample of people with schizophrenia. Results RAS domains with the highest levels of recovery were “Goal/success orientation” and “Reliance on others”, while the domain “Not dominated by symptoms” presented the lowest score. Higher levels of personal recovery in general were associated only with a lower level of depressive symptoms (as measured by Calgary scale) and a higher score for occupational functioning (as measured by SAOF). Methods This cross-sectional study comprised a non-probabilistic sample of 104 people with schizophrenia receiving outpatient care in a university psychiatric centre. Personal Recovery was measured using the short-version of the Recovery Assessment Scale (RAS), comprising 24 items. We first examined the domains of personal recovery in the study sample and their associations of with the following clinical measures were also used: the Self-Assessment of Occupational Functioning Scale (SAOF); the general psychopathology, the Positive and Negative Syndrome Scale (PANSS); the Calgary Depression Scale for Schizophrenia; the Independent Living Skills Survey (ILSS) and the Clinical Global Impression (CGI). The mean scores of RAS domains were compared using ANOVA for repeated measures and clinical measures associated with RAS total score were identified using linear multiple regression. Significance level was p<0.05. Research Article Keywords: Recovery, Mental Health, Schizophrenia, Functioning Posted Date: July 21st, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1855705/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 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. R d F ll Li Page 1/14 Page 1/14 Page 1/14 1. Introduction Schizophrenia is often represented as a psychiatric disorder high rates of functional impairment. Despite this negative view, two-thirds of affected individuals manage to achieve satisfactory levels of clinical recovery [1-4]. However, in the last decades, a new understanding related to the possibility of a personal recovery has also emerged. Personal Recovery has been defined as a subjective process aimed at changing and structuring a positive identity in people affected by severe mental disorders. This process is related to the continuing cultivation of hope and the establishment of new perspectives in life, increasing personally meaningful participation in society. [5,6]. Page 2/14 Page 2/14 Research has systematized measurable dimensions of recovery, such as: connection, both in the community and in personal life; hope and optimism about the future; construction of an identity beyond the disease; purpose and meaning of life; empowerment; and resilience, which is related to the positive management of difficulties that arise during the process of overcoming living with a severe mental disorder [7-9]. Several qualitative studies in recovery, based on the narratives and reports of people with lived experience, have also highlighted the presence of other similar dimensions such as: acceptance of the disease itself, from the subject and his family and friends; learning how to deal with their symptoms and difficulties; the achievement of citizenship; engaging in activities that are meaningful to self and others; insertion into a social and family support network [10-16]. Based on these studies and evidences, several countries have moved their efforts to adopt recovery as the guiding vision for their public policies and mental health services. For example, in 2012, the World Health Organization (WHO) published the “Plan of Action on Mental Health 2013-2020”, advocating recovery as a principle to be incorporated by countries in their mental health policies and routine practice [17, 18]. More recently, WHO [19] launched a series of “Guidance and Technical Packages for Community Mental Health Services: Person-Centered Promotion and Human Rights-Based Approaches”, enphasizing, among others, the increasing adoption of recovery-oriented practices. Along with this incorporation of the concept of recovery into mental health policies and manuals in different countries, there is a growing concern with its translation and operationalization into evidence-based practices in the clinical routine. In this sense, different standardized and validated measures can be used by services to assess its level of recoveryorientation and its implementation. [18,20]. 2.1 Design and setting This is a cross-sectional study, with a non-probability sample that attended a university outpatient mental health service. The study was carried out at PROESQ (Schizophrenia Program), UNIFESP (Federal University of São Paulo), as part of an interdisciplinary project, in which all participants underwent a psychiatric, neuropsychological, occupational and clinical examination interview (blood and DNA tests, and neuroimaging). The study was approved by the Ethics Committee of UNIFESP and all participants agreed to participate in the study and signed the consent form. 1. Introduction Despite that, there are still no published studies using this instrument to assess the level of recovery and its associated factors among individuals with serious mental illnesses in Brazil. In this sense, this study aims to fill this gap. Moreover, to the best of our knowledge, this is the first quantitative research on personal recovery in the country. 1. Introduction While there is a progressive incorporation of recovery into mental health policies internationally, Latin America, specifically, is still in a period of consolidation of community mental health care. Despite the existence of innovative practices in this context, similar in many aspects to those oriented towards recovery, the use and incorporation of this concept is still at an early stage [21-23]. Most of the few published studies are related to the incorporation of the concept of recovery and the lived experience of its processes and dimensions by people with severe mental illnesses [23, 24], as well as the cross-cultural validation of instruments to be used in services and professional practices. [22, 25,26]. In Brazil, there is a growing consensus that the incorporation of recovery-oriented practices may contribute to mental health care policies and care, as well as expand what has already been implemented from the perspective and care practices of psychosocial rehabilitation [27, 28]. In recent years, studies in this field in Brazil have been mostly based on the narratives of people with lived experience of mental illnesses, seeking to understand their understandings and experiences related to the recovery process, pointed to elements such as: the non-linear experience of the recovery process; stigma and social barriers to access work, leisure and relationships with people; experience of self-stigma linked to illness and disability; the importance of reaching economic stability and/or developing meaningful occupational activities, as well as the view of professionals still focused on the disease and not on the perspective of recovery in the services [22, 29, 30]. Page 3/14 Page 3/14 Page 3/14 Among recovery measures, the Recovery Assessment Scale (RAS) [31] stands out as one of the most widely used internationally. It has demonstrated satisfactory psychometric properties in different cultural contexts [32-37] including Brazil. Despite that, there are still no published studies using this instrument to assess the level of recovery and its associated factors among individuals with serious mental illnesses in Brazil. In this sense, this study aims to fill this gap. Moreover, to the best of our knowledge, this is the first quantitative research on personal recovery in the country. Among recovery measures, the Recovery Assessment Scale (RAS) [31] stands out as one of the most widely used internationally. It has demonstrated satisfactory psychometric properties in different cultural contexts [32-37] including Brazil. 2.2 Measures and procedures The short version of the RAS validated in Brazil has 24 items [25,38, 39] with response options available on a Likert scale, ranging from 1 to 5 (strongly disagree=1; disagree=2; not sure=3; agree=4 and strongly agree =5). The RAS is originally divided into five dimensions, which were replicated in an international study using factor analysis. These dimensions are: Goal and success orientation; Personal confidence and hope; No domination by symptoms; Willingness to ask for help; Reliance on others. In the validation study of the Brazilian version, 6 factors were found, through a principal component analysis. The only difference with the original version is that the dimension Personal-confidence and hope was split into two different factors. However, considering that the factor structure found in the Brazilian version of the RAS has not yet been confirmed and aiming at greater comparability of the results of the present study with international findings, the dimensional structure of five factors was maintained. The original scale is self- administered and the items are scored according to the higher the score, the better the patient's perception and perspective on his/her recovery process. The following measures were used to assess symptomatology and functioning: ILSS-SR (Independent Life Skills Inventory) [40,41], SAOF (Self-Assessment Questionnaire of Occupational Functioning) [42,43] and other psychiatric assessment instruments such as PANSS (Positive and Negative Syndrome Scale) [44,45], CGI (Clinical Global Impression) [46,47], and Calgary Depression Scale [48,49]. All the instruments were administered by a trained researcher. 2.3 Data analysis 2.3 Data analysis Page 4/14 Data were analyzed using the IBM Statistical Package for the Social Sciences (SPSS) for Windows, version 23. Descriptive analysis was used to describe the study sample, in terms of percentage, mean and standard deviation. One-way ANOVA for repeated measures, with Bonferroni post-hoc test, was performed to compare the scores of RAS subscales, since they reflect diferent but still related dimensions of a same concept (recovery). Although such analysis is not frequently used for this purpose, it may identify which dimensions would be more important for the study participants. Finally, to determine the correlates of recovery, a multiple linear regression model was performed. The independent variables were: sociodemographic and clinical data, and the total scores of ILSS - SR, SAOF, CGI, Calgary Depression Scale and the three domains of PANSS (positive, negative and general symptoms). The dependent variable was the total score of RAS. A statistical significance of p < 0,05 was set in all the analyses. 3. Results The majority of the sample was male (67.30%), with an average age of 37 years (SD: 11,08), mostly single (78.80%), living with the family or accompanied (97%), with education in most up to high school (67.70%) and without occupation and/or own income (70.70%). The age of onset was 23.78 years and the majority underwent psychiatric hospitalization (65.70%). The level of overall personal recovery, in terms of the RAS scale score, was equal to 92.84 (SD: 12.92), as the sum of the total items, and 3.97 (SD: 0.45), as the mean of the instrument items. The scores of the other clinical and functional measures used in this study are described in Table 1. In terms of bivariate analyses, the variables that showed a significant association (p<0.05) with the level of recovery and that were retained to be included in the regression analysis were the following: occupational performance (SAOF; p< 0.01), independent living skills (ILSS; p=0.01), general clinical impression (CGI; p=0.03), negative symptoms (PANSS negative symptoms; p=0.02). Variables that presented a significance level of p<0.10 were also retained for the regression analysis, which was the case only for the Calgary scale score (p=0.07). The details of sociodemographic, clinical and functional characteristics of the participants, as the results of univariate analysis, are described in Table 1. 4. Discussion This study examines the recovery process and associated factors in a sample of people with schizophrenia in Brazil. To our knowledge, this is the first quantitative study on the level of recovery and its associated factors in a Brazilian sample, since most of the previous studies in the country focused on qualitative data from the narratives and reports of people with mental disorders. The result of the mean score for RAS scale in the present sample (average score = 3.9) replicates findings from international studies. A literature review that included 28 international studies that used the instrument, found the mean score to range between 3.14 and 4.12 [33]. It is important to note that this review included studies from several countries, including non-English-speaking countries (such as Portugal, Spain, Sweden, Sweden, China and Japan). According to the results of the analysis of variance (ANOVA), which compared the means of RAS domains, lower scores were found in the domain “no domination by symptoms” and the highest ones in the domains of “reliance on others” and “goal and success orientation”. The higher score in the domain of trust in others might be related to the sociocultural aspect of this specific population in Brazil, which values the importance of long lasting affective and even financial support (especcially from relatives) for life and, consequently, as support for processes recovery, as pointed out by a previous qualitative studies from Brazil [28-30]. The variables included in the regression model explain only 20% of the variance in the RAS score in the present sample. In addition, only sociodemographic and clinical variables, and measures of symptoms and functioning were introduced into the model as independent variables. On the other hand, factors that have been shown in the literature as more associated with the recovery process, such as hope, empowerment, well-being and quality of life, for example, were not included in this study [50,51]. This might explain the low percentage found in this study. Positive Symptoms (as measured by PANSS) and global symptom and functioning assessment (as measured by CGI) did not demonstrate a significant association with personal recovery. Together with the lower score for the domain “not dominated by symptoms”, compared to other RAS domains, this result is in convergence with international studies. For example, recent metanalysis have pointed that positive, negative and general symptoms usually have small and negative correlation with recovery measures [50,52]. Please, insert Table 1 here Table 2 shows the comparison between the means of the RAS subscales, using the ANOVA test for repeated measures, with post hoc Bonferroni. Results pointed that Sphericity assumption seems to be violated according to Mauchly’s test, but it might be assumed using Greenhouse-Geisser correction. The analysis presented an overall statistically significant result. It was found that subscale 5 (no domination by symptoms) had a lower score than the other subscales, with the exception of subscale 2 (willingness to ask for help). Subscale 3 (goal and success orientation) had a higher average than all subscales, with the exception of subscale 4 (Reliance on others), which, in turn, was also higher than the averages of subscales 2 (willingness to ask help) and 5 (no domination by symptoms). Please, insert Table 2 here Page 5/14 Page 5/14 The regression analysis (Table 3) showed that only measures of occupational performance (SAOF) and of depressive symptoms (Calgary) had a significant association with the level of recovery. Specifically, this result pointed to an association of higher levels of the person's recovery process with a better self- perception of their occupational performance and with lower levels of depressive symptoms. Please, insert Table 3 here Please, insert Table 3 here Please, insert Table 3 here Please, insert Table 3 here 4. Discussion In this sense, this finding suggests that the recovery process takes place beyond symptoms, and Page 6/14 that, even with their presence and manifestation, people are able to have meaningful ,productive lives, as stated in the most cited defnitions of personal recovery [6]. The lack of significant association, in this study by the ILSS, between the clinical parameter of functional independence and recovery processes, could be related to the fact that performance and independence in some activities of daily living might not be consideredin the participants' perception, as important for their recovery processes. Furthermore, almost none of the participants lived alone and therefore, might not need to carry out certain activities in their daily routine at home, for example. In this sense, it could also be associated with the above mentioned Brazilian and Latin-American sociocultural aspect of long lasting affective and financial reliance on family, which has been pointed out in previous studies [21- 24,29, 33]. The significant association found between SAOF and RAS scores reinforces the importance of subjective assessment of the occupational dimension for the recovery process. This association is also in agreement with international studies, which point to the importance of the occupational dimension and performance of work activities such as, the function and role of the person with experience as a peer worker in health services. Accordingly, the perception and satisfaction that the person has of his/her own performance, as assessed by SAOF, in the area of personal causality, personal interests and sense of self- fulfillment, seem to be related to their recovery process (positive identity, purpose and meaning in life and empowerment) [50,54-56, 57]. It was also possible to identify a significant and negative association between personal recovery and depressive symptoms (as measure by Calgary scale). This finding converges with previous studies, which pointed out that affective symptoms are often the only symptoms showing a moderate and negative correlation with recovery [50,52,58]. This might also be explained by the fact that the presence of depressive symptoms as measured by this scale, especially hopelessness and self depreciation, points to a state opposite to recovery, in which hope and positive identity appear as core processes [57]. Some limitations should be mentioned. First, the study included a non-probability sample. Consequently, further quantitative Brazilian and Latin-American research on this topic is required, using a larger and probabilistic sample. 4. Discussion Another limitation is the cross-sectional design, which does not allow causal inferences between the variables. Despite these limitations, this study is the first to explore the factors associated with the recovery level for people with schizophrenia in Brazil, using quantitative data. It replicates, data from the international literature and highlights specificcultural aspects. As such, the study reinforces the idea of recovery as a universal process, and the need for better guidance of health practices and services aimed at promoting strategies to support people's recovery process. Ethics approval The study was approved by the Ethics Committee of UNIFESP and all participants agreed to participate in the study and signed the consent form. (number: 1737/06). Conflict of interests Conflict of interests Page 7/14 RAB has received honoraria for speaking and chairing engagements from Janssen, Torrent and Ache and outside this manuscript. He has also received personal fees and non-financial support from Janssen outside this manuscript. AG has been a consultant and/or advisor to or has received honoraria from Ache, Cristalia, Daiichi-Sankyo and Janssen. The other authors declare that they have no conflict of interest. RAB has received honoraria for speaking and chairing engagements from Janssen, Torrent and Ache and outside this manuscript. He has also received personal fees and non-financial support from Janssen outside this manuscript. AG has been a consultant and/or advisor to or has received honoraria from Ache, Cristalia, Daiichi-Sankyo and Janssen. The other authors declare that they have no conflict of interest. Funding This study was partially funded by FAPESP (São Paulo State Research Support Foundation). Consent to participate Participants provided informed consent. Participants provided informed consent. References 1. Fett, A. K. J., Viechtbauer, W., Penn, D. L., van Os, J., & Krabbendam, L. (2011). 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A method of comparing therating scale for schizophrenics. Schizophr. Res. 3: 247–251. Page 11/14 Page 11/14 Table 1. Descriptive and univariate analysis of the covariables of recovery level Table 1. Table 1. Descriptive and univariate analysis of the covariables of recovery level Descriptive and univariate analysis of the covariables of recovery level Page 12/14 Page 12/14 Variable N (%) or Mean (SD) t or r (p-value) Sex (n = 104) Men Women 70 (67.30%) 34 (32.70%) t = -0.91 (0.37) Age (n = 100) 37.77 (11.08) r = -0.03 (0.78) Marital Status (n = 99) Single Others 78 (78.80%) 21 (21.20%) t = 2.54 (0.01) Living alone (n = 99) Yes No 03 (3%) 96 (97%) --- Education Status (n = 99) Until High school Higher education 67 (67.70%) 32 (32.30% t = -0.51 (0.61) Occupation and/or own income (n = 99) Yes No 29 (29.30%) 70 (70.70%) t = -2.37 (0.02) Age of onset 23.78 (7.88) r = 0.02 (0.80) Psychiatric hospitalization (n = 99) Yes No 65 (65.70%) 34 (34.30%) t = 0.64 (0.52) SAOF (n = 103) 6.06 (0.95) r = 0.37 (p<0.001) Calgary (n = 99) 2.36 (3.41) r = -0.18 (0.07) ILSS (n = 104) 7.62 (0.96) r = 0.25 (0.01) CGI (n = 99) 3.79 (0.99) r = -0.,21 (0.03) PANSS Positive symptoms (n = 98) 13.00 (4.77) r = -0.03 (0.75) PANSS Negative symptoms (n = 98) 16.55 (5.49) r = -0.24 (0.02) PANSS General Psychopathology (n = 98) 28.92 (7.16) r = -0.12 (0.25) Variable N (%) or Mean (SD) t or r (p-value) Sex (n = 104) Men Women 70 (67.30%) 34 (32.70%) t = -0.91 (0.37) Age (n = 100) 37.77 (11.08) r = -0.03 (0.78) Marital Status (n = 99) Single Others 78 (78.80%) 21 (21.20%) t = 2.54 (0.01) Living alone (n = 99) Yes No 03 (3%) 96 (97%) --- Education Status (n = 99) Until High school Higher education 67 (67.70%) 32 (32.30% t = -0.51 (0.61) Occupation and/or own income (n = 99) Yes No 29 (29.30%) 70 (70.70%) t = -2.37 (0.02) Age of onset 23.78 (7.88) r = 0.02 (0.80) Psychiatric hospitalization (n = 99) Yes No 65 (65.70%) 34 (34.30%) t = 0.64 (0.52) SAOF (n = 103) 6.06 (0.95) r = 0.37 (p<0.001) Calgary (n = 99) 2.36 (3.41) r = -0.18 (0.07) ILSS (n = 104) 7.62 (0.96) r = 0.25 (0.01) CGI (n = 99) 3.79 (0.99) r = -0.,21 (0.03) PANSS Positive symptoms (n = 98) 13.00 (4.77) r = -0.03 (0.75) PANSS Negative symptoms (n = 98) 16.55 (5.49) r = -0.24 (0.02) PANSS General Psychopathology (n = 98) 28.92 (7.16) r = -0.12 (0.25) N (%) or Mean (SD) t or r (p-value) Table 2 Description of overall and subscales RAS scores and comparison between subscales Subscales Mean (SD) Bonferroni Post hoc (p) 1 2 3 4 5 1.Personal confidence and hope 3.97 (0.58) -- ,43 ,06 1,00 ,01* 2. Table 1. Descriptive and univariate analysis of the covariables of recovery level Willingness to ask for help 3.82 (0.63) -- -- ,01* ,02* 1,00 3. Goal and success orientation 4.10 (0.57) -- -- -- 1,00 < ,01 4. Reliance on others 4.04 (0,61) -- -- -- -- < ,01 5. No domination by symptoms 3.75 (0.74) -- -- -- -- -- * p < 0.05; Bonferroni Post hoc: significance level for comparison between sub-scales. ANOVA: F = 8.813; p < 0.001. Mauchly's Test of Sphericity: Mauchly´s W =0.581, X2(2) = 55,155, p < 0.001. Grennhouse-Geisser = 0.834. * p < 0.05; Bonferroni Post hoc: significance level for comparison between sub-scales. ANOVA: F = 8.813; p < 0.001. Mauchly's Test of Sphericity: Mauchly´s W =0.581, X2(2) = 55,155, p < 0.001. Grennhouse-Geisser = 0.834. Table 3. Regression analysis of the correlates of RAS total score Correlates Β SE Δβ T p Constant 75,83 11,96 --- 6,34 <0.01 R2 = 0,28 Δ R2 = 0.22 F = 4.70 (p = <0,001) [DW] = 1.92 Marital Status (Single x Others) -2.99 2.80 -0.10 -1.07 0.29 Occupation and/or own income (Yes x No) -1.32 2.73 -0.05 -0.48 0.63 SAOF 3.48 1.16 0.30 3.00 <0.01 ILSS 0.51 1.20 0.04 0.43 0.67 Calgary -0.77 0.31 -0.23 -2.45 0.02 PANSS negative -0.35 0.28 -0.17 -1.24 0.22 CGI -1.30 1.56 -0.11 -0.83 0.41 Table 3. Regression analysis of the correlates of RAS total score Page 14/14 Page 14/14
https://openalex.org/W2964352267	https://hrcak.srce.hr/file/325670	Bosnian	null	Hrvatski za-infinitiv	Fluminensia	2,019	cc-by	8,297	Jozo Vela, Hrvatski za-infinitiv: izvanjsko posuđivanje ili unutarnji jezični razvoj FLUMINENSIA, god. 31 (2019), br. 1, str. 61-83 Jozo Vela, Hrvatski za-infinitiv: izvanjsko posuđivanje ili unutarnji jezični razvoj FLUMINENSIA, god. 31 (2019), br. 1, str. 61-83 Jozo Vela, Hrvatski za-infinitiv: izvanjsko posuđivanje ili unutarnji jezični razvoj FLUMINENSIA, god. 31 (2019), br. 1, str. 61-83 61 This work is licensed under a Creative Commons Attribution 4.0 International License. Ovaj rad dostupan je za upotrebu pod međunarodnom licencom Creative Commons Attribution 4.0. This work is licensed under a Creative Commons Attribution 4.0 International License. Ovaj rad dostupan je za upotrebu pod međunarodnom licencom Creative Commons Attribution 4.0. https://doi.org/10.31820/f.31.1.10 https://doi.org/10.31820/f.31.1.10 Jozo Vela dr. sc. Jozo Vela, Staroslavenski institut, jvela@stin.hr, Zagreb izvorni znanstveni članak UDK 811.163.42’367.625.41 rukopis primljen: 14. ožujka 2019.; prihvaćen za tisak: 31. svibnja 2019. Upotrebu infinitiva u primjeru Imate li sobu za iznajmiti? u suvremenom hrvatskom jeziku obično se naziva „prijedložnim infinitivom” ili „konstrukcijom za + infinitiv”. Infinitiv upotrijebljen sa za smatra se negramatičnom pojavom, posuđenicom iz stranih jezika te se preporuča izbjegavanje takvog izričaja u biranijem književnom izrazu. U radu se predlaže drukčiji pristup i ukazuje na to da je moguće riječ o unutarnjem jezičnom razvoju, kako je to naznačio već i I. Pranjković. Štoviše, za-infinitiv u hrvatskom jeziku slijedi univerzalni put semantičke i formalne gramatikalizacije infinitiva kakav je ocrtao M. Haspelmath. Pokazat će se to na temelju povijesne građe hrvatskog književnog jezika te na temelju određenih usporedbi s drugim europskim jezicima. U skladu s time, na kraju se predlaže novi formalni opis ovakve upotrebe infinitiva u hrvatskom jeziku. Ključne riječi: hrvatski jezik; infinitiv; prijedložni infinitiv; za + infinitiv; jezična promjena; gramatikalizacija; posuđenica HRVATSKI ZA-INFINITIV: IZVANJSKO POSUĐIVANJE ILI UNUTARNJI JEZIČNI RAZVOJ dr. sc. Jozo Vela, Staroslavenski institut, jvela@stin.hr, Zagreb 1. Ne-gramatična posuđenica Svezu riječi za i infinitiva u hrvatskom jeziku, kao u primjerima Imate li sobu za iznajmiti?, Ovo sam uzeo za ponijeti na plažu., Kava za (ponijeti) van., Ovo je teško za ponijeti., To za ponijeti trebaš biti snažan., On je pravi za ponijeti Jozo Vela, Hrvatski za-infinitiv: izvanjsko posuđivanje ili unutarnji jezični razvoj FLUMINENSIA, god. 31 (2019), br. 1, str. 61-83 62 to., Ručak za prste polizati., obično se u jezikoslovnoj literaturi naziva „konstrukcijom za + infinitiv” ili „prijedložnim infinitivom”. Premda se za- infinitiv najčešće ne opisuje dublje od ovih uvriježenih naziva, njihova upotreba otkriva temeljni pristup ovoj svezi kod pojedinih autora, a ujedno govori i o povijesti jezikoslovnog bavljenja njome. Nazivom „konstrukcija” tako se implicitno upozorava na to kako je riječ o „stranom” elementu u hrvatskom jeziku, o kalku nastalom ugledanjem na druge susjedne jezike, talijanski i njemački (i latinski). Svojevrsni, pak, jezikoslovni oksimoron, pojam „prijedložni infinitiv”, upozorava čitatelja na negramatičnost ove sveze – jer prijedlozi kao vrsta riječi prema normativnim pravilima dolaze ispred imenice ili zamjenice, ne i ispred infinitiva – te na potrebu njenog izostavljanja u uglađenijem književnom izričaju. Protiveći se ideji sagledavanja ove sveze isključivo kao inojezičnog kalka i njezina oštrog normativnog uklanjanja u lektoriranim tekstovima, I. Pranjković ukazuje na to da je moguće riječ o inherentnom hrvatskom jezičnom obilježju i kako sveza za + infinitiv može biti rezultat unutarnjeg jezičnog razvoja hrvatskog jezika. Pranjković je u ovom razmišljanju na dobrom tragu, no on za svezu za i infinitiva uvodi termin „hrvatski perifrastični supin”, što može biti problematično. Ostajući, naime, čvrsto pri gramatičkoj odrednici po kojoj je za uz infinitiv prijedlog Pranjković, ne razmatra mogućnost gramatikalizacije prijedloga za u infinitivnu česticu, te slijedom toga, kako bi udovoljio normativnim pravilima, infinitiv promatra kao (glagolsku) imenicu u akuzativu. S obzirom na to da supin kao arhaična glagolska imenica odgovara traženim propozicijama oblika kojim prijedlog za upravlja, povezivanje infinitiva sa supinom čini se opravdanim, pogotovo ako se u obzir uzmu krnje forme infinitva bez završnog -i koje su oblikom jednake supinu. Svezu za + infinitiv najbolje bi bilo nazivati ‘za-infinitivom’, smatrati je vlastitim jezičnim obilježjem hrvatskog jezika i rezultatom njegova pravilnog jezičnog razvoja te ju deskriptivno (pr)opisati i normirati njezinu upotrebu. Ovu ću tvrdnju nastojati potkrijepiti kasnije u nastavku ovog rada, a prije toga, valja nešto opširnije reći o dosadašnjim rezultatima jezikoslovnog bavljenja za-infinitivom. 1.1. Kalk prema talijanskom, njemačkom, latinskom jeziku Iako je sveza za-infinitiv koja se pojavljuje u hrvatskom jeziku u jezikoslovnoj literaturi prisutna već više od stoljeća, zapravo je malo radova koji dublje promišljaju o ovoj pojavi. U studiji objavljenoj davne 1887. Jozo Vela, Hrvatski za-infinitiv: izvanjsko posuđivanje ili unutarnji jezični razvoj FLUMINENSIA, god. 31 (2019), br. 1, str. 61-83 63 godine i naslovljenoj Njekoje većinom sintaktičke razlike između čakavštine, kajkavštine i štokavštine, studiji koja je danas, zbog količine primjera i uočenih sintaktičkih pojava, uglavnom neizostavna u sintaktičkim istraživanjima hrvatskog jezika koja uključuju ili se vode dijakronijskim pristupom jeziku, L. Zima navodi kako je prijedlog za uz infinitiv „izlišan”, tj. suvišan, a njegovu pojavu u čakavštini, kajkavštini i štokavštini tumači „koje talijanskim, koje njemačkim uplivom” (Zima 1887: 306). Mišljenje kako je kod za-infinitiva riječ o inojezičnom kalku redovito se od tog vremena izražava u jezikoslovnoj literaturi. Tako, T. Maretić u studiji o jeziku slavonskih pisaca dvoji „jesu li to slav. pisci uzeli od dalmatinskijeh, u kojih je to italijanizam, ili je to germanizam” (Maretić 1910: 232). Lj. Jonke, odgovarajući na pismo čitatelja, u časopisu Jezik iznosi stav kako „nema sumnje, da je to u tim najzapadnijim područjima hrvatskoga jezika utjecaj talijanske konstrukcije prijedloga per, koji znači za, zbog, radi, i infinitiva”, te kako je „ta konstrukcija prilično proširena i u našim sjeverozapadnim krajevima, ali u njima pod utjecajem njemačkoga jezika, u kojem se s infinitivom upotrebljava prijedlog zu” (Jonke 1952: 151–152). Obrađivači, pak, i urednici Akademijinog Rječnika hrvatskog ili srpskog jezika (1974.) navode kako je „ta [je] veza prema lat. ad s gerundom, tal. per s inf. i njem. um” (ARj XXI 1973-1974: 666). J. Melvinger također navodi kako „slavenski jezici ne poznaju prijedložni infinitiv” te je prema tomu „riječ o inovaciji koja se javila pod utjecajem sintaktičkog modela nekog neslavenskog, po svoj prilici njemačkog jezika” (Melvinger 1980: 228). U kasnijem radu Melvinger ističe kako se „konstrukcija za + infinitiv pojavila u našem jeziku pod utjecajem odgovarajućih modela kakvi postoje u germanskim i romanskim jezicima” (Melvinger 1982: 74). V. Rišner razloge upotrebe prijedloga za ispred infinitiva u suvremenom hrvatskom jeziku vidi u „prepletanju talijanskih, njemačkih i latinskih utjecaja, ali i u ukorijenjenosti prijedloga za ispred infinitiva u hrvatskom književnom jeziku (...) 18. i 19. stoljeća” čemu se „pridružila i širina upotrebe i značenja izraza s prijedlogom za” (Rišner 2006: 100). 1 V. npr. Požar 2018: 298–299, Vrtič 2009: 270, 394, Županović 2008: 43, Despot 2006: 143, 194–195, Kuna 1977, Grickat 1975: 57. 1.1. Kalk prema talijanskom, njemačkom, latinskom jeziku Ocjene kako je kod za-infinitiva u hrvatskom jeziku i književnim djelima njime pisanim riječ o utjecaju njemačkog, talijanskog ili latinskog jezika mogu se susresti i drugdje u jezikoslovnoj literaturi.1 Jozo Vela, Hrvatski za-infinitiv: izvanjsko posuđivanje ili unutarnji jezični razvoj FLUMINENSIA, god. 31 (2019), br. 1, str. 61-83 Jozo Vela, Hrvatski za-infinitiv: izvanjsko posuđivanje ili unutarnji jezični razvoj FLUMINENSIA, god. 31 (2019), br. 1, str. 61-83 64 3 Za-infinitiv potvrđen je u djelima Marulića, Zoranića, Lucića, Hektorovića, Albertija, Luke Bračanina, zatim u Ranjininu zborniku, u djelima Držića, Pelegrinovića, Gundulića, u dubrovačkim dramama 16. – 18. stoljeća, u oporukama i arhivskim spisima iz Dubrovnika iz 17. i 18. stoljeća, te u lekcionarskoj tradiciji od Zadarskog i Bernardinova do Kašićeva lekcionara, u protestantskim svetopisamskim prijevodima (Dalmatin), i u Katančićevu prijevodu Svetog pisma. ‘Za-infinitiv’ raširen je u djelima svih čakavski i štokavskih pisaca, dubrovačkih, slavonskih i bosanskih, odnosno uopće franjevačkih pisaca, 17. i 18. stoljeća (Kanižlić, Reljković, Divković, Matijević, Ančić, Papić, Bandulavić, Posilović, Lastrić, Babić, Lašvanin, Benić, Knežević, Filipović, Grabovac, Kačić). Usp. Vrtič 2009: 270–271, 293–394. 1.2. Barbarizam Jednoznačno povezivanje za-infinitiva s istovjetnim svezama u jezicima koji su u kontaktu s hrvatskim, tj. talijanskom svezom per + infinitiv, i njemačkim um, odnosno zu + infinitiv, imalo je za izravnu posljedicu to da se za-infinitiv našao na udaru jezikoslovnog purizma. Kako to Pranjković zgodno navodi, naprosto „su se neki naši jezikoslovci, i to ne samo stariji, [naprosto] natjecali u tome da gotovo svaku pojavu kojoj se mogla naći analogija u nekom od ‘većih’ europskih jezika proglase rezultatom stranoga utjecaja” (Pranjkovć 1993: 40). T. Maretić u svom je Jezičnom savjetniku za-infinitiv označio njemačkom jezičnom pojavom te predlagao njegovu zamjenu jer je „najveći deo naših rđavih reči načinjen prema nemačkima” (Maretić 1924: XXX, 179). Lj. Jonke smatra za-infinitiv grubom sintaktičkom pogreškom (Jonke 1964: 359–360) i poziva pisce „da se klone tih romanizama i germanizama, koji samo nagrđuju naš jezik” (Jonke 1952: 152). J. Melvinger naziva ovu pojavu supstandardnom sintaktičkom inovacijom te veli: „kad god ta sintaktička konstrukcija nije u funkciji stilskog sredstva, nužna je energična lektorska intervencija” (Melvinger 1982: 74). Ona, međutim, upozorava kako se osim glagolskom imenicom, ova konstrukcija može zamijeniti i drugim sintaktičkim sredstvima hrvatskog standardnog jezika, kao što su: rečenica s veznikom da, odnosna atributna rečenica s modalnim predikatom, infinitiv bez prijedloga, sveza modalnog glagola i infinitiva, i sl.. (Melvinger 1980: 228–232, Melvinger 1982). Nastojanja da se za-infinitiv iskorijeni iz jezične upotrebe, barem u poljima gdje se upotrebljava standardni jezik, nastavljena su i u suvremeno vrijeme. Mada u znatno ublaženijem tonu i uvažajući plodnost i živost za-infinitiva u hrvatskom jeziku, u Hrvatskom se jezičnom savjetniku iz 1999. godine infinitiv iza prijedloga ipak naziva ne-infinitivom te se preporuča njegovo izbjegavanje u biranijem i njegovanijem književnom jeziku, a upotrebu se preporuča tek u nekim uporabnim područjima standardnog jezika gdje se korištenjem tog izraza želi dobiti na ekspresivnosti. (Barić i dr. 1999: 196, 248). Dakako, stav jezikoslovaca prema negramatičnosti i nedopuštenosti za- infinitiva u standardnoj upotrebi hrvatskog jezika ogleda se i u suvremenim gramatikama hrvatskog jezika. U trima gramatičkim priručnicima koji su ponajviše u suvremenoj školskoj i sveučilišnoj upotrebi (Težak – Babić 2000, Barić i dr. 1997, Silić – Pranjković 2005) za-infinitiv uopće se ne spominje, što implicitno znači da ga se ne smatra sastavnim dijelom gramatike hrvatskog jezika. Jozo Vela, Hrvatski za-infinitiv: izvanjsko posuđivanje ili unutarnji jezični razvoj FLUMINENSIA, god. 31 (2019), br. 1, str. 61-83 65 2 Rišner 2007: 98–100, Rišner 2006: 127, Barić i dr. 1999: 248, Pranjković 1993: 40–45, Melvinger 1982: 74. 4 Dakako ni u dijalektološkim radovima ne izostaju ocjene kako je riječ o stranom utjecaju. 2. Normativnom protjerivanju usprkos Unatoč tomu što jezični savjetodavci već dugi niz godina protjeruju za- -infinitiv iz standardnog jezika, njegova je upotreba kontinuirano živa u razgovornom jeziku, a sve češće i u publicističkom, administrativnom, pa i književnoumjetničkom stilu književnog jezika.2 Osim toga, jezikoslovna sintaktička istraživanja posljednjih desetljeća pokazala su da je za-infinitiv imao itekako plodnu upotrebu u djelima starije hrvatske književnosti 16., 17., 18., pa i 19. stoljeća, osobito u jezičnim strukturama gdje se koristio kao ekvivalent namjerne rečenice, ali i u adnominalnoj upotrebi, također namjerno-posljedičnog značenja.3 Prema D. Gabrić-Bagarić uporaba za- -infinitiva kao zamjene za zavisnu, najčešće namjernu, rečenicu je „najizrazitija zajednička osobina svih pisaca 18. st.” (Gabrić-Bagarić 2007: 138). O uvriježenosti za-infinitiva u starijim djelima hrvatskog književno umjetničkog stvaralaštva dovoljno govori i podatak da ga fra Tomo Babić smatra općeknjiževnom crtom, opisuje u svojoj gramatici, a rabi i u metajeziku (Gabrić-Bagarić 2007: 139), te da ga Relković u svojoj gramatici (1767) također navodi, i to kao poseban glagolski oblik (Pranjković 1993: 42). Ne treba zaboraviti ni to da svezu za-infinitiv jezikoslovac J. Mikalja (1649/1951) upotrebljava u naslovu svoje gramatike Gramatika talijanska ukratko ili kratak nauk za naučiti latinski jezik. Usprkos sustavnom normativističkom protjerivanju za-infinitiv česta je pojava i u najvišem stilu hrvatskog jezika u suvremenoj hrvatskoj književnosti, u djelima I. Kušana, Z. Majdaka, I. Mandića, A. Šoljana, S. Šembere, i drugih. (Melvinger 1982: 74). Kako je za-infinitiv ipak najčešći u razgovornom jeziku, to da ga se često bilježi u dijalektološkim radovima, nije potrebno posebno isticati. Njegova je prisutnost u razgovornom jeziku, tj. u organskim govorima Hrvata, tolika da ga se izrijekom navodi kao jedno Jozo Vela, Hrvatski za-infinitiv: izvanjsko posuđivanje ili unutarnji jezični razvoj FLUMINENSIA, god. 31 (2019), br. 1, str. 61-83 66 od bitnih sintaktičkih obilježja na razini narječja, svih triju hrvatskih na rječja, kajkavskog, čakavskog i štokavskog (Lončarić 1996: 123, Lisac 2003: 28, Lisac 2009: 28)4. 5 Pranjković ostaje pri tom da je i dalje riječ o prijedlogu. 6 Usp. Jeffers 1975, Disterheft 1980, Disterheft 1981, Holland 1982, Szemerényi 1996: 324, Ramat 1994: 259, Comrie 1998: 88. 7 Usp. Disterheft 1980, Disterheft 1981. 3. Hrvatski perifrastični supin Zastavši nad činjenicom da „te konstrukcije, unatoč tomu što su jezikoslovci manje-više jednodušni u njihovu normativističkom osporavanju, uporno odolijevaju osporavateljima”, odnosno da se širenjem utjecaja javnih glasila sve više učvršćuju, I. Pranjković promišlja o za-infinitivu na nov i originalan način, po prvi ga put nastojeći dublje analizirati i sagledati iz pozicija razvojnih tendencija hrvatskog jezika. U članku objavljenom 1987. godine (Pranjković 1987), koji je ponovno uz manje izmjene otisnut u knjizi 1993. godine (Pranjković 1993: 40–45), autor između ostalog polemizira s tvrdnjom J. Melvinger kako je za-infinitiv supstandardna sintaktička inovacija navodeći kako je u istočnim oblastima (tj. srpskoj povelji) ta konstrukcija prvi put zabilježena 1198. godine te kako se ne može nazvati inovacijom nešto što ‘živi’ već preko osam stoljeća. Osim toga, unatoč gotovo stoljetnim prosvjedima purista spomenuta je konstrukcija raširena, manje ili više, na cijelom području hrvatskog jezika, i prisutna je u svim funkcionalnim stilovima hrvatskog jezika (premda nije jednako tipična za sve stilove), a „[s]ve su to stilovi književnog (standardnog) jezika (a ne supstandardnog)” (Pranjković 1993: 44). Također, osporavajući tvrdnju J. Melvinger kako se za-infinitiv redovito može adekvatno zamijeniti, upozorava na strukture u kojima takva zamjenjivost nije moguća (Ti si za ubiti ≠ Ti si za ubijanje, Ti si da (se) ubiješ, Ti si takav da te treba ubiti i sl.), te je stoga protiv pravila kako je nužna energična lektorska intervencija kad god ta sintaktička konstrukcija nije u funkciji stilskog sredstva. Pranjković s pravom postavlja pitanje: „Može li se uopće dokazati (i čime, ako može) da je ta konstrukcija uvezena i odakle?” (Pranjković 1993 40). Zatim se, zastupajući, čini se, upravo suprotno, odnosno da pojava za-infinitiva u hrvatskom jeziku počiva na unutarjezičnim uvjetima te da nije rezultat vanjskog utjecaja, autor upušta u složenu argumentaciju njezina razvitka. Pri tom on polazi od naravi prijedloga za. Budući da prijedlog za kad dolazi uz akuzativ ima apstraktno značenje namjere, cilja i sl., značenje Jozo Vela, Hrvatski za-infinitiv: izvanjsko posuđivanje ili unutarnji jezični razvoj FLUMINENSIA, god. 31 (2019), br. 1, str. 61-83 67 koje se obično pridaje i akuzativu samom, jasno je da je prijedlog za mogao poprimiti sposobnost akuzativiranja tj. pridavanja akuzativnog značenja i komponentama koje nemaju akuzativa. Najekonomičniji put pri tom je taj da se obliku kojim se samo imenuje radnja (infinitiv) doda obavijest o namjeni ili cilju (za). 3. Hrvatski perifrastični supin Pranjković nadalje veli: „Takvo akuzativiranje infinitiva moglo bi se ovdje uvjetno nazvati i ‘supinizacijom’” (Pranjković 1993: 41), budući da je, povijesnolingvistički gledano, upravo supin okamenjeni akuzativ glagolske imenice. On, stoga, za-infinitiv naziva supinskom konstrukcijom, odnosno perifrastičnim supinom. 3.1. Ekskurs o supinu Pranjković je neosporno u pravu kada uviđa da je kod prijedloga za došlo do određenog pomaka u značenju i funkciji, no mišljenju kako je nova funkcija koji je riječ za dobila akuzativiranje5 i kako je umjesto infinitiva riječ o supinu mogu se izreći određeni prigovori. Praindoeuropski jezik nije imao infinitiva, nego je on u europskim jezicima rezultat kasnijeg razvoja, odnosno toga da su se različite glagolske imenice, različitih tvorbenih osnova i deklinacijskih razreda nakon raspada praindoeuropskog zajedništva postupno verbalizirale i ulazile u glagolski sustav, gubeći svaku vezu s imenskom paradigmom kojoj su pripadale.6 Što su više glagolske imenice ulazile u glagolski sustav (tj. poprimale sintaktičke funkcije glagola), dva su procesa usporedo tim više napredovala. Jedan je taj da su imenske paradigme kojima su u početku glagolske imenice pripadale postajale neproduktivne, a oblici su infinitiva njihove relikte kao okamenjene forme sačuvali samo u padežu u kojem su se najčešće upotrebljavali. Drugi je proces taj da su neki infinitivni oblici preuzimali uloge drugim infinitivni oblicima (što prije verbalizacije infinitiva nikako ne bi bilo moguće), te je s vremenom došlo do pojednostavljenja u broju infinitiva u sustavu. Najčešće je broj oblika sveden na jedan.7 Ovaj razvoj iz indoeuropskog prajezika moguće je pratiti i u jezicima iz kojih su se razvili suvremeni slavenski jezici, pa i hrvatski. Što se tiče baltoslavenskog prajezika, za njega se još uvijek ne može rekonstruirati Jozo Vela, Hrvatski za-infinitiv: izvanjsko posuđivanje ili unutarnji jezični razvoj FLUMINENSIA, god. 31 (2019), br. 1, str. 61-83 68 jedinstvena tvorba infinitiva, budući da u litavskim dijalektima i staro pruskome imamo potvrde različitih tvorbi infinitiva. (Matasović 2008: 300). U staroslavenskom jeziku, koji se zbog starine spomenika u ovom slučaju smatra vjernim odrazom praslavenskog jezika, dva su oblika za koje se može tvrditi da su okamenjeni ostaci nekoć produktivnih paradigmi glagolskih imenica: jedan je glagolski oblik na -ti, poznat pod nazivom infinitiv, a drugi je glagolski oblik na -tъ, poznat pod nazivom supin.8 I slavenski supin i infinitiv podrijetlom su glagolske imenice, a supin se od infinitiva razlikuje na dva načina: 1) supin se koristio samo uz glagole kretanja te je u slavenskim jezicima gdje je sačuvan, sačuvan samo nakon glagola kretanja; 2) infinitiv je proces verbalizacije iz glagolske imenice prošao potpuno, tj. izgubio je vezu s imenskom paradigmom i verbalizirao se – a supin tek djelomično, tj. 8 Infinitivni nastavak -ti složeni je sufiks koji se sastoji od morfema poimeničenja -t- i starog indoeuropskog dativnog (ili lokativnog) nastavka -ei koji u slavenskim jezicima monoftongizacijom daje -i. Kod supina je riječ o starom obliku akuzativa. Na temelju stanja u starocrkvenoslavenskom može se kao završetak rekonstruirati sufiks poimeničavanja -t- proširen akuzativnim nastavkom -um. Od takvoga će se praslavenskoga završetka normalnim glasovnim razvojem razviti potvrđeni starocrkvenoslavenski nastavak za supin -tъ. (Mihaljević 2014: 183). 3.1. Ekskurs o supinu samo je izgubio vezu s imenskom paradigmom, no nije (do kraja) prošao proces verbalizacije, što se ogleda u činjenici da njegov objekt ne stoji u akuzativu (kao kod glagola), nego u genitivu (kao kod imenica). S obzirom na jezični razvoj, razumljivo je da su infinitiv i supin u biti isti oblici. Staroslavenskom je obliku na -tъ zbog potpune značenjske i etimološke veze s latinskim supinom na -um dan tradicionalni naziv supin, no potpuno ga se ispravno moglo nazvati i infinitivom drugim ili slično. Daljnjim razvojem od praslavenskog prema suvremenim slavenskim jezicima došlo je do očekivanog procesa pojednostavljivanja broja infini tivnih oblika u sustavu, te je infinitiv zamijenio, odnosno potisnuo supin u funkciji izražavanja svrhe ili cilja iza glagola kretanja na većini slavenskog područja. Supin kao poseban oblik očuvan je danas jedino u slovenskom i donjolužičkom jeziku te u kajkavskom narječju hrvatskog jezika. (Lončarić 1996: 26, 108–109, Grošelj 2014, Zakar 2016). Govoriti dakle o za-infinitivu kao o supinskoj konstrukciji i o procesu njegova razvoja u hrvatskom jeziku kao o supinizaciji iz povijesnojezične je perspektive problematično iz dva razloga. Prvo, supinizacija bi kao proces Jozo Vela, Hrvatski za-infinitiv: izvanjsko posuđivanje ili unutarnji jezični razvoj FLUMINENSIA, god. 31 (2019), br. 1, str. 61-83 69 ne samo pretpostavljala suprotstavljanje stoljetnim jezičnim tendencijama uzmaka supina pred infinitivom i oživljavanje supina na područjima na kojima je on davno uklonjen iz sustava, nego i istodobno njegovo širenje na funkcije koje mu povijesno nikad nisu pripadale: dopuna glagola koji nisu glagoli kretanja, adnominalna dopuna i sl. Drugo, argumentacija da je uz za riječ o supinu, a ne infinitivu, čini se opravdanom jedino ako bi govornici koji upotrebljavaju za-infinitiv, pa čak i oni koji upotrebljavaju supin (Idem spat) bili svjesni da je kod supina riječ o akuzativu. Premda se nije do kraja verbali zirao, supin je, naime, proces odvajanja od imenske paradigme kojoj je pripadao prošao u potpunosti, što znači da kod njega nema ni traga akuza tivnog obilježja kojeg je nekoć nosio. Iako supin nije u punom smislu glagolski oblik, jer se po objektu u genitivu razlikuje od drugih glagolskih oblika s akuzativnom rekcijom, on nije niti imenica, nema imensku paradigmu kojoj pripada i nije nositelj padežnog obilježja. Odabirati naziv supin umjesto termina infinitiv iz razloga jer je prvi oblik u akuzativu, problematično je u vremenu u kojem on već stoljećima više nije oblik u akuzativu. Kod za-infinitiva nije, dakle, riječ o perifrastičnom supinu. 9 Treba imati na umu da hrvatski crkvenoslavenski jezik, kao književni jezik svojedobno najvišeg stila, samo uz pažljivo razlučivanje jezičnih pojava može biti podatan za istraživanje hrvatskog jezika, budući da njegova osnovica počiva na starocrkvenoslavenskom jeziku, a 3.1. Ekskurs o supinu To znači da je vjerojatnije da je ipak riječ o infinitivu. Objašnjenje njegove pojave u hrvatskom jeziku treba stoga tražiti u prirodi infinitiva i procesu povijesno jezičnog razvoja infinitiva. Najprije se valja vratiti u jezičnu prošlost prije vremena pojave za-infinitiva u hrvatskom jeziku, kako bismo znali što mu je prethodilo. samo je nadgradnja hrvatska. Ona je nastala jer su pisci, prevoditelji i pisari svjesno u tekstove unosili hrvatske jezične osobine. Kad je riječ o razini sintakse, osim hrvatske nadgradnje, postoji i nadgradnja koja bi se mogla nazvati latinskom, a riječ je o ugledanju na latinski jezik predložaka, što je osobina ne samo hrvatskog crkvenoslavenskog jezika, nego i mnogih djela starije hrvatske književnosti na hrvatskom jeziku koja su prijevodi latinskih izvornika. Ipak, kako je prodor hrvatskih jezičnih osobina u hrvatske crkvenoslavenske tekstove bio slobodan, slobodniji nego u drugim redakcijama crkvenoslavenskog jezika, i provodio se na svim jezičnim razinama, hrvatski crkvenoslavenski tekstovi, mogu nam nuditi i nude nam relevantne podatke i o onodobnom hrvatskom jeziku. 10 V. Vela 2018: 174–175. Podatak se temelji na velikom istraživanju koje je obuhvatilo 5860 primjera upotrebe infinitiva. 4. Za-infinitiv u hrvatskom crkvenoslavenskom jeziku Iz već navedene literature može se zaključiti kako se upotreba za- infinitiva u hrvatskom književnom jeziku može sa sigurnošću pratiti od 15. stoljeća nadalje. Za uvid u jezičnu građu koja je prethodila 15. stoljeću, istražio sam u ovom radu spomenike pisane hrvatskim crkvenoslavenskim jezikom i hrvatsko-crkvenoslavenskim amalgamom. Istraživanje je provedeno na Građi za Rječnik crkvenoslavenskoga jezika hrvatske redakcije, koja je nastala i čuva se u Staroslavenskom institutu u Zagrebu a uključuje tekstove pisane od 12. do polovice 17. stoljeća, s tim da većina spomenika datira od 13. do 15. stoljeća. (Gadžijeva 2012: 11).9 Osim tekstova pisanih hrvatskim crkveno Jozo Vela, Hrvatski za-infinitiv: izvanjsko posuđivanje ili unutarnji jezični razvoj FLUMINENSIA, god. 31 (2019), br. 1, str. 61-83 70 slavenskim jezikom, Građa sadrži i tekstove iz hrvatskoglagoljskih zbornika od. 14. do 17. st. pisane hrvatsko-crkvenoslavenskim amalgamom, koji se može definirati kao hrvatski (čakavski) jezik s manjom ili većom crkveno slavenskom nadgradnjom. U Građi je dosad zabilježen samo jedan primjer upotrebe za-infinitiva u hrvatskim crkvenoslavenskim spomenicima.10 Riječ je o primjeru iz sanktorala II. novljanskog brevijara (BrN2, 1495.), točnije iz sermona sv. Jeronima koji se čita na oktavu sv. Lovre: aĉe ... za utežaniê ihь veĉe potvrditi taki i toliki službi vzdati spodobilь e(stь) – BrN2 472a quod si ... ob merita suorum amplius comprobanda talia et tanta dignatus est exhibere aĉe ... za utežaniê ihь veĉe potvrditi taki i toliki službi vzdati spodobilь e(stь) – BrN2 472a 2 quod si ... ob merita suorum amplius comprobanda talia et tanta dignatus est exhibere Kako vidimo riječ je o brevijaru s kraja 15. stoljeća, dakle o hrvatsko glagoljskom brevijaru mlađeg tipa. Za-infinitivom u njemu je preveden latinski prijedlog ob (zbog, radi) + gerundiv i njime se izriče namjera. Možemo, dakle, zaključiti da se u hrvatskom crkvenoslavenskom jeziku, a ni u hrvatsko-crkvenoslavenskom amalgamu za-infinitiv u biti ne upo trebljava. (HCSJ 2014: 361). Zanimljivo je, međutim, pogledati što se u hrvatskoglagoljskim tekstovima upotrebljava u strukturama koje su slične onima u kojima se u starijoj hrvatskoj književnosti i u suvremenom hrvatskom jeziku upo trebljava za-infinitiv. U prijevodima s latinskog jezika u starijoj se hrvatskoj književnosti za-infinitiv najčešće upotrebljava za prevođenje sveze ad + gerund/gerundiv te se ova sveza čak navodi kao jedan od mogućih ino jezičnih utjecaja na razvoj za-infinitiva. 10 V. Vela 2018: 174–175. Podatak se temelji na velikom istraživanju koje je obuhvatilo 5860 primjera upotrebe infinitiva. 4. Za-infinitiv u hrvatskom crkvenoslavenskom jeziku Jozo Vela, Hrvatski za-infinitiv: izvanjsko posuđivanje ili unutarnji jezični razvoj FLUMINENSIA, god. 31 (2019), br. 1, str. 61-83 71 Latinski ad + gerund/gerundiv u hrvatskom crkvenoslavenskom jeziku prevodi se: 1) glagolskom imenicom u akuzativu s prijedlozima na i vь: 1) glagolskom imenicom u akuzativu s prijedlozima na i vь: egda ap(usto)li na bl(a)govêstovanie c(êsa)rstviê b(o)žiê poslani biše – BrVO 332d posteaquam apostoli ad evangelizandum regnum Dei sunt destinati kьždo bo svoû večeru varêet’ v snêdenie – MVat4 87a uniusquisque enim suam caenam praesumit ad manducandum – 1Kor 11,21 1) glagolskom imenicom u akuzativu s prijedlozima na i vь: egda ap(usto)li na bl(a)govêstovanie c(êsa)rstviê b(o)žiê poslani biše – BrVO 332d posteaquam apostoli ad evangelizandum regnum Dei sunt destinati kьždo bo svoû večeru varêet’ v snêdenie – MVat4 87a uniusquisque enim suam caenam praesumit ad manducandum – 1Kor 11,21 2) glagolskom imenicom u dativu s prijedlogom kь: tьgda nečist(i)vi c(êsa)rь (...) nik(a)kože k vêrovaniû h(rьst)a vzbudi se – BrN2 485d tunc impius (...) nullo modo ad credendum Christo incitatus 2) glagolskom imenicom u dativu s prijedlogom kь: tьgda nečist(i)vi c(êsa)rь (...) nik(a)kože k vêrovaniû h(rьst)a vzbudi se – BrN2 485d tunc impius (...) nullo modo ad credendum Christo incitatus 3) infinitivom bez za: eda hrama ne imate êsti i piti – MVat4 87a numquid domos non habetis ad manducandum et bibendum – 1Kor 11,22 iže težati vinograd’ svoi dêlateli privodit’ – BrVO 128d qui ad excolendam vineam suam operarios conducit iže težati vinograd’ svoi dêlateli privodit’ – BrVO 128d qui ad excolendam vineam suam operarios conducit žl’čь dobra e(stь) pomazati oči – BrN2 222c fel valet ad ungendos oculos – Tob 6,9 žl’čь dobra e(stь) pomazati oči – BrN2 222c fel valet ad ungendos oculos – Tob 6,9 žl’čь dobra e(stь) pomazati oči – BrN2 222c fel valet ad ungendos oculos – Tob 6,9 interrogavit Dominus unde emerentur panes ad turbas pascendas Potonji primjeri (3) sa samim infinitivom osobito su zanimljivi. U njima se infinitiv pojavljuje bez riječi za unatoč tomu što u latinskom stoji gerund/gerundiv s prijedlogom ad. Valja primijetiti kako su navedeni primjeri također oni u kojima bi suvremeni govornik hrvatskog jezika vjerojatno ispred infinitiva upotrijebio za. Latinskom konstrukcijom ad + gerund/gerundiv izriče se namjera. Infinitiv bez za u hrvatskom crkvenoslavenskom upotrebljava se za izricanje Jozo Vela, Hrvatski za-infinitiv: izvanjsko posuđivanje ili unutarnji jezični razvoj FLUMINENSIA, god. 31 (2019), br. 1, str. 4. Za-infinitiv u hrvatskom crkvenoslavenskom jeziku 61-83 72 namjere i kad ne prevodi spomenutu latinsku konstrukciju, ali niti latinski infinitiv namjere: počeše napravlati korabli iti i vzeti û s velikoû sl(a)voû – BrN2 494c posla listi pod’ pečatomь prstena svoego prizvati mučiteli oh(rьst)ь nie – BrLab 161a misit litteras regio anulo signatas per infra iacentes provincias ad omnes rhetores et grammaticos ut ad praetorium Alexandrinum eo studiosius occurrant posla listi pod’ pečatomь prstena svoego prizvati mučiteli oh(rьst)ь nie – BrLab 161a misit litteras regio anulo signatas per infra iacentes provincias ad omnes rhetores et grammaticos ut ad praetorium Alexandrinum eo studiosius occurrant i ne budet’ k tomu vod’ potopa pogubiti z(e)mlû – BrVO 138d neque erit deinceps diluvium dissipans terram poslavь is’pit’niki osuditi i na k’rižь – BrN2 383c mandans hoc questionariis ut ligatis pedibus et manibus quasi in eculeo tenderetur Navedeni primjeri pomno su odabrani iz razloga što u njima hrvatski crkvenoslavenski tekst odstupa od vjerojatnog latinskog predloška, a to pokazuje da je riječ o primjerima koji nisu nastali pod utjecajem jezika predloška, što znači da je vjerojatno riječ o jezičnoj crti piščeva govornog jezika. Svi ovi primjeri sa samim infinitivom također su slučajevi u kojima bi suvremeni govornik hrvatskog jezika, upotrijebivši infinitiv, zacijelo bio ponukan upotrijebiti ga s riječju za. Slično je s hrvatskim crkvenoslavenskim infinitivom u adnominalnoj funkciji: činь kr’stiti dite – RitKlim 152v ritus baptizandi parvulos m(o)litva podstric ̂i otroče – MRoč 223a oratio in prima capillorum detensione m(o)litva podstric ̂i otroče – MRoč 223a oratio in prima capillorum detensione n(i)ne e(stь) vrême ĵis’ti i piti a malo li e(stь) dni b(og)a moliti – CBč 75b Dakako, u Građi nalazimo i primjere upotrebe infinitiva namjere uz glagole kretanja u kojima bi i suvremeni govornik upotrijebio infinitiv bez prijedloga za: Jozo Vela, Hrvatski za-infinitiv: izvanjsko posuđivanje ili unutarnji jezični razvoj FLUMINENSIA, god. 31 (2019), br. 1, str. 61-83 73 vidê li kogda greduĉago krasti – BrVO 348c vidisti aliquem euntem ad furtum Hrvatskoglagoljski tekstovi nude nam dva glavna zaključka. Prvi je taj da se u njima latinska namjerna konstrukcija ad + gerund/gerundiv prevodi samim infinitivom, odnosno infinitivom bez za. Drugi je taj da hrvatski glagoljaši upotrebljavaju infinitiv bez za za izražavanje namjere i u drugim slučajevima, ponekad i u onima u kojima bi suvremeni govornik hrvatskog jezika upotrijebio za-infinitiv. U posebno biranim primjerima nije riječ o nasljedovanju jezika predložaka, što znači da je vjerojatno riječ o osobini govornog jezika hrvatskih glagoljaša. Uvid u hrvatskoglagoljske tekstove pokazuje da u njima za-infinitiv nije prisutan, ali i to da se u barem nekim sintaktičkim i semantičkim funkcijama u kojima se kasnije u hrvatskom jeziku koristi za-infinitiv, u njima koristi jednostavni infinitiv bez za. Pitanje je, dakle, zašto je u određenom trenutku došlo do upotrebe riječi za uz infinitiv koji je nekoć upotrebljavan samostalno. Odgovor na to pitanje tiče se prirode infinitiva, s jedne strane, i prirode riječi za, s druge strane. 11 Jezikoslovna tipologija je disciplina koja se bavi izučavanjem gramatičkih obrazaca koji se sustavno pojavljuju u različitim jezicima različitih jezičnih porodica tražeći moguća objašnjenja tih obrazaca. 12 Gramatikalizacija je proces promjene u kojoj riječi ili sveze riječi u određenim jezičnim kontekstima počinju poprimati gramatičke uloge, te jednom gramatikalizirani, razvijaju nove gramatičke uloge. (Hopper – Traugott 2008: XV). Termin gramakalizacija prvi je sročio i upotrijebio A. Meillet u djelu L’évolution des Formes Grammaticales (1912.) i definirao ga kao „pridavanje gramatičkog karaktera nekoć samostalnoj riječi”. Posljednjih desetljeća gramatikalizacija je, sudeći prema broju radova, postala izrazito popularan termin u jezikoslovlju, priskrbivši si čak i naslov teorije, a za to je među zaslužnijima svakako Ch. Lehmann koji je u svojem djelu Thoughts on Grammaticalization (1982., 1995.) izumio niz parametara po kojima se stupanj gramatikalizacije može mjeriti, sinkronijski i dijakronijski. (Hopper – Traugott 2008: 19–38). Jedna od temeljnih postavki „teorije” gramatikalizacije jest da je ona stupnjevita pojava koja može uključivati nekoliko faza. Prelazak iz jedne faze u drugu očituje se u cijelom nizu različitih parametara koji pripadaju različitim razinama: semantičkoj, pragmatičkoj, morfosintaktičkoj ili fonološkoj. Kad je riječ o glagolima, 11 Jezikoslovna tipologija je disciplina koja se bavi izučavanjem gramatičkih obrazaca koji se sustavno pojavljuju u različitim jezicima različitih jezičnih porodica tražeći moguća objašnjenja tih obrazaca. gramatikalizacija od glagola do klitike, odnosno naveska, prema B. Heine i T. Kutevoj (Heine – Kuteva 2005: 15) uključuje sljedeće faze: 1) ekstenzija, odnosno pojava novog gramatičkog značenja kada se jezični izraz proširi na nove kontekste, 2) desemantizacija, odnosno blijeđenje, gubljenje, značenja, 3) dekategorizacija, odnosno gubljenje dotadašnjih morfosintaktičkih obilježja koja su novom gramatičkom značenju suvišna, 4) fonetska erozija. 13 O mogućem tumačenju da je riječ o lokativu glagolske imenice v. Grković-Mejdžor 2011: 29–30. 5. Razvoj infinitiva U odgovoru na pitanje o prirodi i razvoju infinitiva važna nam saznanja nude jezičnotipološka11 istraživanja. Gramatikalizacija12 oblika kojima se u 11 Jezikoslovna tipologija je disciplina koja se bavi izučavanjem gramatičkih obrazaca koji se sustavno pojavljuju u različitim jezicima različitih jezičnih porodica tražeći moguća objašnjenja tih obrazaca. 12 Jozo Vela, Hrvatski za-infinitiv: izvanjsko posuđivanje ili unutarnji jezični razvoj FLUMINENSIA, god. 31 (2019), br. 1, str. 61-83 74 jeziku izriče namjena/namjera (engl. purpose) u infinitiv ili infinitivne markere osobina je, ne samo indoeuropskih jezika, nego ima i tipološka obilježja, odnosno pokazuje se kao univerzalni put razvoja infinitiva u jezicima svijeta. Nemaju svi svjetski jezici infinitiv kao poseban morfem, no u onima u kojima se razvio, može se pokazati kako se razvio iz oblika kojima se prototipno izražavala namjena. (Haspelmath 1989, Heine – Kuteva 2002: 247–248). Slavenski jezici, kao i ostali indoeuropski jezici potvrđuju ovaj tipološki razvoj, jer se slavenski infinitiv na -ti kao adverbijalna dopuna namjernog značenja najčešće tumači kao okamenjeni dativni13 oblik glagolske imenice. (Grković-Mejdžor 2007: 280). Dativ kao padež namjene i cilja (Težak – Babić 2000: 294), odnosno padež čije je temeljno značenje negranična direktivnost, tj. smjer i namjena (Silić – Pranjković 2005: 219), samim svojim prototipnim značenjem nosi mogućnost izražavanja namjere. Dativom glagolske imenice bilo je moguće izraziti namjeru čak i prije nego je taj oblik verbaliziran u infinitiv, a infinitivu prototipno pripada semantika namjene/namjere. U studiji posvećenoj tipologiji infinitiva M. Haspelmath (Haspelmath 1989) na temelju primjera iz jezika različitih jezičnih porodica (ugro-finske, turkijske, semitske, dravidske, nahsko-dagestanske itd.) pokazuje da se infinitivni oblici razvijaju gramatikalizacijom oblika kojima se u jeziku izražava namjena (engl. purpose), a oni se razvijaju iz posve konkretnih značenja adlativa, beneficijenta i uzroka. Vodeći se Lehmanovim parametrima teorije gramatikalizacije, autor je, naime, na temelju povijesne građe nje mačkog jezika utvrdio da je njemački zu-infinitiv desemantizacijom tijekom povijesti proširio semantički spektar, od onog da izražava namjenu/namjeru na druga (modalna) značenja, te da je proširio svoju ulogu na dopune različitim glagolima. Stoga, Haspelmath kao univerzalnu shemu semantičke gramatikalizacije infinitiva nudi sljedeći stupnjeviti prikaz: Jozo Vela, Hrvatski za-infinitiv: izvanjsko posuđivanje ili unutarnji jezični razvoj FLUMINENSIA, god. 31 (2019), br. 1, str. 61-83 Jozo Vela, Hrvatski za-infinitiv: izvanjsko posuđivanje ili unutarnji jezični razvoj FLUMINENSIA, god. 31 (2019), br. 1, str. 61-83 75 benefactive allative →purposive →irrealis- directive →irrealis- potential →realis non factive →(realis factive) causal Haspelmath razlikuje četiri stupnja modalnosti infinitiva kao dopune glagolima. Uz kauzativne (npr. 5. Razvoj infinitiva ‘narediti’) i deziderativne glagole (npr. ‘željeti’) infinitiv ostvaruje značenje irealno-direktivne modalnosti (irrealis- directive), odnosno infinitivom se iskazuje radnja koja je irealna u smislu da nije ostvarena, ali se njeno ostvarenje očekuje u budućnosti. Ovaj tip modalnosti odgovara onom što M. Ivić (Ivić 1970) naziva obilježjem ekspektativnosti. Irealno-potencijalnu modalnost (irrealis-potential) ostvaruje infinitiv kad je dopuna modalnim glagolima poput ‘moći’, ‘trebati’ i sl., a ima značenje radnje koja nije ostvarena, niti se očekuje da bude ostvarena, nego se njezino ostvarenje pokazuje kao moguće u bilo kojem trenutku. Realno-ne-faktivna (realis-non-factive) je modalnost koju infinitiv ima kad je dopuna glagolima mišljenja (smatrati, vjerovati) ili govorenja (govoriti, navoditi), ali u situacijama kada vršitelj nema saznanja o istinitosti onog što govori ili misli. Konačno, realno-faktivna modalnost (realis-factive) karakterizira infinitiv kao dopunu glagolima spoznavanja i vrednovanja. Ona se ostvaruje u slučajevima kad je vršitelj siguran u istinitost sadržaja vlastite propozicije. Ova modalna značenja, kako Haspelmath primjećuje, drastično su različita od značenja namjene/namjere i pokazuju progresivno slabljenje izvornog konkretnog značenja infinitiva. Dok je kod irealno-direktivne modalnosti značenje namjene/namjere još uvijek prisutno, u realno- faktivnoj modalnosti ono je potpuno izblijedilo. Kako je razvoj infinitiva od toga da izražava namjeru do drugih njegovih funkcija u jeziku rezultat procesa gramatikalizacije, on pokazuje i druga važna obilježja procesa gramatikalizacije. Prvo takvo obilježje jest da je proces jednosmjeran, odnosno da se ne može obrnuti. Drugo obilježje proizlazi kao posljedica prvoga: s obzirom na to da je proces gramatikalizacije jedno smjeran, gramatikalizirani oblici tijekom vremena imaju tendenciju osnaživanja svog izvornog značenja. Budući da dolazi do znatnog proširenja značenja i čestotnosti upotrebe gramatičkog oblika, on više nije dovoljno snažan izraziti svoje originalno značenje te mu se stoga pridružuje drugi Jozo Vela, Hrvatski za-infinitiv: izvanjsko posuđivanje ili unutarnji jezični razvoj FLUMINENSIA, god. 31 (2019), br. 1, str. 61-83 76 jezični element sposoban osnažiti to značenje. U povijesti njemačkog jezika ovo se osnaživanje infinitiva dogodilo dvaput. Najprije je infinitivu dodano zi/zu, a zatim je, kad se zu-infinitiv proširio i na druge upotrebe infinitiva, namjerno značenje infinitiva ponovno osnaženo dodavanjem um. U suvre menom njemačkom jeziku, Haspelmath zaključuje, zu-infinitiv više ne može biti upotrijebljen za izražavanje namjene/namjere nego mu je potrebno dodati um (Er ging nach Amerika, um Arbeit zu finden ≠ Er ging nach Amerika, Arbeit zu finden). U hrvatskom jeziku infinitiv je, čini se, ostvario samo prva dva stupnja gramatikalizacije i desemantizacije od izvorno namjernog značenja: irealno- direktivnu i irealno-potencijalnu modalnost. 14 Infinitivne dopune koje se u suvremenom hrvatskom jeziku pojavljuju uz ograničen broj glagola opažanja pripisuju se stranom utjecaju. (Mihaljević 2009). Produktom stranog utjecaja smatraju se i infinitivne dopune uz glagole govorenja i mišljenja u (starijoj) hrvatskoj književnosti. (Hudečak 2001, Hudeček 2003). Glagoli opažanja, mišljenja i govorenja ovisno o stupnju evidencijalnosti mogu se svrstati u Haspelmathove kategorije realno-ne-faktivne i realno-faktivne modalnosti. 5. Razvoj infinitiva Upotrebu za izražavanje različitih vrsta realne modalnosti infinitiv nije dosegao, izuzev u slučajevima kalkiranja u konstrukciji koja se naziva akuzativ s infinitivom.14 Ipak, proširenost upotrebe i značenja infinitiva bila je čini se i u hrvatskom jeziku dovoljna da se javi potreba za osnaženjem izvorno namjernog značenja infinitiva, i u tu svrhu upotrijebljena je, isto kao u njemačkom jeziku zu, riječ za koja je izvorno prijedlog. Prijedlog za, koji je u hrvatskom jeziku prilično značenjski raspršen te dolazi čak s tri padeža15, gramatikalizirao se dakle proširivši svoju funkciju te je u tom procesu dobio novu gramatičku odrednicu, od prijedloga postavši česticom.16 16 V. Smailagić (2006: 114) također predlaže da se riječ za uz infinitiv u bosanskom jeziku smatra infinitivnom partikulom. 15 Što ga čini idealnim kandidatom za proces gramatikalizacije. 6. Osnaživanje značenja namjere Ponuđeno objašnjenje kako je u hrvatskom jeziku moglo doći do upotrebe za-infinitiva, umjesto prijašnjeg jednostavnog infinitiva ne isključuje mogućnost da su talijanski per + infinitiv i njemački zu + infinitiv odigrali određenu ulogu u oblikovanju hrvatskog za-infinitiva. Suvremene studije o posuđivanju među jezicima u kontaktu uglavnom naglašavaju kako se posuđivanje često odvija kada se u sustavu primatelju stvore 16 V. Smailagić (2006: 114) također predlaže da se riječ za uz infinitiv u bosanskom jeziku smatra infinitivnom partikulom. Jozo Vela, Hrvatski za-infinitiv: izvanjsko posuđivanje ili unutarnji jezični razvoj FLUMINENSIA, god. 31 (2019), br. 1, str. 61-83 77 određeni uvjeti za preuzimanje stranog jezičnog elementa. Tu mogućnost kod za-infinitiva svakako dakle treba uvažiti. No ona se nikako ne smije prenaglašavati. Da se doista sustav hrvatskog jezika od 15. stoljeća nadalje kretao prema tomu da je značenje namjere trebalo dodatno osnažiti pokazuju primjeri u kojima se ista riječ za upotrebljava za pojačavanje značenja namjere kod finitnih rečenica s veznikom da. Kako je veznik da imao znatno raširenu upotrebu i kod finitnih rečenica javila se potreba za osnaživanjem značenja namjere u namjernim rečenicama upotrebom za da. Kako pokazuju primjeri iz literature, u ranijoj hrvatskoj književnosti nisu bile nimalo neobične rečenice tipa Obišli smo mora i polje za da bude ljudem bolje (Marulić)17. Namjerna rečenica uvedena veznikom za da dobro je potvrđena u starijoj hrvatskoj književnosti i starijem hrvatskom jeziku. Nalazimo je u Kašićevu i Škarićevu prijevodu Svetog pisma, u čakavskih pisaca (Marulić, Lucić, Bijanković), u dalmatinskih štokavskih pisaca 18. st. (Banovac, Kadčić, Matić), u dubrovačkim oporukama i različitim arhivskim spisima 17. i 18. stoljeća. (Vrtič 2009: 394). Danas složeni veznik za da više nije u upotrebi, no zato se koristi složeni veznik zato da kad god je govornik hrvatskoga jezika u situaciji da treba dodatno osnažiti značenje namjere kod finitne rečenice s veznikom da. Promotrimo li pobliže takve situacije, postaje nam jasno kako je došlo do potrebe da se za upotrijebi uz infinitiv i uz finitne rečenice s veznikom da. Suvremeni će govornik hrvatskoga, naime, upotrijebiti samo veznik da u situacijama kad je značenje namjere finitne da rečenice samorazumljivo (npr. Išao je prečacem da dođe što prije.). Upotrijebi li se, međutim, glagol kod kojeg namjerna dopuna nije najčešća i samorazumljiva kao što je glagol pisati (npr. Pisala mu je da dođe što prije.) značenje će namjere, ukoliko se upravo ono želi izraziti, trebati dodatno osnažiti upotrebom zato da (npr. 17 Primjer preuzet iz Zima 1887: 306. 6. Osnaživanje značenja namjere Pisala mu je zato da dođe što prije). Prilog zato suvremeni će govornik hrvatskog jezika upotrijebiti kad god iz rečenice kojoj je zavisna namjerna rečenica s veznikom da podređena nije dovoljno jasno kako da koje slijedi ima namjerno značenje. Za razliku od slučajeva u kojima je namjerno značenje da-rečenice očekivano (npr. uz glagole kretanja), uz glagole npr. mišljenja i govorenja da-rečenica neće biti namjerna nego izrična, ako ju se dodatno ne osnaži upotrebom priloga zato. Jozo Vela, Hrvatski za-infinitiv: izvanjsko posuđivanje ili unutarnji jezični razvoj FLUMINENSIA, god. 31 (2019), br. 1, str. 61-83 78 Potreba osnaživanja značenja namjere nije ograničena na njemački, talijanski i hrvatski jezik, nego je prisutna je u drugim europskim jezicima (franc. pour + inf.; rus. čtoby + inf., dlja togo čtoby + inf.; staroeng. to + inf., engl. in order to + inf.; nizoz. om te + inf.; mak. (za) da + prez.; bug. za da + prez.; novogrč. γιά να < διὰ ἵνα + konj.), pa nije promakla opažanju jezikoslovaca. Prvi je, čini se, o tomu pisao 1888. godine A. Potebnja (1958: 338–354), a na njegova se promišljanja naslanja i I. Grickat (1975: 84–116). Haspelmath (1989: 304–308) to također primjećuje te zaključuje: „the grammaticalization from purposive meaning to realis modality complement functions (...) is an even more general phanomenon, of which the evolution of the infinitive is just a special case”. Za donošenje pouzdanijeg zaključka o za-infinitivu u hrvatskom jeziku valjalo bi u obzir uzeti veći korpus tekstova, kako povijesnih, tako i suvre menih, te vidjeti u kojim se funkcionalnostilskim i sintaktičkim kontekstima on pojavljuje.18 Uzmu li se u obzir primjeri iz navedene literature posvećene ovom pitanju, stječe se dojam da za-infinitiv u suvremenom jeziku više nije toliko u upotrebi u značenju namjere – što je ranije bio slučaj, čak i uz glagole kretanja – nego se prvenstveno koristi za izražavanje prototipnog značenja namjene ili značenja semantički blisko povezanog s namjerom, a to je značenje moguće, neostvarene posljedice19. Mogući uzmak za-infinitiva namjere, ukoliko je do njega zaista došlo, mogao bi se objasniti širenjem finitne namjerne da-rečenice, no moguće da je njegovu potiskivanju prido nijela i dugotrajna politika jezikoslovnog purizma, koja je upravo inzistirala na njegovu zamjenjivanju finitnom da rečenicom, što je kod za-infinitiva namjere lakše provedivo nego kod struktura gdje je riječ o značenju namjene ili moguće posljedice. Ipak, to je tema koja nadilazi ovaj rad. 18 Zahvaljujem anonimnom recenzentu ovog rada na ovoj opravdanoj primjedbi. 19 Usp. Vukojević 2008. 19 Usp. Vukojević 2008. 7. Zaključak Za-infinitiv obično se smatra sintaktičkim kalkom prema njem. (um) zu + inf., ili tal. per + infinitiv, a njegova se upotreba smatra negrama tičnom, jer se za shvaća prijedlogom. U ovom radu nastojao sam pokazati na koji se način za-infinitiv mogao razviti u hrvatskom jeziku, a to je tendencija razvoja koju dijele brojni europski jezici, ne samo oni u hrvatskom neposrednom okruženju. Promatramo li hrvatski jezik u ovom pogledu u Jozo Vela, Hrvatski za-infinitiv: izvanjsko posuđivanje ili unutarnji jezični razvoj FLUMINENSIA, god. 31 (2019), br. 1, str. 61-83 79 kontekstu drugih europskih jezika, najbolje bi, čini se, bilo govoriti o arealnim tendencijama razvoja kojih je hrvatski jezik dio. Kad je riječ o formalnom opisu za-infinitiva u hrvatskom jeziku, naziv prijedložni infinitiv trebalo bi odbaciti, jer nema dostatnih razloga da se za smatra prijedlogom, odnosno da se smatra kako je sveza za + infinitiv negramatična. Za uz infinitiv je čestica koja pojačava njegovo prototipno značenje, a za-infinitiv bi trebalo uvrstiti u formalni opis hrvatskog jezika, ne samo zato što pokazuje da je u živoj i plodnoj upotrebi nego i zato što nije riječ ni o kakvoj pojavi koja odudara od sustava hrvatskog jezika, odnosno može se objasniti kao produkt njegova (i) unutarnjeg razvoja. 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Silić, Josip, Ivo Pranjković (2005) Gramatika hrvatskoga jezika za gimnazije i visoka učilišta, Školska knjiga, Zagreb. Smailagić, Vedad (2006) „Konstrukcije za + infinitiv u bosanskom jeziku”, Pismo, 4, 1, 107–115. Szemerényi, Oswald J. L. (41996) Introduction to Indo-European Linguistics, Clarendon Press, Oxford. Težak, Stjepko, Stjepan Babić (122000) Gramatika hrvatskoga jezika – priručnik za osnovno jezično obrazovanje, Školska knjiga, Zagreb. Vela, Jozo (2018) Sintaksa infinitiva i supina u hrvatskome crkvenoslavenskome jeziku, doktorska disertacija, Filozofski fakultet u Rijeci. Vrtič, Ivana (2009) Sintaksa Kašićeva prijevoda Svetoga pisma, doktorska disertacija, Filozofski fakultet u Zagrebu. Vukojević, Luka (2008) „Infinitivne posljedične konstrukcije”, Rasprave Instituta za hrvatski jezik i jezikoslovlje, 34, 449–462. Zakar, Viktor (2016) „Supin vo dolnolužičkosrpskiot jazik”, Croatica et Slavica Iadertina, 12, 2, 529–540. Zima, Luka (1887) Njekoje, većinom sintaktičke razlike između čakavštine, kajkavštine i štokavštine, Djela JAZU, 7. Županović, Nada (2008) „Analiza talijanizama u Hvarkinji Martina Bene tovića”, Fluminensia 20, 1, 33–53. Jozo Vela, Hrvatski za-infinitiv: izvanjsko posuđivanje ili unutarnji jezični razvoj FLUMINENSIA, god. 31 (2019), br. 1, str. 61-83 Jozo Vela, Hrvatski za-infinitiv: izvanjsko posuđivanje ili unutarnji jezični razvoj FLUMINENSIA, god. 31 (2019), br. 1, str. 61-83 83 SUMMARY Jozo Vela CROATIAN ZA-INFINITIVE: EXTERNAL BORROWING VS. INTERNAL LANGUAGE CHANGE The use of the infinitive in sentences such as Imate li sobu za iznajmiti? in the modern Croatian language is usually called “prepositional infinitive” or “za + infinitive construction”. Infinitive used with za is considered to be ungrammatical and a loan from other languages, namely German and/or Italian. It is therefore recommended to avoid using such an expression in a finer literary discourse. i This paper proposes a different approach and points out that the za-infinitive might be an issue of internal language change, as has already been indicated by Pranjković. Furthermore, it shows that the Croatian za-infinitive follows the universal path of semantic and formal grammaticalization of the infinitive, as outlined by Haspelmath. Such a claim is supported by evidence from older texts in the Croatian language, as well as by certain comparisons with other European languages. Consequently, a new formal description of the infinitive use in question is proposed. Key words: Croatian language; infinitive; prepositional infinitive; za + infinitive; language change; grammaticalization; loan
https://openalex.org/W2889594068	https://www.intechopen.com/citation-pdf-url/58145	English	null	A Value Chain Analysis for Bioenergy Production from Biomass and Biodegradable Waste: A Case Study in Northern Norway	InTech eBooks	2,018	cc-by	10,120	Selection of our books indexed in the Book Citation Index in Web of Science™ Core Collection (BKCI) Interested in publishing with us? Contact book.department@intechopen.com Numbers displayed above are based on latest data collected. For more information visit www.intechopen.com Open access books available Countries delivered to Contributors from top 500 universities International authors and editors Our authors are among the most cited scientists Downloads We are IntechOpen, the world’s leading publisher of Open Access books Built by scientists, for scientists 14% 191,000 210M TOP 1% 154 7,200 Chapter 11 Abstract During the past decades, the concerns of the depletion of fossil fuels and global warming caused by excess GHG emissions have become the most important driving force for the development and utilization of renewable energy resources. The successful experiences from the EU-28 have proved that bioenergy production from biomass and biodegradable waste is the most reliable and promising solution in today’s renewable energy market. This chapter presents a general model for value chain analysis of bioenergy production from biomass and biodegradable waste. In addition, a feasibility study for establishing a bioenergy plant in the northern part of Norway is given to discuss the opportunities and challenges of bioenergy production. The feedstock of the planned bioenergy plant is from local agriculture, waste management sector, fishery and livestock industry. Value chain analysis is used to balance the economic and environmental influences of the bio- energy production in the area. Furthermore, suggestions for resolving the challenges and minimizing the potential risks of bioenergy production are also discussed in this chapter. Keywords: value chain analysis, biodegradable waste, biomass, bioenergy, energy Keywords: value chain analysis, biodegradable waste, biomass, bioenergy, energy production Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.72346 1. Introduction Bioenergy production from biomass and biodegradable waste has received increasing focus due to recent acceleration in depletion of fossil fuels. The portion of renewable energies counted only 11% of total energy consumption while 74% is fossil energy in EU-28 in 2012 (Figure 1(a)) [1]. The heavy dependency on fossil fuels has resulted in two critical issues. First, fossil fuel is non-renewable and cannot be replenished by nature within a reasonable © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 0 6 e ut o (s) ce see ec s c apte s d st buted u de t e te s o t e C eat e Co o s ttribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, nd reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, di ib i d d i i di id d h i i l k i l i d Energy Systems and Environment 84 Figure 1. (a) Gross inland energy consumption by source of the EU-28 in 2012 and (b) renewable energy consumption by source of the EU-28 in 2012 [1]. Figure 1. (a) Gross inland energy consumption by source of the EU-28 in 2012 and (b) renewable energy consumption by source of the EU-28 in 2012 [1]. timeframe. Moreover, the over-exploration and exponentially increasing consumption in recent years accelerate the depletion of fossil fuels. The estimated lifespan for the reserves of crude oil, natural gas and coal is approximately 35, 37 and 107 years, respectively [2]. The development of renewable energy resources becomes therefore of significant importance to meet energy demand in the near future. Second, the greenhouse gas (GHG) emission related to energy production and consumption from fossil fuels has played an important role to the global warming and climate change [3]. Both of them are believed to be the most significant challenges in the twenty-first century, which may lead to severe consequences for human’s existence [4]. Due to these reasons, extensive efforts have been devoted in reducing GHG emissions in the past few decades. One of the most promising solutions to the abovemen- tioned challenges is the explosion of renewable resources, i.e. wind, solar, tidal, biomass, etc., which not only provide an attractive alternative for energy production but also contribute to the mitigation of GHG emissions. Bioenergy production from biomass and biodegradable waste is the most reliable renewable energy resource, which occupies predominant share of today’s marketplace [5]. As shown in Figure 1(b), the consumption of energy generated by biomass and biodegradable waste, liquid biomass, hydropower and wind power are 58.3, 8.5, 15.6 and 9.6%, respectively, and the other renewable resources including solar thermal, solar photovoltaic and geothermal constitute only 8% of the total consumption [1]. The reason for this high portion of bioenergy production in Europe is mainly due to the long-term efforts on developing legislative mechanism and tech- nological means for recovering energy from biomass and biodegradable waste. For example, EU Landfill Directive (Council Directive 1999/31/EC [6]) implemented in 1999 sets the periodic target for the member states, and since then the amount of the biodegradable waste ended up in landfill has been dramatically reduced. EU Renewable Directive (Directive 2001/77/EC [7]) was implemented in 2001 and repealed in 2009 (Directive 2009/28/EC [8]) for promoting more appli- cations of renewable energy resources. This has been followed up by Norwegian authority with A Value Chain Analysis for Bioenergy Production from Biomass and Biodegradable Waste… http://dx.doi.org/10.5772/intechopen.72346 185 185 a White Paper on Norwegian climate policies [9]. The White Paper documented particularly the large emissions of GHG from agriculture industry and waste management in Norway, and a specific goal is also stated to develop more bioenergy production plants in Norway. A joint treatment of biodegradable waste from both households and agriculture sections is emphasized. As shown in Figure 2, only 35% of biomass and biodegradable waste are utilized in bioen- ergy production, and this will lead to a significant increase at 2.3 TWh (governmental target). Currently, the bioenergy production from biomass and biodegradable waste in Norway is also relatively small (0.5 TWh [10]). This can partly be explained by existing large energy production from other renewable resources, i.e. hydropower [11], which leads to fairly low energy price in general. Also, the limited infrastructure for bioenergy production and high investment are the main obstacles to an increased bioenergy production in Norway. The political willingness is to change the current situation of bioenergy production and establish more bioenergy plants in Norway. However, the governmental subsides and economic incentives for promoting bioen- ergy production have not been well established yet. In addition, some other institutional and regulative mechanisms should also be considered, i.e. competence enhancement, tax relief for transport utilizing biofuel, lowering gate fee for the delivery of biomass and biodegradable waste for energy production, etc. In order to provide a better understanding of the bioenergy production from biomass and biodegradable waste, a general model for value chain analysis is first formulated and dis- cussed in this chapter, and a feasibility analysis is then given to discuss the opportunities and challenges of establishing a bioenergy production plant in Northern Norway. The reminder of this chapter is structured as follows. Section 2 gives the definition and treatment methods of the feedstock of bioenergy production: biomass and biodegradable waste. Section 3 for- mulates a general value chain model of bioenergy production and performs the value chain analysis of bioenergy production. Section 4 presents a feasibility study for establishing bio- energy production plant in northern part of Norway. Section 5 summarizes the chapter and suggests for future studies. Figure 2. Potential bioenergy production in Norway within 2020 [10]. Figure 2. Potential bioenergy production in Norway within 2020 [10]. Energy Systems and Environment 186 2.1. Biomass and biodegradable waste Biomass is defined in twofold [12]. One is “the total quantity or weight in a given area or volume”, which emphasizes its essential attribute as organic degradable substances. This definition is on a biological ecological basis. Another is “organic matter used as a fuel, especially in a power station for the generation of electricity”, and this definition focuses the use of biomass for energy production. Similarly, Cambridge dictionary [13] defines biomass from both biological and engineering perspectives. From biological perspective, the biomass is defined as “The total mass of living things in a particular area”. Herein, the inherent property of biomass as “living things” is focused. From engineering perspective, the biomass is defined as “dead plant and animal mate- rials suitable for using as a fuel”, and the property of biomass as a type of fuel is emphasized. Biodegradable waste is another commonly used term to describe the feedstock of bioenergy production. Biodegradability is referred as the ability to decay naturally and non-harmfully [14]. According to Basel Convention [15], wastes are defined as “substances or objects, which are disposed of or are intended to be disposed of or are required to be disposed of by provisions of national law”. Viewing from consumers’ perspective, EU Directive 2008/98/EC [16] defines waste as “an object the holder discards, intends to discard or is required to discard”. Based on the definition above, biodegradable waste is the portion of waste that can be decayed by nature.Biomass and biodegradable waste are the most important sources for bioenergy production, which mainly come from five sectors: for- estry and timber, agriculture, fishery, waste management and wastewater treatment (Figure 3). It is noteworthy that the portion of biomass and biodegradable waste contributed by differ- ent sectors may vary dramatically from country to country, and the generation is significantly influenced by seasonality. Therefore, it is necessary to take into account of those variations and uncertainties in forecasting the amount of the feedstock for bioenergy production. The difference between the two concepts is, compared with biodegradable waste, biomass that specifies a broader domain in which not only organic matters discarded by consumers Figure 3. Sources of biomass and biodegradable waste. Figure 3. Sources of biomass and biodegradable waste. A Value Chain Analysis for Bioenergy Production from Biomass and Biodegradable Waste… http://dx.doi.org/10.5772/intechopen.72346 187 187 but also the ones obtained intentionally for bioenergy production is included (e.g. energy crops). 2.1. Biomass and biodegradable waste However, those two terms can sometimes be interchangeable when the feedstock of bioenergy production mainly refers to the organic substances that have lost their usefulness value to the consumers, and this applies to the case study presented latter in this chapter. 2.2. Treatment of biomass and biodegradable waste Based on processing technologies, the treatment of biomass and biodegradable waste can be categorized into four types: direct combustion, thermochemical conversion, biochemical conversion and non-value-added treatment. The first three types are waste-to-energy (WTE) processes aiming to exploit the remaining value of biomass and biodegradable waste, and the last one usually refers to landfill at which the remaining value is eventually lost. Landfill is usually the least expensive but a non-sustainable way for biodegradable waste treatment [17], and the portion of biodegradable waste landfilled has continuously decreasing in Europe due to the rigorous and comprehensive legislations. WTE is the transformation of biomass and biodegradable waste into energy, and it initially specifies the production of power and heat through the combustion of biomass and biodegradable waste [18]. Nevertheless, its meaning has broadened to include other means of bioenergy recovery with the rapid technological development. Bioenergy has been extensively used in different industries, i.e. transport sec- tor, power generation, heating, agriculture and chemistry industry [19]. Table 1 illustrates the alternative means for bioenergy production and non-value-added treatment of biomass and biodegradable waste. 2.2.2. Thermochemical conversion Thermochemical conversion utilizes constant and high temperature combined with catalysts to convert biomass inside the boiler to biofuel and bioenergy through changing their physical prop- erties and chemical structure [24]. The main technologies of thermochemical conversion of bio- mass and biodegradable waste include pyrolysis, gasification, liquefaction and torrefaction [25], among which pyrolysis and gasification are considered the most promising ones [26]. Pyrolysis is a fundamental method to transform biomass into crude-like liquid bio-oil [27], and after the chemical decomposition, the liquid bio-oil can be converted to the combustion fuels mainly used for transport and chemical industry [28]. The principle of pyrolysis process is the combination of thermal and chemical decomposition with the help of catalysts at relatively lower temperature (450–600 ° C ) and longer vapor residence time in absence of oxygen for converting the organic sub- stances to liquid bio-oil with charcoal and gases as the by-products [24, 29]. Gasification is another important thermochemical technology that converts different kinds of biomass into syngas. The main composition of syngas is methane, hydrogen, carbon dioxide and carbon monoxide, which are extensively applied in space heating, power generation, transport and chemical industry [30]. Different from pyrolysis, gasification process requires relatively higher temperature (700–1300°C) with the absence or limited oxygen environment in order to optimize the production of syngas [23, 26]. Recently, with the technological development, the probability of biomass and biodegrad- able waste gasification at lower temperature has also been discussed (e.g. [31]). 2.2.1. Direct combustion Direct combustion has been widely used for over three decades in generating electricity and heat from biomass [20]. The principle is to utilize the heat generated by combustion of biomass for cooking, industrial process, direct home heating [21] or driving the steam power cycle for Technology Method WTE/disposal Product Market Thermochemical conversion Pyrolysis WTE Bio-oil Transport Gasification WTE Syngas Chemistry, transport Direct combustion Incineration WTE Electricity, heat Power, heating Biochemical conversion Composting WTE Fertilizer Agriculture Anaerobic digestion WTE Biogas, fertilizer Transport, agriculture MBT WTE Biogas, fertilizer Transport, agriculture Non-value-added treatment Landfill Disposal N/A N/A Table 1. Alternative technologies for bioenergy production and non-value-added treatment of biomass and biodegradable waste. Table 1. Alternative technologies for bioenergy production and non-value-added treatment of biomass and biodegradable waste. Energy Systems and Environment 188 electricity generation [22]. Due to the high level of moisture content, combustion promoters are usually used in order to improve the conversion efficiency. Coal is the most frequently used promoter, and co-combustion of biomass/biodegradable waste with coal has dominated the bioenergy production market in some countries, i.e. Sweden, Japan, etc. However, envi- ronmental challenges, i.e. emission of CO2, SOx and NOx, from the co-combustion process are the major bottleneck for this method for bioenergy recovery [23]. Furthermore, as the common challenge of incineration plant, the flying ash is another pollutant that needs lots of efforts and costs to deal with so that the environmental influence is minimized. 2.2.3. Biochemical conversion Biochemical conversion utilizes biological and chemical processes with the help of aerobic or anaerobic microorganism to transform biomass into biogas and bio-rest, and the main biochem- ical technologies are composting, anaerobic digestion and mechanical biological treatment (MBT). Composting is an aerobic digestion process and a popular method for the treatment of biomass and biodegradable waste (e.g. Finland). The basic principle is to use biochemi- cal process with the help of aerobic microorganism under open air environment for convert- ing biomass into environmentally friendly bio-rest, which can be used as fertilizer. Anaerobic digestion is the most popular biochemical technology for bioenergy production, and thousands of anaerobic digestion bioenergy production plants have been established all over the world [32]. Through the biochemical decomposition process with the help of anaerobic bacteria at constant temperature in the absence of oxygen, the biomass can be transformed into not only bio-rest but also energy-rich biogas [33]. Biogas is mainly comprised by methane (60%) and carbon dioxide (40%), and it is mainly used as vehicle fuels after cleaned and upgraded [34]. In A Value Chain Analysis for Bioenergy Production from Biomass and Biodegradable Waste… http://dx.doi.org/10.5772/intechopen.72346 1 189 some cases, aerobic digestion and anaerobic digestion are combined for the treatment of bio- degradable waste, e.g. wastewater sludge [35]. MBT combines mechanical pre-treatment and anaerobic digestion. The pre-treatment of biomass through different physical and mechanical processes breaks the physical structure and improves the quality of the input organic sub- stances for anaerobic digestion [36], and the efficiency of bioenergy production is improved as well. Comparing with aerobic composting, both anaerobic digestion and MBT processes have much higher requirement for creating thermostatic and anaerobic environment. 3.1. A value chain model for bioenergy production The concept of value chain was originally proposed by Porter from financial perspective to account the sequential value creation and appreciation through the whole network com- prised by different companies and enterprises [37]. This concept is usually accompanied with another word with similar meaning: supply chain (i.e. in [38, 39]). The difference between those two concepts is sometimes negligible especially when the value creation and apprecia- tion process over the material flow are predominately accounted. However, a recent study by Holweg and Helo [40] has explicitly distinguished the two concepts from the perspective of their focuses. Supply chain management focuses on the links and interactions among different companies from the operational level considering strategies, methodologies, design, planning and operation of an efficient and effective multi-stakeholder inner- and/or inter-company net- work. However, value chain mainly concerns the value-added activities from one company to another within the network and the opportunities and challenges for maximizing the overall value creation and appreciation through the whole network. The value chain of bioenergy production from biomass and biodegradable waste has been extensively modelled in the literature. Balaman and Selim [41] proposed a biomass-to-energy value chain model and a decision support tool for maximizing the overall profit generated through bioenergy production. A simplified value chain model is developed by Parker et al. [42], and the primary target of the model is to improve the economic value of biofuel produc- tion from biomass. An et al. [43] formulated a computational model to optimize the overall profit of a lignocellulosic biofuel value-added chain. Kim et al. [44] developed a four-echelon value chain framework for biofuel production through fast pyrolysis conversion. The maxi- mization of the overall value creation from bioenergy production is focused by Kim et al. [45] and Dal Mas et al. [46]. The maximization of the value creation is sometimes formulated in an opposite way that minimizes the system cost. Chen and Fan [47] formulated a bioethanol production value chain model that applies mixed integer programming for minimizing the overall system cost. Aksoy et al. [48] developed an optimization model for minimizing the transportation cost of bioenergy production from woody biomass and mill waste. The envi- ronmental benefits of bioenergy production have been increasingly focused in recent years. A value-added chain of bioenergy production from biomass is modelled by Lam et al. [49], which focuses on the mitigation of carbon footprint of bioenergy production. 3.1. A value chain model for bioenergy production Energy Systems and Environment 190 Figure 4. A general value chain model of bioenergy-from-biomass and biodegradable waste. Figure 4. A general value chain model of bioenergy-from-biomass and biodegradable waste. Bioenergy production from biomass yields economic benefits while reduces waste. However, it is not focused in the literature to account both economic and environmental benefits in the value chain of bioenergy production. In order to fill the literature gap, a general value chain model of bioenergy production from biomass and biodegradable waste is given in Figure 4. A typical value-added process of bioenergy production consists of the following activities: • Harvesting and collection of biomass and biodegradable waste from different sectors: The main feedstock of bioenergy production includes agricultural residues, forestry residues, unban woody residues, fishery residues, slaughter wastes, animal manure, biodegradable munici- pal wastes and wastewater sludge, which are usually collected by different companies and/ or public service departments. • Intermediate storage and distribution of biomass and biodegradable waste: Road transport is the most flexible and commonly used way for the distribution of biomass and biodegradable waste; however, other means, i.e., train and ship, are also applicable especially for large amount of biomass and biodegradable waste transported over very long distance due to their relatively low costs. • Bioenergy production or proper disposal of biomass and biodegradable waste: It is the most im- portant value creation process that transforms the “raw materials” into “semi-finished • Bioenergy production or proper disposal of biomass and biodegradable waste: It is the most im- portant value creation process that transforms the “raw materials” into “semi-finished A Value Chain Analysis for Bioenergy Production from Biomass and Biodegradable Waste… http://dx.doi.org/10.5772/intechopen.72346 191 191 product”. The main technologies are gasification, pyrolysis, aerobic composting, anaerobic digestion, direct combustion and landfill. • Purification and upgrade of the generated bioenergy: This step is a critical value-added process that converts the “semi-finished product” into “finished product”. The biogas and bio-fuel produced from previous step cannot be directly sent to market due to their complex chemi- cal composition and low efficiency, so thermal and chemical decomposition, purification and upgrade are necessary in this phase. Besides, the model aims at maximizing the value creation of bioenergy production, so the utilization of landfill gas is taken into account in this value chain model. 3.1. A value chain model for bioenergy production • Distribution and sales of the bioenergy as well as other by-products in the market: This is the final step of the value chain of bioenergy production, where the value creation from the bio- energy production is eventually realized. The biogas, bio-fuel and bio-rest can be used in many different sectors including transport, aviation industry, chemical industry, agricul- ture, power generation and space heating. 3.2. Value chain analysis of bioenergy production Value chain analysis has been widely used for investigating, through qualitative and/or quan- titative methods, the value-added process in many different fields, i.e. mining industry, fish- ery industry, aviation industry, dairy industry, catering, production and manufacturing, etc. Conventionally, value chain analysis only emphasizes the value creation and appreciation from financial point of view. However, the increased concern on environmental challenges has led to much more focuses on the “green value-added process” in which not only eco- nomic value creation but also environmental value contribution through the entire material flow is accounted (e.g. in [50]). Bioenergy production is a value-added process from both economic and environmental perspectives, so value chain analysis is a reasonable basis for regarding pro et contra for bioenergy production. The value chain of bioenergy production comprises all joints in the flow from materials to products, and it can be analysed in such a way that all important joints are balanced out of a combination of economic and sustainable aspects all the way from cradle to grave. Value chain analysis of bioenergy production provides decision makers with fundamental basis to divert biomass and biodegradable waste from landfill to WTE process. Previously, the value of biomass and biodegradable waste does not get enough attention, and a large portion is treated through non-value-added method that leads to great potential environmental prob- lems. The model streamlines the value creation and appreciation of bioenergy production from biomass and biodegradable waste, and both economic advantages and environmental benefits are discussed. Bioenergy production takes a different point from waste management perspective to consider biomass as the “raw material” of the value chain and realize the trans- formation from “waste” to “financial and environmental value”. The utilization of biofuel and biogas can dramatically decrease the high dependency on fossil fuels and improve the energy security of a country; further, the nature of self-replenishment of biomass and biodegradable waste makes them become one of the most important renewable energy resources. Besides, Energy Systems and Environment 2 192 Decision level Decisions Strategic decision • Selection of WTE and treatment technologies • Selection of network configuration: location, capacity, etc. • Selection of potential suppliers and markets Tactical decision • Selection of equipment at different facilities • Aggregate production planning • Policy making for supplier and customer management Operational decision • Execution of the policies made in previous step • Scheduling and route planning Table 2. 3.2. Value chain analysis of bioenergy production Some strategic, tactical and operational decisions in the planning of a value chain for bioenergy production. Table 2. Some strategic, tactical and operational decisions in the planning of a value chain for bioenergy production. ble 2. Some strategic, tactical and operational decisions in the planning of a value chain for bioenergy producti the GHG and hazardous gas emission can be reduced by the utilization of biofuel and biogas as the substitutes of fossil fuels in land transport and aviation industry [51]. The realization of an effective and efficient value-added chain of bioenergy production from biomass and biodegradable waste requires sophisticated decision tools for planning and developing an optimal and robust logistical network, and several strategic, tactical and opera- tional decisions that have to be made are summarized in Table 2. In order to provide reliable support for decision-making, great efforts should be spent in the development of theoretical and computational models and decision support systems. Besides, the inherent characteristic of the seasonal availability of biomass and biodegradable waste generation makes the predic- tion of the feedstock of bioenergy production becoming extremely complicated. Therefore, the aforementioned factors have become the most challenging obstacles for realizing the value-added process of bioenergy production, and inappropriate decisions will hinder the achievement of maximum value creation and appreciation. 4.1. Bioenergy production and bioenergy plants in Norway The most significant energy consumption in Norway is electricity, which constitutes 88.8% of the total energy consumption in 2012, and it is approximately 17 times higher than the second largest one: petroleum products [52]. The main reason for the high dependency on electri- cal power is the lower price than other types of energy resources due to the rich reserves of hydropower for electricity generation. Hydroelectric power once contributed more than 99% electricity production in Norway [53], and the situation has not been changed until the latest years when energy production from biomass and biodegradable waste takes a small share from hydroelectric power. A Value Chain Analysis for Bioenergy Production from Biomass and Biodegradable Waste… http://dx.doi.org/10.5772/intechopen.72346 193 193 All the Scandinavian countries, such as Denmark, Sweden, Norway and Finland, support the use of renewable energy resources for power generation and space heating, among which Norway has expelled itself as one of the best countries in Europe for renewable energy gen- eration and consumption (58%) due to the high contribution from hydroelectric power [54]. However, the contribution from bioenergy production is extremely insignificant. Furthermore, compared with other Scandinavian countries where bioenergy has already played an impor- tant role in power generation, the share of electricity production by biomass and biodegrad- able waste in Norway is much smaller as shown in Figure 5. All the Scandinavian countries, such as Denmark, Sweden, Norway and Finland, support the use of renewable energy resources for power generation and space heating, among which Norway has expelled itself as one of the best countries in Europe for renewable energy gen- eration and consumption (58%) due to the high contribution from hydroelectric power [54]. However, the contribution from bioenergy production is extremely insignificant. Furthermore, compared with other Scandinavian countries where bioenergy has already played an impor- tant role in power generation, the share of electricity production by biomass and biodegrad- able waste in Norway is much smaller as shown in Figure 5. The government in Norway has made an ambitious strategic plan for dramatically increasing the bioenergy production by 2020 through policy measures and financial supports [55]. For example, waste regulation has been implemented in Norway since 2009 implementing a ban, which specifies alternative ways for the treatment of biodegradable waste other than land- fill. 4.1. Bioenergy production and bioenergy plants in Norway Besides, the forestry and agricultural legislation promote sustainable economic and envi- ronmental development in forest management and agriculture industry [53], so bioenergy production from forest and agricultural residues is encouraged. Further, the use of biofuels in land transport to replace fossil fuels is also encouraged in Norway. The road tax charges carbon emission for the vehicles using petroleum and natural gas products, but the cars using biofuels or biogas as the main power are exempt from this charge. In addition, plans for increasing the use of bioenergy for space heating of public and commercial buildings are also under development in order to reduce the fossil fuel consumption for heating. In Norway, bioenergy production has two characteristics. First, compared with electricity generation, the use of biofuels and biogas in transport sector seems more attractive, because it decreases both the high dependency on fossil fuels and GHG emissions. Besides, Norway Figure 5. Power generation by sources in Scandinavian countries [54]. Figure 5. Power generation by sources in Scandinavian countries [54]. Energy Systems and Environment 194 implemented the report of biofuels’ usage in transport sector from 2014, and it has become one of the most important criteria to assess sustainability in transport sector. Therefore, ther- mochemical technologies, anaerobic digestion and MBT are widely used methods in Norway for biogas and biofuel production. Figure 6 illustrates the established biogas and biofuel production plants as well as the demo- graphic distribution, forestry and agricultural areas and road transport network in Norway. Currently, all the established bioenergy production plants are geographically located around the largest cities in the southern and central parts of Norway, and the northernmost bioen- ergy production plant is located at Verdal (North Trøndelag County). The established biogas and biofuel production plants in Norway have maintained well economic performance and contributed to the mitigation of GHG emissions. The critical success factors for bioenergy production in this region are summarized as follows: • Dense population, agricultural and industrial clusters provide enough feedstock of bio- mass and biodegradable waste for bioenergy production. • Dense population, agricultural and industrial clusters provide enough feedstock of bio- mass and biodegradable waste for bioenergy production. • Well-developed road transport network provides easy access to the collection and distribu- tion of biomass and biodegradable waste. • Well-developed road transport network provides easy access to the collection and distribu- tion of biomass and biodegradable waste. 4.1. Bioenergy production and bioenergy plants in Norway • Short distance between the bioenergy plants and collection points of biomass and biode- gradable waste reduces the transportation and logistics cost. • Short distance between the bioenergy plants and collection points of biomass and biode- gradable waste reduces the transportation and logistics cost. • Governmental support for developing biogas and biofuel market, i.e. biogas used as the main fuel for the public transport in Fredrikstad, tax relief for biogas and biofuel in trans- port sector [53]. • Incentives for bioenergy production from biomass and biodegradable waste. Figure 6. (a) Established biogas and biofuel production plants in Norway; (b) demographic distribution of southern and central parts of Norway; (c) forestry and agricultural industry in southern and central parts of Norway; and (d) road transport network of southern and central parts of Norway [11, 52]. Figure 6. (a) Established biogas and biofuel production plants in Norway; (b) demographic distribution of southern and central parts of Norway; (c) forestry and agricultural industry in southern and central parts of Norway; and (d) road transport network of southern and central parts of Norway [11, 52]. A Value Chain Analysis for Bioenergy Production from Biomass and Biodegradable Waste… http://dx.doi.org/10.5772/intechopen.72346 195 195 The other characteristic of bioenergy production in Norway is the feedstock mainly comes from forestry/wood industry and waste management. Figure 7 illustrates the sources of bio- mass and biodegradable waste for bioenergy production in Nordic countries. As shown in the figure, black liquor from chemical industry and wood residues from forestry industry are the most important resources for bioenergy production in Sweden and Finland, while the feed- stock for bioenergy production in Norway and Denmark is mainly from waste management sector and forestry industry. Utilization of wood and forestry residues has been well-devel- oped in Norway, and previous studies have discussed the policy structure [56], impact [57] and future potential [58] of bioenergy production from forestry biomass and waste. Besides, it is also noteworthy that energy crops, i.e. poplar, reed canary grass, willow, etc. [59], are not commercially cultivated in Norway, and the portion of bioenergy production from energy crops in the other Scandinavian countries is small as well. The main reason is that the long winter and cold climate in Scandinavian countries make it becoming economically unafford- able for cultivation of energy crops for bioenergy production in this area. 4.2. A feasibility study of bioenergy production in Nordland county Bioenergy production in Northern Norway has been focused for many years. However, the negotiation between different stakeholders has been difficult reaching an implementation plan due to the conflicting interests involved. Earlier, the locations of landfill and composting plant were focused due to the consideration of the costs for waste collection and environmen- tal risks of treatment facilities of biodegradable waste. Recently, the treatment plants have been equipped with closable ports, ventilation system, air cleaning system as well as other upgrades, which tremendously reduce the negative impact on the environment, and the focus on the energy recovery of biodegradable waste has been increasingly discussed. Figure 7. Bioenergy production by sources in Nordic countries [54]. Figure 7. Bioenergy production by sources in Nordic countries [54]. Energy Systems and Environment 196 In order to promote bioenergy production from biomass and biodegradable waste, Nordland County has drafted an initial plan for establishing a bioenergy plant at the costal-town Leknes. Leknes is the geographic centre of the famous Lofoten archipelago, and the close proximity to local agricultural areas, fishery and livestock industry is the main reason for the strategic decision of plant location. Besides, Leknes also has long-term experience in aerobic technologies, and the biomass and biodegradable waste generated at this region are treated at the composting plant. The upgrade of the aerobic composting plant to a MBT plant for producing biogas has been dis- cussed for several years, and the strongest support is from local agriculture and livestock industry. In order to promote bioenergy production from biomass and biodegradable waste, Nordland County has drafted an initial plan for establishing a bioenergy plant at the costal-town Leknes. Leknes is the geographic centre of the famous Lofoten archipelago, and the close proximity to local agricultural areas, fishery and livestock industry is the main reason for the strategic decision of plant location. Besides, Leknes also has long-term experience in aerobic technologies, and the biomass and biodegradable waste generated at this region are treated at the composting plant. The upgrade of the aerobic composting plant to a MBT plant for producing biogas has been dis- cussed for several years, and the strongest support is from local agriculture and livestock industry. Leknes is one of the largest agricultural municipalities in Nordland County, and the meat production from poultry and livestock is on a large scale. The treatment of animal’s manure is one of the most challenges in this area. 4.2. A feasibility study of bioenergy production in Nordland county Traditionally, the animal’s manure is stored during winter time in barn and used as fertilizer for pasture grass production, but the emissions of methane and other hazardous gases from animal’s manure are harmful to the environment. The planned MBT plant can effectively resolve this problem through converting the animal’s manure into biogas and bio-rest, which can be used as vehicle fuel and fertilizer, so the plan is welcomed by local agriculture and livestock industry. Another supporter for bioenergy pro- duction from biodegradable waste is the local waste management company: LAS. Currently, LAS operates a landfill and a composting plant, and the anaerobic digestion facility planned in Leknes will be an attractive alternative for the treatment of biodegradable waste from LAS. Figure 8 illustrates the value-added process of bioenergy production. The feedstock in this area is mainly from agriculture residues, livestock residues, fishery residues and municipal waste; however, biomass from forestry industry is not included in the current plan. The tech- nology applied for bioenergy production in Leknes is MBT, and the main products are biogas and bio-rest. The biogas can be used as the fuels at land transport sector, and the bio-rest can be used as fertilizer for agriculture industry. Both biogas and bio-rest can either be used locally or sold in domestic/international markets. The bioenergy production plant in Leknes aims at providing a sustainable solution in deal- ing with biomass and biodegradable waste from the municipalities in Nordland County. The most important factor of bioenergy production is to maintain economy of scale, so knowledge Figure 8. Value chain architecture of bioenergy production plant at Leknes [11, 52]. Figure 8. Value chain architecture of bioenergy production plant at Leknes [11, 52]. A Value Chain Analysis for Bioenergy Production from Biomass and Biodegradable Waste… http://dx.doi.org/10.5772/intechopen.72346 197 Biodegradable waste Estimated biogas output (m3/year)(4) Average factor(4) Total biogas output (m3/year) Conversion factor (KWh/ton)(4) Total estimated energy output (GWh) BMW(1) 204 180 2,340,000 1120 14.5 BWS(2) 93 102 142,800 444 0.62 AM(3) 19 19 95,000 190 0.95 Sum N/A N/A 2,577,800 N/A 16.3 (1)BMW: biodegradable municipal waste. (2)BWS: biodegradable waste from slaughterhouse. (3)AM: animal’s manure. (4)Basic data from Refs. [8, 63, 64]. Table 3. Estimated output amounts of biogas and bioenergy. A Value Chain Analysis for Bioenergy Production from Biomass and Biodegradable Waste… http://dx.doi.org/10.5772/intechopen.72346 1 Table 3. Estimated output amounts of biogas and bioenergy. 4.2. A feasibility study of bioenergy production in Nordland county of annual generation of biomass and biodegradable waste is of importance. Estimation of the annual amount of three types of biodegradable waste is presented as follows: of annual generation of biomass and biodegradable waste is of importance. Estimation of the annual amount of three types of biodegradable waste is presented as follows: 1. Biodegradable municipal waste collected by regional waste management companies: 13,000 ton/year. 2. Biodegradable waste from slaughterhouse: 1400 tons/year. 3. Animal’s manure from livestock industry: 5000 tons/year. Calculation of the output of bioenergy production based on input amount provides valuable criteria for value chain analysis. Previous studies have formulated different models for the calculation of biogas output from different types of feedstock. Calculation in this chapter is based on the established models for biodegradable municipal waste and livestock residues from Norway [8], Sweden [60] and Denmark [61]. Table 3 gives the estimated output of biogas from the three types of feedstock. The estimated output amount may be different from the actual amount, and this is due to the following reasons: • The calculation of the outputs from the three types of biodegradable waste is conducted through the combination of different literatures. However, as a general rule, the biogas output in reality is usually lower than the estimated value. • It is a common situation that different types of biomass are mixed in the digester for an- aerobic digestion, and this will lead to different output amount with the separate digestion that estimated in the table. It is therefore impossible to foresee the exact output due to the biochemical decomposition of protein, carbohydrates, lipid differs and other substrates. • Animal’s manure has the least conversion efficiency, but its presence has great meaning for the conversion of mixed biomass and biodegradable waste due to the content of bacteria, nourishment and trace elements, which provide a stabilizing effect for the microbial digestion in the reactor tank. Researches on the optimization of biogas production have revealed that Energy Systems and Environment 198 compound mixtures of different substrates result in a microbial society with a larger diversity and are more stable [62], and that counts for an optimistic view of the calculated output. Environmental challenges in high north arctic regions have been attracted much more focuses due to the potentially severe consequences such as melting glaciers and sea-level rise caused by global warming. Table 4. Emissions of methane from stored animal’s manure. (1)Data provided from Farmer’s Interest Organization, Lofoten region. 4.2. A feasibility study of bioenergy production in Nordland county GHG emissions have been proved to be the most important driving force for the global warming and climate change, and it has more influences on the vulnerable eco- environment. Thus, the environmental impact of bioenergy production is assessed by GHG emissions. Two types of GHG emissions are estimated in this chapter, one is the emissions of methane from stored animal’s manure in livestock industry, and the other is the CO2 emis- sions from the transportation to the MBT plant at Leknes. The storage of animal’s manure in winter not only occupies great space but also releases a large amount of methane. The treatment of animal’s manure through bioenergy production can effectively resolve this problem. From the national perspective, Norwegian strategic document for bioenergy development estimates that the overall amount of animal’s manure from livestock industry for potential bioenergy production in Norway is approximately 3.92 million tons, and this will lead to 305,000 tons CO2-equivalent reduction of GHG emissions [10]. The amount of different animals in local livestock industry is given by the Farmer’s Interest Organization at Lofoten region. Besides, we also presupposed an input amount of 5000 tons of animal’s manure to a co-substrate-mixture at the bioenergy production plant. Table 4 illustrates the annual emissions of methane from the animal’s manure by sources. Based on the method provided in the governmental calculation, an overall 0.39 tons CO2- equivalent reduction of methane emissions from local livestock industry can be estimated. Besides, the calculation also implies a reduction of fertilizer for pasture grass production; however, this can also be solved by utilizing the bio-rest from bioenergy production at Leknes. Development for utilizing wet bio-rest in agriculture has recently been proved to be a good fertilizer [64–66], and the close distance from local agriculture and livestock industry to the planned location of the MBT plant is another advantage for the utilization of bio-rest. Animal Amounts(1) Individual methane emission factor (ton/animal/ year)(2) Total emission of methane (ton/year) Cow (milk) 830 0.018 14.9 Cow (meat) 2100 0.0027 5.67 Hen 2600 0.0009 2.34 Pig 80 0.0071 0.568 Sheep 6800 0.0002 1.36 Goat 975 0.00012 0.117 Horse 69 0.00295 0.204 Sum 25.2 (1)Data provided from Farmer’s Interest Organization, Lofoten region. (2)Basic data from statistical yearbook of Norway [52]. 4.2. A feasibility study of bioenergy production in Nordland county A Value Chain Analysis for Bioenergy Production from Biomass and Biodegradable Waste… http://dx.doi.org/10.5772/intechopen.72346 199 199 GHG emissions associated with the transportation of biomass and biodegradable waste to Leknes are another important problem when the bioenergy production network is designed. In Nordland County, four regional waste management companies are involved in the collec- tion, transportation and treatment of biodegradable waste in different municipalities: HRS, RENO VEST, IRIS and LAS. Currently, RENO VEST and LAS send their waste to Leknes for aerobic composting, and the organic waste collected at Narvik municipality is treated at the incineration plant at Kiruna in northern Sweden. Bodø municipality has its own compost- ing plant for dealing with biodegradable waste. Obviously, both aerobic digestion and direct combustion are less sustainable than bioenergy production through MBT, so the planned MBT plant at Leknes becomes a suitable alternative for HRS and IRIS. Table 5 presents the annual generation of biodegradable waste at different municipalities in Nordland County, and the distance from each municipality to Leknes is also given in this table. As shown in the table, transportation of small amount of biodegradable waste over long distance is necessary when the MBT plant is located at Leknes, and this will lose the advantage of economy of scale and increase GHG emissions. However, it is also the situa- tion in today’s waste management system. For example, the biodegradable waste collected at Narvik municipality travels 178 km to Kiruna for incineration. Compared with today’s situation, only the transportation of biodegradable waste from Narvik and Bodø to Leknes will increase GHG emissions. Table 6 illustrates the estimation of CO2 emissions from the transportation of biodegradable waste to Leknes. The estimated CO2 emissions are calculated based on the information provided by the waste management companies, which include the monthly generation of biodegradable waste in each community and the unit fuel consump- tion of waste collection and further distribution. Compared with the current waste manage- ment system, the CO2 emissions of biomass and biodegradable waste transportation of the planned bioenergy production system will be increased by 19.3%. Although it is difficult to formulate the complete value-added process of bioenergy produc- tion, we can still see a large potential of a sustainable solution for both energy production and waste management in Northern Norway. 4.2. A feasibility study of bioenergy production in Nordland county However, decisions must be made based on the consideration and justification of both benefits and challenges, and the interests of dif- ferent stakeholders within the value chain of bioenergy production should also be taken into account. Table 7 summarizes the opportunities and challenges of bioenergy production in Nordland County. As shown in the table, bioenergy production from biomass and biodegrad- able waste will deliver a great amount of biofuels and fertilizer to the market; besides, it will decrease GHG emissions as well as other environmental impacts from local agriculture, live- stock industry and waste management. However, the costs and CO2 emissions of the trans- portation in the overall bioenergy production network will be increased, and the operating costs of the MBT plant are higher in the cold regions due to more energy consumption for maintaining a constant temperature for anaerobic digestion. Further, there are uncertainties about the potential markets for biofuels and fertilizer, and the cost and CO2 emission will be increased for the transportation of them to potential markets. In order to resolve those challenges, policy support must be first formulated accordingly by the government so that the value added through the bioenergy production can be shared by all the stakeholders within the value chain. For example, governmental incentives or tax relief Energy Systems and Environment 200 Waste management company Estimated waste (ton/year) Distance to Leknes (km)(3) Leknes (LAS)(1) 3400 0 Bodø (IRIS)(2) 5000 160(4) Harstad (HRS)(2) 2300 263 Sortland (RENO VEST)(2) 2200 183 Narvik (HRS)(2) 2200 305 (1)Including the biodegradable waste from slaughterhouse. (2)Data given by relevant waste management companies. (3)Calculated on Google map. (4)Including ferry boat transport. Table 5. Generation of biodegradable waste in Nordland County. Waste management company Estimated waste (ton/year) Distance to Leknes (k Table 5. Generation of biodegradable waste in Nordland County. Unit fuel consumption (L/km) Annual transport distance (km/year) Fuel consumption (L/year) CO2 emission (ton/ year)(1) LAS (local area) 0.58 24,000 13,920 37.4 RENO VEST 0.42 38,896 16,336 44 HRS, Harstad 0.48 62,660 30,077 39 HRS, Narvik 0.48 64,480 30,950 40 IRIS, Bodø 0.48 21,538 10,333 13 Total 211,564 101,616 173.4 (1)Calculated based upon Energilink [67]. Table 5. Generation of biodegradable waste in Nordland County. stimated fuel consumption and CO2 emissions from the transportation. 4.2. A feasibility study of bioenergy production in Nordland county Opportunities Challenges • Large amount production of biofuels • Production of bio-rest/fertilizer • Increased use of biofuels in transport • Less GHG emissions from agriculture, livestock industry and waste treatment • Less environmental impact • Increased costs for the transportation of biodegradable waste • Increased CO2 emission from the transportation of biodegradable waste • Increased investment on bioenergy production in cold climate • Uncertain market demands • Cost and CO2 emission related to the distribution of biofuels and bio-rest to potential market T bl 7 O t iti d h ll f bi d ti i N dl d C t Table 7. Opportunities and challenges of bioenergy production in Nordland County. should be given to HRS and IRIS to compensate their increased transportation cost to deliver the biodegradable waste to Leknes, and this may also be achieved through implementing a lower entrance fee of the MBT plant. Besides, the successful example of using biofuels in the A Value Chain Analysis for Bioenergy Production from Biomass and Biodegradable Waste… http://dx.doi.org/10.5772/intechopen.72346 2 201 public transport sector in Southern Norway is also applicable for Nordland County, and the upgrade of buses for using biofuels should be subsidized by the local government. Further, the incentives or lower purchase price should also be given to the agriculture and pasture grass production in order to promote the local use of bio-fertilizer, which will tremendously decrease the cost and GHG emissions for delivering the bio-fertilizer to remote markets. 5. Conclusion During the past decades, the concerns of the depletion of fossil fuels and global warming caused by excess GHG emissions have become the most important driving force for the development and utilization of renewable energy resources. The successful experiences from the EU-28 have proved that bioenergy production from biomass and biodegradable waste is the most reliable and promising solution in today’s renewable energy market. This chapter studies bioenergy production from the perspective of value chain analysis. The method of value chain analysis has been developed for over three decades and extensively applied in analysing the value-added process of many different industries. In this chapter, a theoretical architecture for value chain analysis of bioenergy production from biomass and biodegrad- able waste is first formulated for streamlining the value-added activities in the bioenergy production network. In order to give a deep insight of the developed value chain model, we investigated the current situation of bioenergy production in Norway and compared that with other Nordic countries. The feasibility study for establishing a bioenergy production plant in Nordland County, which is located in the northern part of Norway, is also performed. The value chain analysis of bioenergy production from biomass and biodegradable waste in Nordland County estimates both the potential amount of bioenergy output and the envi- ronmental impact, and suggestions for overcoming the challenges of bioenergy production are also given in this section. In order to better achieve the value-added process in bioen- ergy production in Nordland County, future studies are suggested from three aspects. First, information from other local industries should be investigated so that a complete value chain of bioenergy production can be formulated. Second, forestry residues are very impor- tant feedstock for bioenergy production in other places; however, it is not included in cur- rent plan for bioenergy production. Thus, further studies with inclusion of forestry waste as the feedstock of the MBT plant at Leknes should be carried out. Third, decision sup- port tools for optimal design of integrated network for biodegradable waste transportation should be developed in order to balance both transportation costs and GHG emissions in an optimal manner. Author details Hao Yu1, Elisabeth Román2 and Wei Deng Solvang1 Address all correspondence to: hao.yu@uit.no Hao Yu1, Elisabeth Román2 and Wei Deng Solvang1 Address all correspondence to: hao.yu@uit.no 1 Department of Industrial Engineering, UiT—The Arctic University of Norway, Narvik, Norway 2 Department of Building, Energy and Material Technology, UiT—The Arctic University of Norway, Narvik, Norway Acknowledgements This project is supported by the Norwegian Research Council/VRI-Nordland (Commitment 2012-0446) under strategy to promote renewable energy production in Nordland County. 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https://openalex.org/W2093700138	https://informesdelaconstruccion.revistas.csic.es/index.php/informesdelaconstruccion/article/download/2152/2460/2900	Spanish; Castilian	null	Ampliación del hangar n° 2 Aeropuerto de Madrid-Barajas España	Informes de la construcción	1,982	cc-by	1,896	AMPLIACIÓN DEL HANGAR N.° 2. AEROPUERTO DE MADRID-BARAJAS ESPAÑA^ SINOPSIS SINOPSIS La necesidad de realizar trabajos de mantenimien de los aviones AIRBUS de Iberia obligaron a esta compañía a ampliar el hangar n. ^ Z hasta ahora destinado a aviones de tipo medio. Después del estudio de diversas propuestas: viga biapoyada isostática, pórtico mixto de hormigón armado y viga metálica, etc., y teniendo en cuen las limitaciones de altura y la de servidumbre de espacio aéreo, que obligaban a una estructura de 6,50 m de canto máximo, se adoptó la solución de una viga metálica de celosía de 125,60 m de longitud (110,60 m entre ejes de apoyos y dos voladizos de 7,5 m anclados al cimiento), 3,00 m de ancho y 6,50 m de canto. Antonio Tabera García, ingeniero de Caminos** Alfonso Mauleón López-lbarrondo, Ingeniero Industriar* * Premio SERCOMETAL 1981. ** Del Departamento Técnico de Huarte y Cía., S. A. Empresa Constructor 545-34 La viga se prefabricó parcialmente en taller y e izado duró siete días. * Premio SERCOMETAL 1981. ** Del Departamento Técnico de Huarte y Cía., S. A. Empresa Constructo © Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://informesdelaconstruccion.revistas.csic.es © Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://informesdelaconstruccion.revistas.csic.es 26 Informes de la Constrycción/339 antecedente tánea para 3 aparatos. La dimensión en planta del hangar era de 100 m de ancho, 53 m de. profun» didad y una altura libre de 12 m. La estructura principal de la cuÉierta estaba resuelta mediante 4 vigas-cajón metálicas en celosía que, ancladas en la línea de cerramiento posterior del edificio y apoyadas en estructuras de hormigón ubicadas en el interior del hangar, se proyectaban en ménsulas de 38 m hasta la fachada frontal cerrada por puer- tas corredizas de 12 m de altura, lo que dejaba una embocadura totalmente diáfana de 100 x 12 m^. tánea para 3 aparatos. La dimensión en planta del hangar era de 100 m de ancho, 53 m de. profun» didad y una altura libre de 12 m. La estructura principal de la cuÉierta estaba resuelta mediante 4 vigas-cajón metálicas en celosía que, ancladas en la línea de cerramiento posterior del edificio y apoyadas en estructuras de hormigón ubicadas en el interior del hangar, se proyectaban en ménsulas de 38 m hasta la fachada frontal cerrada por puer- tas corredizas de 12 m de altura, lo que dejaba una embocadura totalmente diáfana de 100 x 12 m^. Con objeto de realizar el mantenimiento de los nuevos aviones Airbus A300-B4 adquiridos por IBERIA LINEAS AEREAS DE ESPAÑA, esta Com^ pañía convocó un concurso de proyecto y ejecu- ción de una ampliación para el HANGAR N.^ 2 situado en la zona industrial n..^ 1 del Aeropuerto de MADRID-BARAJAS. El HANGAFi N.^ 2 estaba destinado hasta ese mo- mento a\ mantenírniento de aviones de tipo medio (Boeing 727, Douglas DC»9) con capacidad simul- Las dimensiones de los nuevos aviones^ que se querían mantener en este hangar, obligaban a una doble ampliación del edificio: por un lado debía aumentarse su dimensión en profundidad en 12 m y, por otro lado, la mayor altura de cola de los aviones exigía una altura libre de 19,50 m en una franja en planta de 100 x 19 ni^, adyacente a la linea de puertas. Esta segunda ampliación exigía el desmontaje de una franja de 100 x 7 m^ en planta de la cubierta existente, así como de las puertas. b T' ••' ' * H ''•-•. l : < i ; 1 ) ] f ,...--w - ] ^ 1 planta de cubiertas t:- SECCiON^A.A sección A-A b T' ••' ' * H ''•-•. antecedente l : < i ; 1 ) ] f ,...--w - ] ^ 1 planta de cubiertas t:- Era condición impuesta para cualquier solución el respetar la diafanidad de la embocadura que, una vez ampliado el edificio, debía ser de 100 m de ancho y de 19,50 m de altura. planta de cubiertas Esta embocadura inicial tuvo que ser ampliada posteriormente a 110 m libres, ya que la posición de los apoyos interfería en los alojamientos de ios andamies móviles que se Iban a utilizar en el mantenimiento de los aviones» Otra condición previa a adoptar, que afectaba a la solución estructural, era la existencia de una limitación en la altura máxima del edificio, im- puesta por la Subsecretaría de Aviación Civil y motivada por razones de servidumbre del espacio aéreo, y que señalaba como cota máxima en altura sección A-A cercha-1 7^'AC i - -V 4^ © Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://informesdelaconstruccion.revistas.csic.es cercha-1 7^'AC i - -V 4^ cercha-1 cercha-1 © Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://informesdelaconstruccion.revistas.csic.es 27 Informes de la Construcción7339 la + 26,00 respecto al nivel de la pista, lo que limitaba a 6,50 el canto máximo posible para la estructura. proceso de montaje estudio de alternativas A la vista de los condicionantes impuestos, se iniciaron los estudios de las posibles alternativas estructurales para cubrir el vano de 110 m con un elemento de 6,50 m de canto máximo. Desde un principio quedaron eliminadas todas aquellas soluciones que no fueran de estructura metálica, igualmente de entre éstas últimas tan sólo se consideraron las de tipo celosía. Un problema que se manifestó como determinante desde los primeros tanteos fue el de las deforma- ciones verticales de la estructura bajo las diversas combinaciones de cargas (nieve, viento, etc.), da- das las estriótas tolerancias que imponían las puertas del hangar. La solución estructural más elemental, una viga biapoyada isostática, hacía incompatibles las limi- taciones de deformación con unos razonables cri- terios económicos de aprovechamiento del mate- rial empleado. Era, pues, necesario encontrar un diseño que minimizara las citadas deformaciones de la viga, y para ello se analizaron diversas for- mas de coaccionar los giros de la viga en sus apoyos. proceso de montaje http://informesdelaconstruccion.revistas.csic.es Dado que para los apoyos de la viga principal se pensó en disponer una serie de pantallas de hor- migón armado, cabía la posibilidad de formar un pórtico mixto con uniones rígidas entre hormigón armado y viga metálica, pero la magnitud de los esfuerzos desarrollados en la unión aconsejó bus- car otra solución más favorable. Después de varias tentativas la solución evolucio- nó hacia una compensación del vano de 110 m con dos pequeños voladizos de 7,50 m que po- drían pretensarse directamente al cimiento. © Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://informesdelaconstruccion.revistas.csic.es © Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) 28 informes de la Construcción/339 ^^^^^K^^Sk 1 1 /«l\|i I l / A I /MTAOTiÇfiVi Mt'tvafllii Í ^ 1 . 1/ mml/Wil^Ml%mi • ''^^iZ^Ë^^^SflÉ^fl^^H descripción de la solución adoptada El elemento fundamental de la estructura a efecto de cargas verticales es una viga metálica de celo- sía con sección en cajón de 6,50 m de canto, 3,00 m de ancho y 125,60 m de longitud total. Esta viga tiene un vano central de 110,60 m de luz entre ejes de apoyos y dos voladizos de 7,50 m que tienen su extremo anclado al cimiento me- diante cables de pretensado que, una vez situada aquélla en su posición, fueron puestos en carga. Los apoyos de la viga sobre las pantallas de hor- migón están materializados en aparatos de apoyo fijos o deslizantes, según las respectivas direccio- nes, para permitir el funcionamiento de la estruc- tura. Como perfiles básicos para la conformación de la celosía se eligieron perfiles HEB para las barras principales y tubos cuadrados de perfil en frío para las diagonales y barras secundarias de arrios- tramiento. El resto de la cubierta responde a un concepto tradicional, a base de cerchas que apoyan en la viga principal y en la estructura antigua; este últi- mo apoyo se realiza a través de una viga lucerna- rio de 5,00 m de canto. El nivel inferior de toda la cubierta coincide en un plano único que se en- cuentra convenientemente triangulado con el fin de funcionar como viga horizontal apoyada en las detalle de unión de estructura nueva y existente © Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://informesdelaconstruccion.revistas.csic.es detalle de unión de estructura nueva y existente http://informesdelaconstruccion.revistas.csic.es detalle de unión de estructura nueva y existente http://informesdelaconstruccion.revistas.csic.es http://informesdelaconstruccion.revistas.csic.es © Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) 2B Informes de la Coristryccíón/339 alzado alzado A Ipaníalial http://informesdelaconstruccion.revistas.csic.es pantallas de hormigón y resistir los esfuerzos hori- zontales de viento transmítídos por la proyección de la estructura y, principalmente, por las puertas del hangar. La viga principal de la estructura se apoya en un conjunto de pantallas de hormigón armado de 60 cm de espesor. Su geometría, en parte cerrada, garantiza no sólo la transmisión de las cargas ver» tícales sino los componentes horizontales debidos al viento y a los rozamientos de ios apoyos. descripción de la solución adoptada Este conjunto de pantallas descansa a su vez en el terreno a través de una zapata superficial, cuyas dimensiones medias son de 11,00 x 9,00 rrf, un espesor de 2 m y su fondo está situado a la cota —3,00 respecto al nivel de solera del hangar. El atirantado de los voladizos se realiza medíante 2 cables en cada extremo de 18 0 7, con un tesado inicial de 215 t El extremo inferior lleva un anclaje ciego embebido en la cimentación, míen- tras que el anclaje superior es activo. © Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://informesdelaconstruccion.revistas.csic.es proceso constryctíwo Las dimensiones de ios elementos metálicos acon- sejaron su fabricación parcial en taller y su en» samblaje en obra. El ensamblaje en obra se realizó sobre el suelo, justamente en la vertical de su posición definitiva. Este proceso facilitó notablemente la ejecución, asi como todas las operaciones de control que simultáneamente se fueron realizando. arriostramieriío mtefmr entre lineas 1 f 2 " O Y E arriostramieriío mtefmr entre lineas arriostramieriío mtefmr entre lineas 1 f 2 " O Y E alzado A Ipaníalial © Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) © Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://informesdelaconstruccion.revistas.csic.es 30 Informes de la Construcción7339 Durante el tiempo que duró el ensamblaje de la viga se fueron desmontando las puertas y estruc- tura sobrante del hangar antiguo, así como tam- bién se construyeron la cimentación y arranque de las pantallas de hormigón. La operación de izado de la viga se asoció a la construcción de las pantallas de hormigón, me- diante un sistema de encofrado deslizante, que al mismo tiempo y de una manera continua lograba el crecimiento de las pantallas y la elevación de la viga, que descansaba provisionalmente sobre las plataformas de trabajo del encofrado. La operación de deslizar las pantallas se realizó de forma inin- terrumpida durante 7 días, al final de los cuales quedó la viga situada en su cota definitiva. Con posterioridad al deslizamiento se montaron los aparatos de apoyo de la viga y se completó el resto de la estructura de cubierta. Una vez que la viga principal alcanzó la totalidad de sus cargas permanentes, se procedió al montaje y tesado por fases de los cables de pretensado. sección B-B (planta armado de pantallas) •X- -X- ^ sección B-B (planta armado de pantallas) sección B-B (planta armado de pantallas)
https://openalex.org/W4240729833	https://www.researchsquare.com/article/rs-98596/v1.pdf?c=1631873618000	English	null	Addressing Key Gaps in Implementation of Mosquito Larviciding to Accelerate Malaria Vector Control in Southern Tanzania: Results of a Stakeholder Engagement Process in Local District Councils	Research Square (Research Square)	2,020	cc-by	10,708	Addressing Key Gaps in Implementation of Mosqu Larviciding to Accelerate Malaria Vector Control in Southern Tanzania: Results of a Stakeholder Engagement Process in Local District Councils Salum Abdallah Mapua (  smapua@ihi.or.tz ) Environmental Health and Ecological Sciences Department, Ifakara Health Institute, P. O. Box 53, Morogoro, Tanzania https://orcid.org/0000-0002-3309-4328 Marceline F. Finda Ifakara Health Institute Ismail H. Nambunga Ifakara Health Institute Betwel J. Msugupakulya Ifakara Health Institute Kusirye Ukio Predisent's Office-Regional Administration and Local Government. Morogoro Regional Secretariat. Prosper P. Chaki Ifakara Health Institute Frederic Tripet Centre for Applied Entomology and Parasitology, School of Life Sciences, Keele University Ann H. Kelly Department of Global Health and Social Medicine; King's College London Nicola Christofides School of Public Health, Faculty of Health Sciences, University of Witswatersrand Javier Lezaun Institute for Science, Innovation and Society; School of Anthropology and Museum Ethnography, Un of Oxford Fredros O. Okumu Ifakara Health Institute Research Addressing Key Gaps in Implementation of Mosqu Larviciding to Accelerate Malaria Vector Control in Southern Tanzania: Results of a Stakeholder Engagement Process in Local District Councils Salum Abdallah Mapua (  smapua@ihi.or.tz ) Environmental Health and Ecological Sciences Department, Ifakara Health Institute, P. O. Box 53, Morogoro, Tanzania https://orcid.org/0000-0002-3309-4328 Marceline F. Finda Ifakara Health Institute Ismail H. Nambunga Ifakara Health Institute Betwel J. Msugupakulya Ifakara Health Institute Kusirye Ukio Predisent's Office-Regional Administration and Local Government. Morogoro Regional Secretariat. Prosper P. Chaki Ifakara Health Institute Frederic Tripet Centre for Applied Entomology and Parasitology, School of Life Sciences, Keele University Ann H. Kelly Department of Global Health and Social Medicine; King's College London Nicola Christofides School of Public Health, Faculty of Health Sciences, University of Witswatersrand Javier Lezaun Institute for Science, Innovation and Society; School of Anthropology and Museum Ethnography, Un of Oxford Fredros O. Okumu Ifakara Health Institute Research Addressing Key Gaps in Implementation of Mosqu Larviciding to Accelerate Malaria Vector Control in Southern Tanzania: Results of a Stakeholder Engagement Process in Local District Councils Salum Abdallah Mapua (  smapua@ihi.or.tz ) Environmental Health and Ecological Sciences Department, Ifakara Health Institute, P. O. Box 53, Morogoro, Tanzania https://orcid.org/0000-0002-3309-4328 Marceline F. Finda Ifakara Health Institute Ismail H. Nambunga Ifakara Health Institute Betwel J. Msugupakulya Ifakara Health Institute Kusirye Ukio Predisent's Office-Regional Administration and Local Government. Morogoro Regional Secretariat. Prosper P. Chaki Ifakara Health Institute Frederic Tripet Centre for Applied Entomology and Parasitology, School of Life Sciences, Keele University Ann H. Kelly Department of Global Health and Social Medicine; King's College London Nicola Christofides School of Public Health, Faculty of Health Sciences, University of Witswatersrand Javier Lezaun Institute for Science, Innovation and Society; School of Anthropology and Museum Ethnography, Un of Oxford Fredros O. Okumu Ifakara Health Institute R h Research Keywords: Malaria control, malaria elimination, larviciding, larval source management, biolarvicides, stakeholders, public perception, Tanzania, Ifakara Health Institute Posted Date: October 30th, 2020 Posted Date: October 30th, 2020 Posted Date: October 30th, 2020 Page 1/25 DOI: https://doi.org/10.21203/rs.3.rs-98596/v1 DOI: https://doi.org/10.21203/rs.3.rs-98596/v1 DOI: https://doi.org/10.21203/rs.3.rs-98596/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on March 2nd, 2021. See the published version at https://doi.org/10.1186/s12936-021-03661-x. Page 2/25 Abstract Background: Larval source management was historically one of the most effective malaria control methods but is now widely deprioritized in Africa, where insecticide treated nets (ITNs) and indoor residual spraying (IRS) are preferred. However, in Tanzania, following initial successes in urban Dar-es-Salaam starting early- 2000s, the government now encourages larviciding in both rural and urban councils nationwide to complement other efforts; and a biolarvicide production-plant has been established outside the commercial capital. This study investigated key obstacles and opportunities relevant to effective rollout of larviciding for malaria control, with a focus on the meso-endemic region of Morogoro, southern Tanzania. Methods: Key-informants were interviewed to assess awareness and perceptions regarding larviciding among designated health officials (malaria focal persons, vector surveillance officers and ward health officers) in nine administrative councils (N = 27). Interviewer-administered questionnaires were used to assess awareness and perceptions of community members in selected areas regarding larviciding (N = 490). Thematic content analysis was done and descriptive statistics used to summarize the findings. Results: A majority of malaria control officials had participated in larviciding at least once over the previous three years. A majority of community members had neutral perceptions towards positive aspects of larviciding, but overall support for larviciding was high, although several challenges were expressed, notably: i) insufficient knowledge for identifying relevant aquatic habitats of malaria vectors and applying larvicides, ii) inadequate monitoring of program effectiveness, iii) limited financing, and iv) lack of personal protective equipment. Although the key-informants reported sensitizing local communities, most community members were still unaware of larviciding and its potential. Conclusion: The larviciding program was widely supported by both communities and malaria control officials, but there were gaps in technical knowledge, implementation and public engagement. To improve overall impact, it is important to: i) intensify training efforts, particularly for identifying habitats of important vectors, ii) adopt standard technical principles for applying larvicides, iii) improve financing for local implementation and iv) improve public engagement to boost community awareness and participation. These lessons could also be valuable for other malaria endemic areas wishing to deploy larviciding for malaria control or elimination. Background The world has witnessed a significant reduction in malaria burden since 2000 [1], most of these gains being attributed to insecticide-treated bed nets (ITNs), indoor residual spraying (IRS) and effective case management [2,3]. Yet, there were still more than 200 million cases, and 405,000 deaths globally in 2018, 90% in sub-Saharan Africa [1]. Ongoing malaria control efforts are increasingly compromised by several factors, chief among them, parasite resistance to anti-malarial drugs [4,5], behavioral adaptation of mosquitoes to ITNs and IRS [6,7] and growing insecticide resistance in malaria vectors [8,9]. Anthropological factors also play a crucial role in mediating transmission, as human behaviors, economic practices and perceptions of risk can increase dangers of infectious malaria vectors [10–13]. Malaria vector Anthropological factors also play a crucial role in mediating transmission, as human behaviors, economic practices and perceptions of risk can increase dangers of infectious malaria vectors [10–13]. Malaria vector Page 3/25 control in Tanzania has also focused mainly on provision and use of ITNs and IRS [14–18]. This is complemented with other efforts such as increased access to reliable and affordable diagnostics and treatment [19], and universal distribution of prophylaxis for pregnant women [20]. These efforts, combined with a general improvement in economic opportunity, have led to a tremendous decline in malaria burden throughout the country [20,21]. Environmental management to eliminate mosquito breeding habitats was among the first malaria control strategies attempted in Tanzania. Efforts included improving drainage systems and the elimination of the permanent bodies of stagnant water near large human settlements [22,23]. In recent times, the first major use of larviciding in Tanzania was in Dar-es-Salaam in early 2000s [24,25], when regular application of biolarvicides by community-owned resources persons (CORPs) achieved as much benefit as ITNs [25]. The Tanzania National Malaria Strategic Plan, 2014-2020 recommended implementation of larviciding in selected urban settings [26], in line with guidance from the World Health Organization to consider only settings where aquatic habitats of malaria vectors are few, fixed and findable [27]. This policy initially focused on just urban populations, but in recent years the government has encouraged extension of larviciding to include rural settings [28]. The nationwide expansion of larviciding follows the creation in 2014 of Tanzania Biotech Products Limited (TBPL), which is responsible for production and distribution of biolarvicides [29]. Since 2017, TBPL has been manufacturing two types of biolarvicides, Bacillus thuringiensis var. israelensis (Bti) and Bacillus sphaericus (Bs) [29]. Background These products are procured by the district councils across the country, and distributed to all administrative wards. Councils often reserve budgets to compensate community-health workers (CHWs) and volunteers involved in community initiatives such as larviciding [30]. The recent developments by Tanzania to expand larviciding are excellent examples of the much-needed ownership for sustainable vector control, especially given the use of the domestic resources. If sustained, it could yield significant gains over current accruals from the core interventions, and in the process generate important lessons for other countries. Unfortunately, given its extensive scale and novelty as well as the inclusion of predominantly rural councils, there are still multiple challenges that must be addressed to achieve maximum impact. For example, the major malaria vectors in the country use a wide variety of aquatic habitats, which still need to be sufficiently characterized [31]. Moreover, larviciding is also labor intensive and requires active community involvement. This study therefore aimed to identify and characterize important gaps as well as opportunities for improving the implementation of larviciding in Tanzania. The study examined perceptions and experiences of key actors of larviciding in different district and municipal councils. The study focused on the mostly meso-endemic region of Morogoro, southeastern Tanzania. Study area Page 4/25 Page 4/25 The study was conducted in nine administrative councils in the Morogoro region in southern Tanzania between October 2019 and March 2020 (Figure 1). The area has a total population estimated at 2.2 million people [32], and is currently classified as meso-endemic, with malaria prevalence estimated at ~10% according to the most recent estimates [33]. The councils were: Gairo, Mvomero, Kilombero, Ulanga, Kilosa, Morogoro and Malinyi district council, Morogoro municipal council and Ifakara town council (Figure 1). The community members surveyed were from Ulanga and Kilombero districts only. Selection of stakeholders Stakeholders selected for this study included district health officials and community members. The health officials included district malaria focal persons (MFPs), vector surveillance officers (VSOs) and ward health officers. Malaria focal persons were either medical doctors or environmental health specialists in charge of all malaria related-matters at the district level. In this study, all the MFPs had been at their current position for at least two years. They are responsible for all aspects of malaria control, including monitoring trends of malaria cases, deaths and control. Vector surveillance officers on the other hand were environmental health specialists with a diploma in environmental health science and a special training in disease-vector control. The VSOs are responsible for organizing, supervising and executing disease-vector control programmes at the district level. Lastly, the ward health officers were also environmental health specialists and were responsible for all health-related issues at the ward level. They had a diploma or certificate training in environmental health science, and their responsibilities included planning, supervising, monitoring and evaluating overall health services at the ward level. Each district has one MFP, one VSO and multiple ward health officers, but in some cases one ward health officer could serve multiple wards within the district. Malaria focal persons and VSOs were recruited from all districts as well as the municipal and town councils within Morogoro region. However, the ward health officers were recruited from a randomly selected ward in each district, municipal or town council. Each of seven districts had between eight and thirty-eight wards. For the community survey, households were randomly selected from ten randomly selected wards in Ulanga and Kilombero districts in the region (Figure 1), and the survey was administered to the household heads Study design and procedures A concurrent triangulation mixed method study design was used [34], incorporating key informant interviews (KII) and survey questionnaires. Key informant interviews were done with MFPs, VSOs and ward health officers to obtain information on the degree of awareness as well as experiences and perceptions of these officers regarding larviciding. These interviews were conducted by the authors, SAM, MFF and IHN, between February and March of 2020 at the respective council offices. The interviews were audio-recorded following consent of the participants. The audio recordings were supplemented by hand-written notes. Each interview lasted between 15 and 60 minutes and were done in Swahili language. Page 5/25 The questionnaire surveys were conducted with community members from Ulanga and Kilombero district. These were done in Swahili language, and used to gather data on awareness and perceptions of larviciding Page 5/25 as a malaria control intervention. KobotoolboxTM software [35] was used to administer the surveys via electronic tablets, between November and December 2019. The individual-level perception of community members towards larviciding was assessed by measuring the level of agreement towards positive statements on larviciding using a 5-point Likert-scale, ranging from strongly agree (1) to strongly disagree (5). The main statements were as follows: i) larviciding will be effective for malaria control, ii) larviciding will fill gaps left by other interventions, iii) larviciding is safe for humans, animals and the environment, iv) larviciding will be easy to perform, v) larviciding supplies and equipment will be easily accessible, vi) larviciding will be affordable to community members and vii) larviciding will be acceptable in the community. The final perception level was determined by comparing individual perception scores against the median score (see data analysis section). In addition, one joint stakeholder engagement meeting was conducted at the regional office, where all the MFPs and VSOs from the nine administrative councils participated, together with Ifakara Health Institute researchers. Discussions at this meeting involved options for improving larviciding operations in the respective councils, and what roles different stakeholders could play. Data processing and analysis Audio recordings of the key informant interviews were transcribed immediately following the discussions and translated from Swahili to English language. Field notes were added in the written transcripts. The written transcripts were analyzed using NVIVO 12 Plus software [36]. Deductive and inductive coding were used to categorize the codes items. A KII guide was used to develop the deductive codes while the inductive codes were generated based on thorough reviews of the transcripts. Similar codes were grouped and emergent patterns used to identify themes. The extracted themes included: i) knowledge about larval habitats of malaria vectors, ii) awareness of larviciding as a malaria control intervention and iii) challenges facing the implementation of larviciding. Direct quotation from participants were used to support the themes. Information from the key informant interviews and survey were triangulated during the discussion of the findings [37]. The quantitative data on the other hand was analyzed using R statistical software version 4.0.0 [38]. The sum of the scores of the seven statements was calculated for each survey respondent, after which a median of these scores calculated. Perception level was determined by comparing individual perception scores against the median perception score; scores above the median were considered negative while those at or below the median were considered positive. Internal validity of the scale was measured by calculating Cronbach’s alpha [39]. Univariate analysis was used to determine influence of the respondent sex, age group, education level and degree of previous awareness of larviciding on the main outcome variable, i.e. their perceptions of larviciding. Binary logistic regression was used to determine the association between the independent variables and the outcome variable; odds ratio was calculated at 95% confidence intervals (CIs). Results Page 6/25 Characteristics of study respondents A total of 517 people (43% men and 57% women) participated in this study. These included the 27 key informants who participated in the in-depth interviews, and 490 community members responding to the administered questionnaires. Nineteen of the 27 KII participants were men, and all participants had a college or university degrees. The average age of participants in KII was 45 years, ranging from 33 to 60 years. Average duration of employment in their current position and at their current location was 7 years, ranging from six months to 35 years (Table 1). Average age of the community members who participated in the survey was 42 years (range: 18 – 88 years) and two thirds (66%, n=321) were married. About three quarters (73.1%, n=358) had primary school education, 8.8% (n=43) had no formal education, 13.9% (n=68) had secondary education and 4.3% (n=21) had college-level education. A majority (84.3%, n=413) of the respondents reported small-scale farming as their main income-generating activity, but people also practiced small retail businesses, fishing, animal husbandry or had formal employment. Table 1: Characteristics of Key Informant Interviewees Key Informants Mean age (Years) Average no. years in service Males Females Total Malaria Focal Persons 40.1 4.5 6 3 9 Vector Surveillance Officers 47.9 7.4 6 3 9 Ward Health Officers 47.2 9.2 7 2 9 All Participants 45.1 7.0 19 8 27 able 1: Characteristics of Key Informant Interviewees Perception regarding malaria burden Perception regarding malaria burden Table 2 summarizes the respondent perceptions regarding malaria burden in Tanzania. Nearly a half of the survey respondents reported not knowing the current malaria prevalence range in Tanzania. Only 15.3% identified correct range of nation-wide prevalence (6-10% based on 2018 Malaria Indicator Survey [33]). Two thirds believed that rural communities or poor households suffer the heaviest burden. More than a half of respondents believed the country was progressing well towards elimination, and that it could achieve elimination with current interventions. However, a majority of the survey respondents noted that alternative interventions would be necessary to speed up these efforts (Table 2). areness of community members regarding larviciding as a malaria interventio Only a quarter of survey respondents were aware of the government policy to include larviciding as a malaria intervention (Table 3), and more than half did not know whether the intervention was ongoing in their districts. Three quarters also did not know the mode of action of larvicides despite knowing what the Page 7/25 intervention itself is. Older respondents (46 - 55 years) were more aware of larviciding than those 25 years or younger. Table 2: Community perceptions regarding malaria risk and burden (N = 490) General perception of larviciding and its potential as a malaria intervention Perception of community members towards larviciding was assessed based on levels of agreement towards positive statements on a 5-point Likert-scale, ranging from strongly agree to strongly disagree. The median score of the seven statements was 21. Reliability assessment of the perception scale yielded a Cronbach alpha score of 0.77, indicating acceptable reliability of the scale and minimum redundancy. Of all survey participants, 40.4% agreed that larviciding would be acceptable in their community as new intervention. The rest of the community members had neutral perceptions on effectiveness, safety, feasibility, accessibility, affordability or acceptability of larviciding (Table 4). Community members who were already aware of larviciding were more likely to welcome larviciding compared to respondents without previous knowledge prior to the survey (p = 0.029), Table 5). However, three quarters (74.2%, n=364) of respondents said they would support larviciding if introduced to their communities. Table 2: Community perceptions regarding malaria risk and burden (N = 490 Table 2: Community perceptions regarding malaria risk and burden (N = 490) Page 8/25 Questions asked Variables Percentage (n) Which settings are at highest risk of malaria? Rural settings 65.1% (319) Urban settings 7.6% (37) Equal in rural and urban settings 23.7% (116) Do not know 3.7% (18) Which communities are most affected by malaria? Low-income communities 63.9% (313) All communities are equally affected 33.7% (165) Do not know 2.5% (12) Where does most malaria transmission occur? Outdoors 61.3% (300) Indoors 36.7% (180) Do not know 2.0% (10) What is your opinion regarding country’s progress towards malaria elimination Very good 51.6% (253) Good but slow 43.9% (215) Very slow 4.5% (22) Can malaria be eliminated Possible 59.6% (292) Not possible 40.4% (198) Do we need alternative interventions? Table 4: Perception of community members regarding effectiveness, feasibility, affordability and acceptability of larviciding for malaria prevention (N = 490). Awareness, perceptions and experiences of district and ward-level health officials regarding larviciding for malaria control Awareness, perceptions and experiences of district and ward-level health officials regarding larviciding for malaria control Important aquatic habitats of malaria vectors: In the initial analysis, most KII participants reported that they knew the general characteristics of mosquito aquatic habitats, but not all were able to distinguish between habitats of key malaria vectors and habitats of other mosquitoes. When asked to describe the aquatic habitats of important malaria vectors, respondents used terminologies such as fresh waters, standing waters, pit latrines, trash pits, septic pits, used tires, long grass and bushes. When considered separately, most malaria focal persons and vector surveillance officers were able to distinguish between aquatic habitats of malaria vectors. They pointed out that Anopheles mosquitoes prefer fresh waters. A small number of MFPs however were unable to make this distinction, despite knowing that some mosquitoes preferred fresh water. They were unable to specify key characteristics of the actual malaria vectors as distinguishable from the habitats of non-vectors. On the other hand, a majority of the ward health officers were not aware of the differences in breeding habitats between malaria and non- malaria vectors. This group only knew that mosquitoes breed in water. They identified ponds, streams and river banks, septic tanks and pit latrines as possible breeding habitats for all mosquitoes. They conceded that differentiating larval habitats was too technical a task for their capacities; their focus was on identifying places with standing water and treating them with larvicides. “It is not too easy to differentiate between the larval habitats, except if you see a place with a lot of water, then you just know that there will be mosquito larvae there, because we know mosquitoes like to lay their eggs in water. In my ward, for example, we have water ponds that last a whole year, so I know mosquitoes breed there. There are also communities where people still use pit latrines, but the holes are not covered and the toilets do not have doors or roofs. So I also know that mosquitoes can breed in those.” (Ward Health Officer, Male). The term ‘fresh water’ generated great discussion among the key informants. Those who reported that malaria vectors preferred clean and fresh water also listed water storage buckets or pots and morning dew as potential habitats for malaria vectors. General perception of larviciding and its potential as a malaria intervention There is a need 86.1% (422) No need 13.9% (68) Table 3: Knowledge and awareness of larviciding in the communities (N = 490) Questions asked Questions asked Page 9/25 Page 9/25 Page 9/25 Variable assessed Response Percentage (n) Awareness of larviciding (n=490) Yes 26.1% (128) No 73.9% (362) Sources of information (n=128) Friends/family 48.1% (76) Radio/TV 21.5% (34) IHI scientists 10.8% (17) Community meetings 7.6% (12) Saw on a visit in Dar es Salaam 7.6% (12) Community health workers 4.4% (7) Has larviciding been implemented in the community (n=490) Yes 4.5% (22) No 43.5% (213) Do not know 52.2% (255) Larviciding works by killing mosquitoes in their juvenile stage (n=490) Agree 23.9% (117) Do not agree 2.0% (10) Do not know 74.1% (363) Variable assessed Table 4: Perception of community members regarding effectiveness, feasibility, affordability and acceptability of larviciding for malaria prevention (N = 490). Table 4: Perception of community members regarding effectiveness, feasibility, affordability and acceptability of larviciding for malaria prevention (N = 490). Page 10/25 Statement Strongly agree (1) Agree (2) Neutral (3) Disagree (4) Strongly disagree(5) Will be effective 29.8% 14.7% 54.5% 0.4% 0.2% Will fill gaps left by ITNs 28.4% 13.1% 56.1% 1.2% 1.2% Will be safe for humans, animals and environment 7.1% 8.4% 76.9% 3.9% 3.7% Will be easy to use 19.6% 4.7% 72.5% 2.0% 1.2% Will be easily accessible 2.6% 2.2% 84.1% 4.1% 6.9% Will be affordable to residents 2.9% 1.4% 86.7% 1.6% 7.4% Will be acceptable in community 34.3% 6.1% 56.7% 2.2% 0.6% Table 5: Association between the community perception towards larviciding and their socio-demographic characteristics. The odds and p values represent likelihood of certain groups having a favorable opinion of larviciding as a malaria intervention. Category Variable Odds ratio (95% CI) p-value Sex Male 1.00 - Female 0.74 (0.32, 1.70) 0.470 Age category (in years) 18-25 1.00 - 26-35 0.53 (0.14, 2.58) 0.382 36-45 0.56 (1.34, 2.76) 0.428 46-50 0.42 (0.07, 2.36) 0.300 Above 50 0.60 (0.14, 3.04) 0.497 Education Level No formal education 1.00 - Primary (7 years) 2.09 (0.41, 38.20) 0.478 Secondary (12 years) 1.94 (0.24, 39.90) 0.752 Tertiary (>12 years) 7.00 (0.83, 146.87) 0.102 Awareness of larviciding Aware 1.00 - Not aware 0.40 (0.17, 0.93) 0.029* Page 11/25 Page 11/25 Awareness, perceptions and experiences of district and ward-level health officials regarding larviciding for malaria control “What I know is that there are different types of mosquitoes; I know there are Anopheles, Culex and Aedes mosquitoes. I know that Anopheles prefers to breed in clean and fresh water, so they can be found in buckets of clean water, in the clean morning dew. Culex on the other hand likes dirty water; they like to lay their eggs in septic pits and in other dirty places.” (Vector Surveillance Officer, Male). Knowledge of larviciding: All MFPs, VSOs and ward health officers knew that larviciding involved killing mosquitoes with chemicals during their larval stages. They also knew of two types of biolarvicides (i.e. Bti and Bs) available for large-scale implementation in Tanzania, one used to treat fresh and clean water, and the other one used to treat dirty water. Many could however not name the biolarvicides, nor specify which types were applicable for malaria-vector control. Page 12/25 “Larviciding it is the killing of the second stage of mosquito’s life cycle using chemicals called larvicides. In Tanzania we have biological larvicides, so they are called biolarvicides. I understand that these biolarvicides are some kind of bacteria; when they are put in water that contains mosquito larvae, the larvae feed on the bacteria, which kills them.” (Malaria Focal Person, Male). Supply and distribution of larvicides: MFPs reported having received two types of biolarvicides (totaling 720 litres per council) from the government to distribute to the wards within their districts through ward health officers. The first supply was delivered in 2018, and another supply delivered in 2019. It was noted that the distribution of the biolarvicides had been prioritized on wards with the highest reported malaria cases compared to others. Implementation of larviciding: To support larviciding, the ward health officers recruited and trained community health workers (CHW), local residents who had previously participated in a community health training course. Where no CHWs were available, the ward health officers recruited volunteers, who were typically young male residents. The CHWs or volunteers were responsible for actual application of larvicides, with supervision from the ward health officers. The ward health officers would accompany the implementers to identify water bodies within their wards and during the first application. Unfortunately, a majority of the ward health officers had received no specific training on how to implement the larviciding. Awareness, perceptions and experiences of district and ward-level health officials regarding larviciding for malaria control Moreover, in some districts one ward health officer was responsible for overseeing larviciding in up to four wards, thus they were unable to effectively supervise the CHWs. “I supervised this work throughout. I recruited community health workers from different communities in my ward and gave them larvicides. This way I made sure that every community in my ward had larvicides.” (Ward Health Officer, Male). “We were told to involve the community when we received the larvicides, so we spoke with village and community leaders, and with their help we found young men in the communities to help with this work. We then instructed the young men on how to apply the larvicides.” (Ward Health Officer, Male). Training on application of bio-larvicides: Malaria focal persons reported that they had participated in at least one seminar on how to apply the larvicides, in 2018 and or 2019. Some of the MFPs were not holding their current positions in 2018 and had therefore only received one training session. The training, provided jointly by the Muheza College of Health and Allied Sciences [40] at Muheza district and Kibaha Biotech Products Limited (TBPL) [29], was described as largely theoretical, providing information on the two types of biolarvicides and where to use them. There had been no practical training on identification of aquatic habitats, application of larvicides or monitoring of program effectiveness. Fortunately, all MFPs had been given written guidelines for biolarvicides application. “I participated in this year’s [2019] seminar. We were given a formula on how to calculate the amount of larvicides per liter, and they promised to share with us the template with the specific formula for the amount of diluted larvicides to apply in a breeding habitat. It was a PowerPoint presentation; it was all theoretical.” (Malaria Focal Person, Male). Page 13/25 Page 13/25 Unlike the MFPs, the VSOs and ward health officers reported not to have participated in the training programs, but had instead received information on dilution and application methods from the MFPs. Ward health officers then passed on the information to the CHWs and the community volunteers who were responsible for the hands-on implementation of the larviciding. Unlike the MFPs, the VSOs and ward health officers reported not to have participated in the training programs, but had instead received information on dilution and application methods from the MFPs. Awareness, perceptions and experiences of district and ward-level health officials regarding larviciding for malaria control Ward health officers then passed on the information to the CHWs and the community volunteers who were responsible for the hands-on implementation of the larviciding. “I called the volunteers to my office and explained how to dilute the larvicides and how to apply them to the breeding habitats. I did the training in my office. Then I provided them with the larvicides as well as masks to protect themselves.” (Ward Health Officer, Female). Monitoring efficacy of the larvicides: There was no formal mechanism of monitoring effectiveness of the larviciding. Some ward health officers stated that they kept track of the number of malaria cases at the health centers, and assumed that reduced cases meant that the larviciding was working. Other ward health officers reported that they asked community members if they had experienced a reduction in mosquito annoyance. Others relied on their own experience living in the communities to detect a reduction in mosquito abundance. All respondents reported that they believed that larvicides were effective based on these factors. Challenges during implementation of larviciding: Key challenges that district and ward health control officers faced during implementation of larviciding are summarized on table 6 below. The challenges listed included insufficient technical knowledge on identifying habitats of malaria vectors and application of the larvicides, insufficient knowledge on safety of the larvicides, inadequate funding, inadequate supply of larvicides, some resistance from community members, late-involvement of VSOs and ward health officers and inadequate collaboration from non-governmental organizations in the districts or wards. Table 6: Key challenges facing larviciding programs in Morogoro region, southern Tanzania. The table provides a brief description of each identified challenge, as well as examples of direct statements from the study respondents. Page 14/25 Page 14/25 Page 14/25 Challenges Description Examples of respondent quotes “it is not easy to differentiate mosquito breeding sites, however, there are areas that you can recognize as breeding sites upon seeing. For example, we have areas with ponds that last the whole year and a great example is an area close to the secondary school where brick laying created ponds which obvious attract mosquitoes as a breeding site.” (Ward Health Officer, Male). “We do monitoring by asking community members, they are the ones who report sleeping comfortably.” (Ward Health Officer, Female). 2 Lack of knowledge regarding safety of the larvicides “I know that it is safe on humans, but I really do not know if they pose any harm on other insects in the water, on animals or on vegetation around the water. I only know that it does not have any harm on humans.” (Malaria Focal Person, Male). “We look at the statistics, as to whether number of malaria patients increasing or decreasing.” (Ward Health Officer, Female). Awareness, perceptions and experiences of district and ward-level health officials regarding larviciding for malaria control 1 Insufficient technical knowledge on habitat identification and larviciding Malaria Focal Persons, District Surveillance Officers and Ward Health Officers reported that they did not have adequate technical knowledge for assessing whether specific water bodies were likely to contain mosquito larvae, and whether those larvae were likely to belong to Anopheles species or other mosquitoes. As a result, ward health officers reported that they often treated all the water bodies they could find in their wards. The MFPs also reported that they did not have accurate information on the proper amount of larvicides to apply in specific water bodies. Instead, they often just guessed the amount, based on their perceived volumes of the habitats. “Like I said, we lack knowledge on this aspect. We do not even know how much larvicides to spray in a water pond for example. Even if you ask the VSO he will tell you the same. So then we do a lot of guess work, but we do not know for sure if we are putting too much or too little.” (Malaria Focal Person, Female). There was also no uniformity on methods of monitoring efficacy of the larvicides. Some reported that they used number of malaria cases at the health centers as an indicator of efficacy and some used community testimonials on reduced mosquito nuisance bites. “We look at the statistics, as to whether number of malaria patients increasing or decreasing.” (Ward Health Officer, Female). “I know that it is safe on humans, but I really do not know if they pose any harm on other insects in the water, on animals or on vegetation around the water. I only know that it does not have any harm on humans.” (Malaria Focal Person, Male). There were also inconsistencies in knowledge about risks posed by the larvicides. MFPs and VSOs claimed that the larvicides did not pose any harm to people or their livestock, but were not sure whether the larvicides could cause harm to other aquatic organisms. In contrast, most ward health officers believed the larvicides could harm people or animals, since they smelled like poison and turned the color of the water. Page 15/25 Page 15/25 “It has to have harm, I can just tell from the smell that comes when you apply it, the water also turns milky, so it just looks poisonous. Awareness, perceptions and experiences of district and ward-level health officials regarding larviciding for malaria control So I advise people to not use the water immediately after the application, but if they wait after a while the smell disappears and the color goes back to normal.” (Ward Health Officer, Female). “When you ask people in the community to help with this exercise, they expect to get a wage. But when we were implementing this there wasn’t any money set aside for paying the volunteers or the CHWs. Sometimes I had to give them my own money, because I saw how hard they were working.” (Ward Health Officer, Male). All participants reported that lack of sufficient funding was a significant obstacle for successful implementation of larviciding. Funding was needed to provide compensations and wages to the CHWs or the volunteers, procure personal protective gear and application equipment and for transportation. In some cases the participants reported limiting larviciding activities due to limited financial support. “In my district we had to stop before finishing because we just did not have any money to implement this project. We had the larvicides only, but nothing else. We requested money for protective gear, transportation, or for paying people that were doing the application but we did not receive it, so after some time we just had to stop.” (Vector Surveillance Officer, Female). “For an example, my district has 31 wards, and it is not like the breeding habitats are at the headquarters of the wards. You have to go deep into the villages. It is hard to walk with a can containing 20-liters of larvicide. There is only one car at the district, and even that is currently not functioning.” (Vector Surveillance Officer, Male). nadequate upply of arvicides: Some of the ward health officers reported that the larvicides they received were not enough to treat all mosquito breeding habitats in their “I will tell you that the larvicides were not enough. In all the breeding habitats that I had “I will tell you that the larvicides were not enough. In all the breeding habitats that I had Page 16/25 area of jurisdiction. In particular, communities living in swampy areas, needed a lot more supplies than they received. surveyed, we could not cover all of them before running out of the larvicides. We needed more, but there was none.” (Ward Health Officer, Female). “In 2018, I have received two cans of twenty liters which cannot be enough for my ward. area of jurisdiction. In particular, communities living in swampy areas, needed a lot more supplies than they received. surveyed, we could not cover all of them before running out of the larvicides. We needed more, but there was none.” (Ward Health Officer, Female). 5 Some resistance from members of the community Discussion Larviciding is considered as complementary option to be used alongside current major malaria control approaches, notably ITNs, IRS and case management [41]. To accelerate malaria elimination efforts, the Tanzanian government has invested significantly in larviciding, including the establishment of a national production capacity and adoption of larviciding in both rural and urban settings [26]. This study investigated some of the practical obstacles that limit the effective roll-out of this strategy across the country, with a particular focus on the perceptions and experiences of key stakeholders of malaria control i southern Tanzania. The key-informant interviews revealed significant knowledge inadequacies among MFPs, VSOs and ward health officers towards implementation of the larviciding. For instance, all participants knew that mosquitoes have an aquatic habitat stage; but a majority could not easily differentiate the aquatic habitats typical of malaria vector species. Moreover, these health officials reported that malaria vectors do prefer “fresher” water compared to other mosquitoes, but what majority meant by fresh water was any water that looked clean such as water in clay pots or buckets. Ward health officers, who are closely anchored in the community and provide guidance to the community health workers and volunteers during the larviciding, could not differentiate between malaria and non-malaria vectors’ aquatic habitats and reported to use different methods to apply and monitor effectiveness of the larvicides. This lack of adequate knowledge and uniformity might be attributable to the lack of training on how, where and when to apply the larvicides as accorded by WHO guidelines [41]. Some of these malaria control officials particularly MFPs and VSOs reported to have attended at least one theoretical training on larviciding. However, those training proved to be insufficient as acquiring necessary expertise would require practical, “on the job” training rather than a presentation of theoretical principles [42]. No formal training to the actual implementers (i.e. ward health officers, CHWs and volunteers) was reported, this could undermine the overall impact of the programme. Insufficient funding to assist with implementation of larviciding was one of the practical obstacles reported by the MFPs, VSOs and ward health officers. Funding was needed to offer incentives, cover transportation and larvicides costs, and provide personal protective gears to the CHWs and volunteers who did the actual job of applying the larvicides. A successful large scale larviciding trials conducted in Dar-es-Salaam [25,43] in early 2000s had demonstrated the cost-effectiveness of the approach [44]. Awareness, perceptions and experiences of district and ward-level health officials regarding larviciding for malaria control In another round, I had received two cans of twenty liters per village which was not enough either, so we decided to prioritize the most significant settings.” (Ward Health Officer, Female). “The uptake was not very good in the beginning as people were not educated on what larvicides are, how they work or their safety. So they were always reluctant to let people spray near their homes.” (Vector Surveillance Officer, Female). Key informants reported initially facing resistance from some community members who feared that the larvicides would be poisonous to chicken, livestock or fish. This was mostly due to the smell of the larvicides, and by the fact that the water turned milky immediately after application. This initial resistance was however reported to ease once the health officials spent time explaining the benefits and safety of the larvicides. y Community sensitization was primarily done by ward health officers with assistance from CHWs. “Once people were sensitized, the uptake improved. People would even follow us and ask when we would be spraying again, or point me to breeding habitats that I had missed.”(Ward Health Officer, Male). VSOs and ward health officers reported to not being involved in the initial planning of the larviciding programme at the district level, but rather receiving implementation plan from malaria focal person. This overshadows their significant inputs as they have spent more time in the settings on average compared to malaria focal persons. “I was not involved in the planning and these larvicides are new which requires training but we have only been given pamphlets. Only if we can be involved from the early stages, I think it will improve the practice.” (Vector Surveillance Officer, Female). “Providing awareness to the community, maybe we could try but even Boresha Afya indicated disease prevention is not in their priorities but rather case management. SolidarMed priorities are in behavioral change, so we have no stakeholders in disease prevention.” (Malaria Focal Person, Male). Key informants reported inadequate involvement of the non-governmental organizations (NGOs) in the implementation of the larviciding programme. This has been attributed to larviciding not being priority among these NGOs. Page 17/25 Discussion However, larviciding is deemed operationally and financially infeasible in the rural settings [41]. Fortunately, a recent study by Nambunga et al in rural Tanzania highlighted the possibility of minimizing the unnecessary costs, if larviciding could be species-specific [31]. In Kilombero valley, An. funestus accounts for over 80% of the ongoing malaria transmission [8], its aquatic habitats have found to be few and highly distinctive [31]. Thus, effective targeting of An. funestus aquatic habitats alone could potentially reduce malaria transmission by 80% in Kilombero valley. In this valley, An. funestus aquatic habitats adhere to WHO criteria (i.e. few, fixed and findable) for larviciding implementation [41]. The application of larvicides for malaria control in Morogoro region is often directed towards all stagnant water bodies, thus undermining the intended amount of larvicides. Understanding the ecology of major malaria vectors in each district within Morogoro region could cut the unnecessary costs and provide Page 18/25 Page 18/25 Page 18/25 effective larviciding approach. However, studies shows that control of Culicine mosquitoes that are responsible for enormous biting nuisance could maximize community acceptance and support towards malaria control programme [45,46]. This present study also revealed the need to strengthen engagement of key stakeholders including the community. Despite efforts by district-level malaria control officials to inform and sensitize the residents, a majority of the community members surveyed were not aware of larviciding, did not know its function within malaria control efforts, and were not aware whether or not it had been implemented in their settings. This finding was in agreement with a previous study by Mboera et al (2014) in Mvomero district within Morogoro region, where only 17% of the survey respondents were aware of larviciding as a malaria control intervention [47]. Both findings indicate inadequate community engagement methods during the implementation stage. However, community members in both studies showed willingness to support the implementation of larviciding in their communities. In our present study, age, sex and educational level of the survey respondents did not seem to influence their level of awareness and perception towards larviciding, but the contrary was observed in other studies [48,49]. Majority of the districts in Morogoro region have at least one local radio station, which may be relied upon to further strengthen the community engagement. Insufficient support from local stakeholders within Morogoro region might have been among the obstacles towards effective implementation of larviciding. Ethics approval and consent to participate Ethical approvals for this project were obtained from Ifakara Health Institute’s Institutional Review Board (Protocol ID: IHI/IRB/EXT/No: 007 - 2018) and the Medical Research Coordinating Committee (MRCC) at the National Institute for Medical Research, in Tanzania (Protocol ID: NIMR/HQ/R.8a/Vol.IX/2895), as well as University of the Witwatersrand (UW) in South Africa (Clearance certificate No. M180820). Written consents were sought from all participants of this study, after they had understood the purpose and procedure of the discussions. Conclusion Both communities and district-level malaria control officials widely supported the larviciding programme, however, there were gaps in technical knowledge, implementation and stakeholder engagement. To maximize the overall impact of the programme, training efforts should be intensified, particularly for identification of aquatic habitats for important vectors and formal training should be given to the actual implementers (i.e. CHWs and volunteers) not just MFPs, VSOs and ward health officers. Standard technical principles for application of larvicides should strictly be adopted and improvement on financing at a district- level implementation. Furthermore, engagement of community members and other stakeholders such as NGOs should be improved to maximize awareness, participation and sustainability of the programme. These lessons learnt from Morogoro region shed the light for other malaria endemic areas on the possibility of deploying larviciding for malaria control or elimination. Abbreviations KI: Key informant; KII: Key Informant Interview; MFP: Malaria focal person; VSO: Vector surveillance officer; NMSP: National Malaria Strategic Plan; ITN: Insecticide-Treated bed Net; IRS: Indoor Residual Spraying; Bti: Bacillus thuringiensis var. israelensis; Bs: Bacillus sphaericus; IHI: Ifakara Health Institute. Discussion Engagement of other stakeholders particularly non- government organizations (NGOs) have shown to yield fruitful impact in the malaria control. For instance, collaboration between Urban Malaria Control Programme (UMCP) and Ifakara Health institute (IHI) in Dar- es-Salaam during early 2000s towards malaria control through larval source management led to a significant impact [25]. Thus, effective engagement of these NGOs such as IHI will somewhat ensure smooth implementation of larviciding through resources provision and/or capacity building. Insufficient support from local stakeholders within Morogoro region might have been among the obstacles towards effective implementation of larviciding. Engagement of other stakeholders particularly non- government organizations (NGOs) have shown to yield fruitful impact in the malaria control. For instance, collaboration between Urban Malaria Control Programme (UMCP) and Ifakara Health institute (IHI) in Dar- es-Salaam during early 2000s towards malaria control through larval source management led to a significant impact [25]. Thus, effective engagement of these NGOs such as IHI will somewhat ensure smooth implementation of larviciding through resources provision and/or capacity building. Our study also revealed insufficient “early-on” involvement of VSOs and ward health officers during the budgeting and implementation planning. MFPs attend all council’s meeting that involve malaria control initiatives through district technical committee [50], and often instruct the VSOs and ward health officers on the way forward. This could lower the sense of ownership towards the larviciding programme. Adequate involvement of VSOs and ward health officers could strengthen the implementation of the programme, apart from VSOs holding a special training on disease-vectors control but also majority have spent significant number of years in the localities. The study results should be interpreted in the light of several limitations. A response bias may have resulted partially inaccurate responses on the survey. Social desirability bias may have resulted in respondents saying ‘I don’t know’ to most of the statements that assessed their perceptions of larviciding as a majority had early-on indicated that they were not aware of this intervention. Demand characteristics may have also resulted from both the key informants who may have reported insufficient knowledge or lack of resources hoping that these would be provided to them. In addition, our present study did not include district medical officers (DMO) who also plays a crucial role in planning, coordinating and implementing the delivery of health services at the district level [30]. Page 19/25 Page 19/25 Consent for publication Permission to publish this study was obtained from National Institute for Medical Research, in Tanzania (NIMR/HQ/P.12 VOL XXXI/67). Availability of data and materials The data will be made available by the corresponding author upon reasonable request. The data will be made available by the corresponding author upon reasonable request. Author contributions SAM, MFF, FOO, NC, AHK and JL were involved in study design. SAM, MFF and IHN were involved in data collection. SAM and MFF conducted data analysis. SAM and MFF wrote the manuscript. KU and BJM facilitated initial meeting with Morogoro region malaria control officials. FOO, NC, AHK, JL, FT, PC, KU and BJM provided thorough review of the manuscript. All authors read and approved the final manuscript. Acknowledgments We would like to thank the malaria focal persons, vector surveillance officers, ward health officers and community members for their participation in this study. We would also like to extend our sincere gratitude to Ms. Anna Nyoni for her assistance in transcribing and translating audio recordings verbatim. We are also grateful to Mr. Augustino Mwambaluka and Ms. Rukiyah Njalambaha for their transport and administrative supports respectively. Our sincere gratitude goes to Dr. Gwakisa John for facilitating a meeting between IHI researchers and district-level malaria control officials from all nine administrative councils of Morogoro region. We also thank Ms. Najat Kahamba for the map of the study area. Competing interests The authors declare no competing interests. Page 20/25 This study was supported by the Wellcome trust International Masters Fellowship in Tropical Medicine and Hygiene (Grant No. 212633/Z/18/Z) awarded to SAM, Bill and Melinda Gates Foundation (Grant Number: OPP1177156) and the ANTI-veC Network (Grant Number: AVPP0027/1) all held at Ifakara Health Institute. MF was also supported through a Consortium for Advanced Research Training grant awarded by Wellcome Trust (Grant No: 087547/Z/08/Z). FOO was also funded by Howard Hughes Medical institute (Grant number: OPP1099295). elimination. Malar J. 2014; 8. Kaindoa EW, Matowo NS, Ngowo HS, Mkandawile G, Mmbando A, Finda M, et al. Interventions that effectively target Anopheles funestus mosquitoes could significantly improve control of persistent malaria transmission in south-eastern Tanzania. PLoS One. 2017;12. 9. Matowo NS, Munhenga G, Tanner M, Coetzee M, Feringa WF, Ngowo HS, et al. 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Challenges of implementing a large scale larviciding campaign against malaria in rural Burkina Faso - lessons learned and recommendations derived from the EMIRA project. BMC Public Health. 2016; 42. Dambach P, Traoré I, Kaiser A, Sié A, Sauerborn R, Becker N. Challenges of implementing a large scale larviciding campaign against malaria in rural Burkina Faso - lessons learned and recommendations derived from the EMIRA project. BMC Public Health. 2016; 43. Chaki PP, Dongus S, Fillinger U, Kelly A, Killeen GF. Community-owned resource persons for malaria vector control: Enabling factors and challenges in an operational programme in Dar es Salaam, United Republic of Tanzania. Hum Resour Health. 2011;9. 43. Chaki PP, Dongus S, Fillinger U, Kelly A, Killeen GF. Community-owned resource persons for malaria vector control: Enabling factors and challenges in an operational programme in Dar es Salaam, United Republic of Tanzania. Hum Resour Health. 2011;9. 44. Maheu-Giroux M, Castro MC. Cost-effectiveness of larviciding for urban malaria control in Tanzania. Malar J. 2014;13. 44. Maheu-Giroux M, Castro MC. elimination. Malar J. 2014; Cost-effectiveness of larviciding for urban malaria control in Tanzania. Malar J. 2014;13. 45. Stephens C, Masamu ET, Kiama MG, Keto AJ, Kinenekejo M, Ichimori K, et al. Knowledge of mosquitos in relation to public and domestic control activities in the cities of Dar es Salaam and Tanga. Bull World Health Organ. 1995;73:97–104. 45. Stephens C, Masamu ET, Kiama MG, Keto AJ, Kinenekejo M, Ichimori K, et al. Knowledge of mosquitos in relation to public and domestic control activities in the cities of Dar es Salaam and Tanga. Bull World Health Organ. 1995;73:97–104. 46. Chavasse DC, Lines JD, Ichimori K. The relationship between mosquito density and mosquito coil sales in Dar es Salaam. Trans R Soc Trop Med Hyg. 1996;90:493. 46. Chavasse DC, Lines JD, Ichimori K. The relationship between mosquito density and mosquito coil sales in Dar es Salaam. Trans R Soc Trop Med Hyg. 1996;90:493. 47. Mboera LEG, Kramer RA, Miranda ML, Kilima SP, Shayo EH, Lesser A. Community knowledge and acceptance of larviciding for malaria control in a rural district of east-central Tanzania. Int J Environ Res Public Health. 2014; 47. Mboera LEG, Kramer RA, Miranda ML, Kilima SP, Shayo EH, Lesser A. Community knowledge and acceptance of larviciding for malaria control in a rural district of east-central Tanzania. Int J Environ Res Public Health. 2014; 48. Finda MF, Kaindoa EW, Nyoni AP, Okumu FO. “The mosquitoes are preparing to attack us”: Knowledge and perceptions of communities in south-eastern Tanzania regarding mosquito swarms. Malar J. 2019; 48. Finda MF, Kaindoa EW, Nyoni AP, Okumu FO. “The mosquitoes are preparing to attack us”: Knowledge and perceptions of communities in south-eastern Tanzania regarding mosquito swarms. Malar J. 2019; 49. Finda MF, Christofides N, Lezaun J, Tarimo B, Chaki PP, Kelly AH, et al. Opinions of key stakeholders on alternative interventions for malaria control and elimination in Tanzania. 2020; Available from: https://malariajournal.biomedcentral.com/articles/10.1186/s12936-020-03239-z 49. Finda MF, Christofides N, Lezaun J, Tarimo B, Chaki PP, Kelly AH, et al. Opinions of key stakeholders on alternative interventions for malaria control and elimination in Tanzania. 2020; Available from: https://malariajournal.biomedcentral.com/articles/10.1186/s12936-020-03239-z 50. Welfare S, Government L. Functions of Regional Health Management System [Internet]. 2008 [cited 2020 Aug 24]. p. 1–50. Available from: http://hssrc.tamisemi.go.tz/storage/app/uploads/public/5eb/3e2/cd3/5eb3e2cd379e7997437848.pdf 50. Welfare S, Government L. Functions of Regional Health Management System [Internet]. 2008 [cited 2020 Aug 24]. p. 1–50. Available from: http://hssrc.tamisemi.go.tz/storage/app/uploads/public/5eb/3e2/cd3/5eb3e2cd379e7997437848.pdf Figures Figures Page 24/25 Page 24/25 Page 24/25 Page 24/25 Figure 1 Map of Morogoro Region, Tanzania, showing the districts, wards and villages where the study was conducted. Map prepared by Najat Kahamba. Figure 1 Map of Morogoro Region, Tanzania, showing the districts, wards and villages where the study was conducted. Map prepared by Najat Kahamba. Map of Morogoro Region, Tanzania, showing the districts, wards and villages where the study was conducted. Map prepared by Najat Kahamba. Page 25/25 Page 25/25 Page 25/25 Page 25/25
https://openalex.org/W2804641001	http://pdf.blucher.com.br/designproceedings/eneac2018/107.pdf	Portuguese	null	AVALIAÇÃO DA ACESSIBILIDADE EFETIVA DO BLOCO “G” DA UNIVERSIDADE CATÓLICA DE PERNAMBUCO	Blucher Design Proceedings	2,018	cc-by	953	ABSTRACT This work contemplates the study of Effective Accessibility with the object "Block" G of the Universidade Católica de Pernambuco. Techniques and procedures proposed by the Theory of Effective Accessibility were applied in order to obtain a preliminary assessment of the conditions of physical access to the building. Keywords: Effective Accessibility; Ergonomics; Universal Design. Keywords: Effective Accessibility; Ergonomics; Universal Design. RESUMO Este trabalho contempla o estudo de Acessibilidade Efetiva tendo como objeto o Bloco “G” da Universidade Católica de Pernambuco. Foram aplicados técnicas e procedimentos propostos pela Teoria da Acessibilidade Efetiva com o intuito de obter uma avaliação preliminar das condições de acesso físico à edificação. Palavras chave: Acessibilidade Efetiva; Ergonomia; Desenho Universal. 2. CONCEITUAÇÃO TEÓRICA A Acessibilidade Efetiva pode ser compreendida como resultado das interações entre as aptidões de um elemento acessante, as exigências das atividades realizadas, a configuração do elemento acessado e o contexto circunstancial em que este se encontra, configurando um SATA - Sistema Acessante - Tarefa - Acessado. A Teoria é baseada em três pilares: Ergonomia, Desenho Universal e na Classificação Internacional de Funcionalidade Incapacidade e Saúde, que defende que o desempenho de uma pessoa só pode ser analisado se for considerando a influência de fatores ambientais, que podem ser barreiras ou facilitadores (BAPTISTA, 2010) 3. PROCEDIMENTOS METODOLÓGICOS Nesta etapa do desenvolvimento do trabalho, foi realizada uma apreciação subjetiva pelo próprio pesquisador considerando três tipos de Sistema Acessante (Usuários/Pessoa), e dois tipos de tarefas associados a um contexto específico, são eles:  Acessante 01 - Pessoa sem nenhum tipo de limitação física (grupo de controle para comparações);  Acessante 01 - Pessoa sem nenhum tipo de limitação física (grupo de controle para comparações);  Acessante 02 - Pessoa com restrição físico-motora, usuário de cadeira de rodas;  Acessante 03 - Pessoa com restrição sensorial – visual, usuária de bastão de rastreamento;  Contexto positivo - Utilização a passeio (dia sem muita movimentação e tarefas amenas a passeio – ênfase nas barreiras fixas);  Contexto negativo - Utilização a trabalho (dia/horário de grande movimentação e tarefas realizadas apressadamente – barreiras fixas e móveis). 1. INTRODUÇÃO O Bloco “G” é a principal edificação da Universidade Católica de Pernambuco (UNICAP). Está localizado em uma área privilegiada do centro da cidade, e é desfrutado diariamente por um número significativo de pessoas. Não apenas por alunos, professores e funcionários, mas também, por transeuntes que optam por acessar o local seja para utilização de seus serviços ou como espaço de passagem para seus destinos ou outras áreas da UNICAP. Estas pessoas possuem qualidades, funções e limitações diferentes, trazendo a necessidade de se existir um espaço que possa ser acessível por todos de forma igualitária, independente de suas limitações físicas, sensoriais ou cognitivas. Com este intuito, o trabalho busca a verificação da Acessibilidade Efetiva do bloco em estudo, para entender em uma abordagem ergonômica o nível de acessibilidade e buscar possíveis soluções visando um Desenho Universal. 4. LEVANTAMENTO E CONSIDERAÇÕES O bloco em estudo (Bloco G) é um dos maiores blocos da instituição e o que comporta o maior número de alunos. Para a maioria dos transeuntes que não possuem limitações físicas (Ambulantes) quase não encontram dificuldades para acessar o local. A pessoa cadeirante, por outro lado, pode vir a obter dificuldades com algumas barreiras físicas existentes. A pessoa cega, dentre todas, é definitivamente o mais prejudicado, tendo em vista que, apesar de não haver grandes barreiras físicas, não foram encontrados grandes facilitadores táteis ou sonoros que suprissem a sua restrição visual dificultando a passagem ou permanecia no local (ver quadro 01). As portas de algumas salas estão obstruídas por papéis, o que impede a visibilidade, e as aberturas possuem 1,40 m de altura, o que mesmo que não houvesse obstrução ainda não seria possível a visibilidade de cadeirantes ou pessoas com baixa estatura. Não foi encontrado nenhuma identificação em braile em nenhuma das portas (Banheiros, secretaria ou salas de aula). O corredor possui 2,15 m, o que pela NBR 9050/2015 seria uma boa distância, até mesmo para duas cadeiras de rodas (Que segundo a norma seria de 1,50 m a 1,80m), mas em dias de e horários de muita movimentação as lixeiras, os bebedouros e os bancos podem vir a se tornar possíveis barreiras (ver figuras do quadro 01). A partir da análise no local e dos conceitos de Acessibilidade Efetiva, o trabalho visa um entendimento das problemáticas e suas possíveis soluções. Nas próximas etapas, o processo de avaliação será aperfeiçoado para um diagnóstico mais preciso. Quadro 01- Análise da Acessibilidade Efetiva. Fonte: Autores (2018) Quadro 01- Análise da Acessibilidade Efetiva. Fonte: Autores (2018) Quadro 01 Análise da Acessibilidade Efetiva. Fonte: Autores (2018) 5. REFERÊNCIAS BIBLIOGRÁFICAS ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS - ABNT. NBR 9050; Acessibilidade de pessoas portadoras de deficiências a edificações, espaço, mobiliário e equipamentos urbanos. Rio de Janeiro: ABNT, 2015 BAPTISTA, A. H. N. Proposição da Teoria da Acessibilidade Efetiva com plano de verificação para estruturas de circulação de pedestre. Tese (Doutorado em Desenvolvimento 5. REFERÊNCIAS BIBLIOGRÁFICAS 5. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS - ABNT. NBR 9050; Acessibilidade de pessoas portadoras de deficiências a edificações, espaço, mobiliário e equipamentos urbanos. Rio de Janeiro: ABNT, 2015 ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS - ABNT. NBR 9050; Acessibilidade de pessoas portadoras de deficiências a edificações, espaço, mobiliário e equipamentos urbanos. Rio de Janeiro: ABNT, 2015 BAPTISTA, A. H. N. 4. LEVANTAMENTO E CONSIDERAÇÕES Proposição da Teoria da Acessibilidade Efetiva com plano de verificação para estruturas de circulação de pedestre. Tese (Doutorado em Desenvolvimento Urbano). Universidade Federal de Pernambuco, Recife: o autor, 2010. BAPTISTA, A. H. N. Proposição da Teoria da Acessibilidade Efetiva com plano de verificação para estruturas de circulação de pedestre. Tese (Doutorado em Desenvolvimento Urbano). Universidade Federal de Pernambuco, Recife: o autor, 2010.
https://openalex.org/W3008540973	https://ojs.unud.ac.id/index.php/buletinvet/article/download/55558/33824	Indonesian	null	Perkembangan Folikel dan Munculnya Estrus setelah Penyuntikan GnRH pada Sapi Bali yang Mengalami Anestrus Postpartum dengan Body Condition Score Berbeda	Buletin Veteriner Udayana	2,020	cc-by	2,980	ABSTRAK Penelitian ini bertujuan untuk mengetahui perkembangan folikel dan munculnya estrus setelah penyuntikan GnRH pada sapi bali yang mengalami anestrus postpartum lebih dari tiga bulan dengan body condition score (BCS) berbeda di Kabupaten Badung, Bali. Rancangan yang digunakan pada penelitian ini adalah rancangan acak lengkap (RAL) yang terdiri dari tiga kelompok perlakuan yaitu kelompok I : sapi bali yang mengalami anestrus postpartum dengan BCS 2, kelompok II : sapi bali yang mengalami anestrus postpartum dengan BCS 3 , kelompok III : sapi bali yang mengalami anestrus postpartum dengan BCS 4 dan masing-masing kelompok terdiri dari 9 ulangan . Ketiga kelompok sapi ini disuntik GnRH (Fertagyl,Intervet Inc) dengan dosis 500µg/im/ekor. Hasil penelitian menunjukkan rataan ukuran folikel sebelum penyuntikan GnRH adalah 4.03 + 0.21 mm, 4.60 + 0.18 mm, 4.56 + 0.22 mm masing-masing untuk BCS 2, BCS 3 dan BCS 4 dan rataan ukuran folikel setelah penyuntikan GnRH adalah 7.08 + 0.42mm, 11.06 + 0.40mm dan 11.99 + 0.33 masing-masing untuk BCS 2, BCS 3 dan BCS 4 dan secara statistik menunjukkan perbedaan yang nyata (P<0,05). Waktu munculnya estrus pada ketiga kelompok perlakuan adalah 7.75 + 0.89 hari, 4.63 + 0.52 hari dan 3.63 + 0.52 hari, masing- masing untuk BCS 2, BCS 3 dan BCS 4 dan secara statistik menunjukkan perbedaan yang nyata (P<0,05). Ukuran diameter folikel terbesar diperoleh pada sapi bali yang mempunyai BCS 4 yaitu sebesar 11,99 + 0,33 mm dengan waktu munculnya estrus 3.63 + 0.52 hari. Hasil penelitian dapat disimpulkan bahwa penyuntikan GnRH 500µg, mampu meningkatkan perkembangan folikel dan menginduksi munculnya estrus pada sapi bali yang mengalami anestrus postpartum. Kata kunci: Folikel ovarium; estrus; GnRH, sapi bali; BCS Perkembangan Folikel dan Munculnya Estrus setelah Penyuntikan GnRH pada Sa Bali yang Mengalami Anestrus Postpartum dengan Body Condition Score Berbed I Nyoman Oka Widiarta¹, Tjok Gde Oka Pemayun², I Gusti Ngurah Bagus Trilaksana² ¹Puskeswan Sobangan Dinas Pertanian dan Pangan Kabupaten Badung, Bali. ²Laboratorium Reproduksi Veteriner Fakultas Kedokteran Hewan Universitas Udayana, Jl. PB. Sudirman, Denpasar, Bali. Email: oka.widi015@gmail.com Buletin Veteriner Udayana pISSN: 2085-2495; eISSN: 2477-2712 Online pada: http://ojs.unud.ac.id/index.php/buletinvet Terakreditasi Nasional Peringkat 3, DJPRP Kementerian Ristekdikti No. 21/E/KPT/2018, Tanggal 9 Juli 2018 Buletin Veteriner Udayana pISSN: 2085-2495; eISSN: 2477-2712 Online pada: http://ojs.unud.ac.id/index.php/buletinvet Terakreditasi Nasional Peringkat 3, DJPRP Kementerian Ristekdikti No. 21/E/KPT/2018, Tanggal 9 Juli 2018 Volume 12 No. 1: 92-97 Pebruari 2020 DOI: 10.24843/bulvet.2020.v12.i01.p16 Volume 12 No. 1: 92-97 Pebruari 2020 DOI: 10.24843/bulvet.2020.v12.i01.p16 Keywords: Development of follicles ovary; estrus; GnRH; bali cattle; BCS. PENDAHULUAN 2001). Menurut Hafez (2000) bahwa anestrus akibat hipofungsi ovarium sering berhubungan dengan gagalnya sel- sel folikel menanggapi rangsangan hormonal, adanya perubahan kuantitas maupun kualitas sekresi hormonal, menurunnya rangsangan yang berhubungan dengan fungsi hipotalamus- pituitaria-ovarium yang akan menyebabkan menurunnya sekresi gonadotropin sehingga tidak ada aktivitas ovarium setelah melahirkan. Beberapa peneliti telah melaporkan bahwa untuk merangsang aktivitas ovarium pada kasus anestrus postpartum dengan penyuntikan hormon gonadotropin pada sapi (Hafez, 2000). Penyuntikan GnRH pada sapi potong (Yavas and Walton, 2003) dan penyuntikan GnRH pada sapi perah (Pemayun, 2009) Penelitian ini bertujuan untuk mengetahui perkembangan folikel, dan munculnya estrus setelah penyuntikan GnRH 500µg pada sapi bali yang mengalami anestrus postpartum dengan body condition score berbeda Masalah yang banyak dihadapi oleh peternak di Bali adalah panjangnya calving interval akibat tidak munculnya estrus lebih dari tiga bulan setelah melahirkan, tingginya kasus anestrus postpartum mengakibatkan lambatnya laju populasi (Laksmi et al., 2019). Nitis dan Pemayun (2000) melaporkan kasus anestrus postpartum pada sapi bali yaitu rata-rata 4,11 bulan dan panjangnya calving interval dengan rata-rata 14,83 bulan. Kegagalan reproduksi merupakan salah satu faktor utama yang dapat menghambat laju perkembangan populasi ternak. Untuk dapat melahirkan satu anak dalam setahun setiap induk sapi umumnya calving interval tidak lebih dari 365 hari dan kembali estrus tidak lebih dari tiga bulan setelah melahirkan (Opsomer and de Kruif, 1999). Anestrus postpartum sering merupakan penyebab infertilitas pada sapi. Gangguan reproduksi ini umumnya terjadi pada sapi induk sesudah partus atau inseminasi / perkawinan tanpa terjadi konsepsi. Menurut Kesler and Garverick (1982), anestrus pada sapi disebabkan oleh berbagai faktor diantaranya gangguan hormonal, perubahan lingkungan, manajemen pakan yang kurang baik dan penyakit. ABSTRACT This study aim to determine the development of follicles and onset of estrus due to GnRH induction in bali cattle which has anestrus postpartum more than three month with differnt body condition score in the Badung regency of Bali.The designd used in this study was a complete random design were divided into three treatment groups, namely Group 1 : cattle with BCS 2, Group 2 : cattle with BCS 3, Group 3 : cattle with BCS 4, each group consisted of 9 replication. All of group of bali cattle are induced by GnRH (Fertagyl, Intervet,Inc) at a dose of 500µg/IM/cattle. The result of this study indicate the development of follicles averages before induction of GnRH is 4.03 + 0.21 mm, 4.60 + 0.18 mm, 4.56 + 0.22 mm, each for BCS2, BCS 3 and BCS 4 and development of follicles averages after induction of GnRH is 7.08 + 0.42mm, 11.06 + 0.40 mm and 11.99 + 0.33 each for BCS 2, BCS 3 and BCS 4 and statistically shows that there are significant differences (P<0,05). The averages onset of estrus in the three treatment group is 7.75 + 0.89 day, 4.63 + 0.52 day dan 3.63 + 0.52day, each for BCS 2, BCS 3 and BCS 4 and statistically shows that there are significant differences (P<0,05). The largest follicular diameter in BCS 4 is 11,99 + 0,33 mm with the time of onset of estrus 3,63 + 0,52 day. The result can be concluded that the induction of GnRH 500ug in bali cattle can increase follicular development and accelerate the oset of estrous. Keywords: Development of follicles ovary; estrus; GnRH; bali cattle; BCS. 92 Buletin Veteriner Udayana Widiarta et al. METODE PENELITIAN Penelitian ini menggunakan 27 ekor sapi bali yang mengalami anestrus postpartum lebih dari tiga bulan dengan body condition score (BCS) yang berbeda di peternakan yang ada di kabupaten Badung. Penentuan anestrus postpartum dilakukan dengan melakukan palpasi rektal pada sapi yang tidak menunjukkan tanda estrus yang ditandai dengan ovarium berukuran normal dengan permukaan yang licin karena tidak ada perkembangan folikel dan corpus luteum. Pakan yang diberikan adalah jerami padi, batang pisang, rumput raja, hijauan dan konsentrat. Sistem pemeliharaan dengan kandang koloni dimana sapi tidak pernah dikelurkan dari kandang dan tanpa diberikan exercise. Nutrisi merupakan faktor penyebab terjadinya gangguan reproduksi sapi potong di daerah tropis (Besung et al., 2019). Nutrisi dan cadangan energi tubuh dibutuhkan dalam proses metabolisme, sintesis hormon reproduksi, pertumbuhan, laktasi dan aktivitas reproduksi. Arthur (1982) menyatakan bahwa body condition score yang rendah, kondisi tubuh yang kurang baik dan stress saat laktasi dapat memperpanjang periode anestrus. Hal ini akan mempengaruhi fungsi hipofisa anterior sehingga produksi dan skresi hormon FSH dan LH rendah yang menyebabkan ovarium tidak berkembang atau mengalami hipofungsi (Noakes et al., Rancangan yang digunakan pada penelitian ini adalah rancangan acak lengkap (RAL) yang terdiri dari tiga kelompok perlakuan yaitu kelompok I: 93 Buletin Veteriner Udayana pISSN: 2085-2495; eISSN: 2477-2712 Online pada: http://ojs.unud.ac.id/index.php/buletinvet Buletin Veteriner Udayana Volume 12 No. 1: 92-97 Pebruari 2020 DOI: 10.24843/bulvet.2020.v12.i01.p16 Volume 12 No. 1: 92-97 Pebruari 2020 Volume 12 No. 1: 92-97 Pebruari 2020 DOI: 10.24843/bulvet.2020.v12.i01.p16 Volume 12 No. 1: 92-97 Pebruari 2020 DOI: 10.24843/bulvet.2020.v12.i01.p16 y pISSN: 2085-2495; eISSN: 2477-2712 p Online pada: http://ojs.unud.ac.id/index.php/buletinvet p Online pada: http://ojs.unud.ac.id/index.php/buletinvet sapi bali yang mengalami anestrus postpartum dengan BCS 2, kelompok II: sapi bali yang mengalami anestrus postpartum dengan BCS 3, kelompok III: sapi bali yang mengalami anestrus postpartum dengan BCS 4 dan masing- masing kelompok terdiri dari 9 ulangan. Ketiga kelompok sapi ini disuntik GnRH (Fertagyl,Intervet Inc) dengan dosis 500µg/im/ekor. Analisis Statistik Data yang diperoleh di tabulasi dan dianalisis statistik dengan program excel 2017 for windows. HASIL DAN PEMBAHASAN Hasil penelitian menunjukkan bahwa rataan ukuran folikel sebelum penyuntikan GnRH adalah 4.03 + 0.21 mm, 4.60 + 0.18 mm, 4.56 + 0.22 mm masing-masing untuk BCS 2, BCS 3 dan BCS 4 dan rataan ukuran Folikel setelah penyuntikan GnRH adalah 7.08 + 0.42mm, 11.06 + 0.40mm dan 11.99 + 0.33 masing-masing untuk BCS 2, BCS 3 dan BCS 4 (Tabel 1). Dari hasil paparan diatas secara statistik menunjukkan perbedaan yang nyata (P<0,05) terhadap diameter folikel sebelum dan sesudah penyuntikan GnRH antara kelompok I,II dan III. Perbedaan diameter folikel ovarium sebelum dan sesudah penyuntikan GnRH lebih jelas dapat dilihat pada gambar 1. Parameter yang diukur pada penelitian ini adalah perkembangan folikel yang diukur dengan USG (KX 5200, KAIXIN), yaitu sebelum penyuntikan GnRH dan pada saat muncul estrus. Munculnya estrus setelah penyuntikan GnRH yang ditandai dengan keluarnya lendir, perubahan kondisi vulva (merah, bengkak, basah), gelisah dan nafsu makan menurun, menaiki dan diam dinaiki sesamam sapi betina (Hafez, 2000). Pengamatan terhadap munculnya estrus dilakukan dua kali sehari yaitu pagi pukul 06.00-07.00 WITA dan sore hari pukul 17.00-18.00 WITA. Tabel 1. Rata-rata ( 𝑥̅+ SD) Diameter Folikel Ovarium (mm) sebelum dan sesudah pemberian Gn-RH Kelompok Diameter Folikel (mm) Awal ( 𝑥̅+ SD) Akhir ( 𝑥̅+ SD) BCS 2 4.03 + 0.21 ͣ 7.08 + 0.42 ᵇ BCS 3 4.60 + 0.18 ͣ 11.06 + 0.40 ᵇ BCS 4 4.56 + 0.22 ͣ 11.99 + 0.33 ᵇ Ket: Huruf yang berbeda pada baris yang sama adalah perbedaan yang nyata pada tatanan 5 % (P<0,05). . Rata-rata ( 𝑥̅+ SD) Diameter Folikel Ovarium (mm) sebelum dan sesudah pemberian Tabel 1. Rata-rata ( 𝑥̅+ SD) Diameter Folikel Ovarium (mm) sebelum dan sesudah p Gn-RH Ket: Huruf yang berbeda pada baris yang sama adalah perbedaan yang nyata pada tatanan 5 % (P<0,05). Ket: Huruf yang berbeda pada baris yang sama adalah perbedaan yang nyata pada tatanan 5 % (P<0,05). Gambar 1. diameter folikel sebelum dan sesudah diberikan GnRH 500 µg (a.folikel ovarium, b. Ovarium) Sebelum Sesudah Gambar 1. diameter folikel sebelum dan sesudah diberikan GnRH 500 µg (a.folikel ovarium, b. Ovarium) 94 94 Buletin Veteriner Udayana Widiarta et al. penelitian yang dilakukan oleh Putro (2014 ) menunjukkan bahwa pemberian Gn-RH mampu meningkatkan diameter folikel ovulasi secara nyata dengan diameter folikel 14,40 mm. Adanya perbedaan ukuran diameter folikel dipengaruhi status nutrisi. Status nutrisi dan cadangan energi tubuh dapat dievaluasi secara klinis melalui BCS. HASIL DAN PEMBAHASAN Body condition score erat hubungannya dengan cadangan lemak tubuh, dimana diketahui jumlah simpanan lemak tubuh mempengaruhi fertilitas dan proses steroidogenesis. Hal ini mengindikasikan bahwa adanya kaitan erat antara jaringan adiposa dan sistem reproduksi (Leifers,2004). Perbaikan nutrisi yang meliputi kuantitas dan kualitas harus dilakukan pada sapi sapi yang memiliki BCS < 2 sebelum terapi hormonal akan memberikan hasil yang optimal. Adanya interaksi yang komplek antara antara faktor lingkungan atau manajemen (nutrisi), respon individual dan derajat keparahan akan menimbulkan respon kesembuhan yang berbeda (Budiyanto et al., 2016). Rataan terhadap munculnya estrus pada ketiga kelompok perlakuan setelah penyuntikan GnRH adalah 7.75 + 0.89 hari, 4.63 + 0.52 hari dan 3.63 + 0.52 hari, masing-masing untuk BCS 2, BCS 3 dan BCS 4.Waktu munculnya estrus secara statistik menunjukkan perbedaan yang nyata (P<0,05) sesudah penyuntikan GnRH antara Kelompok I, II dan III. Perbedaan waktu munculnya estrus sesudah penyuntikan GnRH antara kelompok I, II dan III lebih jelas dapat dilihat pada diagram 1. p g Diagram 1. waktu munculnya estrus sesudah penyuntikan GnRH 0 2 4 6 8 BCS 2 BCS 3 BCS 4 Waktu Munculnya Estrus (Hari) p g Diagram 1. waktu munculnya estrus sesudah penyuntikan GnRH 0 2 4 6 8 BCS 2 BCS 3 BCS 4 Waktu Munculnya Estrus (Hari) Diagram 1. waktu munculnya estrus sesudah penyuntikan GnRH Aktivitas ovarium setelah melahirkan merupakan hal yang sangat penting harus diperhatikan untuk bisa meningkatkan perfoman reproduksi. Berkembang dan berfungsinya organ reproduksi setelah melahirkan tergantung dari kadar LH dan FSH dari hipofisa anterior yang dikontrol oleh GnRH yang diskresikan oleh hypothalamus dan selain itu status pakan setelah melahirkan sangat berpengaruh terhadap sekresi hormon gonadotrophin (Miller et al., 1998). Mihm and Bleach (2003), menyatakan perbedaan gizi dan status reproduksi dapat mempengaruhi pelepasan GnRH dan LH selama periode postpartum pada sapi perah akibat keseimbangan energy negatif. Keseimbangan energi negatif dapat terjadi pada pada sapi perah dengan skor kondisi tubuh yang rendah. Status metabolisme hewan akibat keseimbangan energi negatif akan mempengaruhi sirkulasi berbagai hormon seperti leptin, insulin, GH, IGF-1, kortisol atau tiroksin yang berpengaruh terhadap proliferasi sel-sel folikuler sapi dan steroidogenesis. Hormon metabolik seperti IGF-1 merupakan mediator intake makanan dan / atau keseimbangan energi terhadap fertilitas sapi yang berperan dalam perkembangan folikel (Diskin et al., 2013). Kurangnya asupan nutrisi akan mempengaruhi senyawa metabolisme Hasil penelitian ini menunjukkan bahwa terjadi peningkatan perkembangan folikel dan munculnya estrus pada sapi bali yang mengalami anestrus postpartum setelah pemberian GnRH. HASIL DAN PEMBAHASAN Hal ini sesuai dengan kerja hormon seperti yang dilaporkan oleh Hafez (2000) bahwa GnRH berfungsi mengiduksi pelepasan FSH dan LH di Hipofisa anterior sehingga dapat menyebabkan folikel dan terjadinya estrus. Sesuai dengan 95 Volume 12 No. 1: 92-97 Pebruari 2020 DOI: 10.24843/bulvet.2020.v12.i01.p16 Volume 12 No. 1: 92-97 Pebruari 2020 Buletin Veteriner Udayana y pISSN: 2085-2495; eISSN: 2477-2712 p Online pada: http://ojs.unud.ac.id/index.php/buletinvet DOI: 10.24843/bulvet.2020.v12.i01.p16 dan hormon seperti insulin dan insulin like growth-I yang mempengaruhi hipothalamus dan hipofisa terhadap respon ovarium dan sensitifitas GnRH pada hipofisis sehingga energy tubuh akan menekan pelepasan GnRH dan mempengaruhi pulsatile LH yang diperlukan untuk pertumbuhan folikel. (Budiyanto et al., 2016). Ukuran folikel de graff yang berkembang cepat dan mencapai ukuran maksimal akan meningkatkan konsentrasi estrogen dalam cairan folikuli folikel ovarium menjadi optimal sehingga menginisiasi terjadinya estrus dengan tampilan birahi lebih jelas (Budiyanto, 2018) estrus yang lebih cepat dipengaruhi karena kadar estrogen lebih tinggi yang disebabkan oleh jumlah folikel yang tumbuh dan berkembang lebih banyak dan lebih cepat (Pemayun,2009). Saran Perlu dilakukan perbaikan nutrisi pada sapi dengan BCS 2 sebelum dilakukan penyuntikan GnRH dan perlu diberikan exercise pada sapi dengan BCS 3 dan BCS 4. UCAPAN TERIMAKASIH Penulis mengucapkan terimakasih kepada Departemen Reproduksi FKH Udayana, dan Dinas Pertanian dan Pangan Kabupaten Badung atas dukungannya dalam memfasilitasi penelitian ini. Simpulan Dari hasil penelitian dapat disimpulkan bahwa pemberian GnRH 500µg mampu meningkatkan perkembangan folikel dan menginduksi munculnya estrus pada sapi bali yang mengalami anestrus postpartum, dimana sapi dengan BCS 4 menunjukkan diameter folikel terbesar sebesar 11,99±0,33 mm dengan waktu munculnya estrus tercepat yaitu 3,63 ± 0,52 hari. Timbulnya estrus pada penelitian ini setelah penyuntikan GnRH 500 µg tidak jauh berbeda seperti yang dilaporkan sebelumnya munculnya estrus rata-rata 7,17 ± 3,24 hari dengan kisaran hari 5-10 hari (Pemayun, 2009). Panjang pendeknya waktu munculnya estrus sangat dipengaruhi oleh peningkatan perkembangan folikel dimana faktor nutrisi sangat berperan penting dalam metabolisme dan sintesis hormon. Semakin tinggi BCS respon terhadap pemberian hormon semakin bagus, ini berkaitan dengan nutrisi dimana kekurangan pakan dalam kurun waktu yang lama pada sapi akan menyebabkan GnRH tidak di respon secara aktif di hipofisa anterior sehingga terjadi penurunan pertumbuhan folikel dominan secara bertahap sehingga tidak mencapai folikel maksimum (Diskin et al., 2003). DAFTAR PUSTAKA Arthur GH. 1982. Veterinary Reproduction and Obstetrics. 5th Ed. Bailleire Tindall, London, UK. Pp. 616. Menurut Bossis et al. (1999) dan Butler (2000), konsumsi energi meningkatkan glukosa darah dan insulin yang dapat meningkatkan getaran sekresi LH dan memperbaiki tanggap ovarium terhadap stimulasi LH, LH diperlukan untuk pertumbuhan folikel. Peningkatan perkembangan folikel ini membawa konsekuensi peningkatan kadar estrogen dalam darah. Estrogen selain menimbulkan estrus pada sapi, juga memacu efek umpan balik positifnya terhadap LH (Hafez,2000). Munculnya Besung INK, Watiniasih NL, Mahardika IGNK, Agustina KK, Suwiti NK. 2019. Mineral levels of Bali cattle (Bos javanicus) from different types of land in Bali, Nusa Penida, and Sumbawa Islands (Indonesia). Biodiversitas. 20(10): 2931-2936. Bossis I, Wettemann RP, Welly SD, Vizcarra JA, Spiceer LJ, Diskin MG. 1999. Nutionally induced anovulation in beef heifers ovarian and endocrine 96 Buletin Veteriner Udayana Widiarta et al. University, Wanginengen and Division of Animal Resources Development, Animal Science Group, Lelystad. ISBN 90-5808-998- 3. function presceeding cessation of ovulation. J. Anim. Sci. 77: 1536- 1546. Budiyanto A, Thopianong TC, Triguntoro, Dewi HK. 2016. Gangguan reproduksi sapi bali pada pola pemeliharaan semi intensif di daerah sistem integrasi sapi – kelapa sawit. Acta Veterinaria Indonesiana. 4(1): 14-18 Mihm M, Bleach ECL. 2003. Endocrine regulation of ovarian antral follicle development in cattle. Anim. Reprod. Sci. 78: 217-237. Miller DW, Blanche D, Boukhliq R, Curlewis JD, Martin GB. 1998. Central metabolic messenger and the effect of nutrition on gonadotrophin scretion in sheep. J. Reprod. Fertility. 112: 347-356. Budiyanto A. 2018. Faktor Gangguan Reproduksi Di Indonesia Dalam Manajemen Breeding Sapi Potong. Disampaikan dalam acara refreshing untuk Dosen Pembimbing Lapangan Koassistensi, Departemen Reproduksi FKH UGM, 21 September 2018. Nitis IM, Lana K, Sukanten W, Pemayun TGO, Puger AW. 1994. Growth and Reproductive Performance of Bali Heifer under Three Strata Forage System. Report to FAO. Project No. AGAP-653AN 40/5. Rome. Buttler WR. 2000. Nutritional interaction with reproduktive perfomance in dairy cattle. Anim. Reprod. Sci. 60- 61: 449-457. Noakes DE, Geoffrey HA, Timothy JP, Gary CWE. 2001. Arthur’s Veterinary Reproduction and Obstetrics, l Eighth Editions. Elsevier Health Sciences. Diskin MG, Mackey DR, Roche JF, Sreenan JM. 2003. Effects of nutrition and metabolic status on circulating hormones and ovarian follicle develpoment cattle. Anim. Reprod. Sci. 78: 345-370. Opsomer G, Grohn YT, Hertl J, Coryn M, Deluyker H, de Kruif A. 2000. Risk factors for post partum ovarian dysfunction in high producing cows in Belgium: A field study. Theriogenology. 53: 841–857. DAFTAR PUSTAKA Hafez ESE. 2000. Reproduction in farm animal. Edition 7th Ed. Lippncott Williams & Wilkins. Maryland. USA. Kesler DJ, Garverick HA. 1982. Ovarian cysts in dairy cattle: A review. J. Anim. Sci. 55: 1147-1159. Pemayun TGO. 2009. Induksi estrus dengan PMSG dan GnRH pada sapi perah anestrus postpartum. Buletin Veteriner Udayana 1(2): 83-87. Laksmi DNDI, Trilaksana IGNB, Darmanta RJ, Darwan M, Bebas IW, Agustina KK. 2019. Correlation between body condition score and hormone level of Bali cattle with postpartum anestrus. Indian J. Anim. Res. 53(12): 1599-1603. Putro P. 2014. Dinamika folikel ovulasi setelah perlakuan sinkronisasi estrus dengan implan progesteron intravagina pada sapi perah. J. Sain Vet. 31(2): 128-137. Liefers S. 2004. Physiologi and Genetics of Leptin in Periparturient Dairy Cows. (Ph.D. Thesis). Animal Breeding and Genetics, Wageningen Yavas Y, Walton J. 2003. Postpartum acyclicity in suckled beef cows: A review Theriogenology. 54(1): 25– 55. 97
https://openalex.org/W4317877606	https://www.researchsquare.com/article/rs-2493409/latest.pdf	English	null	Trace metals contamination in varieties of cereal-based pediatric foods sold in parts of Accra, Ghana	Research Square (Research Square)	2,023	cc-by	4,825	Trace metals contamination in varieties of cereal- based pediatric foods sold in parts of Accra, Ghana Ghana Atomic Energy Commission Trace metals contamination in varieties of cereal- based pediatric foods sold in parts of Accra, Ghana A. Domfeh (  Adomfeh2012@gmail.com ) School of Nuclear and Allied Sciences, University of Ghana A. K. Anim Ghana Atomic Energy Commission A. Asamoah Ghana Atomic Energy Commission Research Article Keywords: Pediatric, Cereal-based foods, Contamination, Cadmium, Lead Posted Date: January 24th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2493409/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Trace metals contamination in varieties of cereal- based pediatric foods sold in parts of Accra, Ghana A. Domfeh (  Adomfeh2012@gmail.com ) School of Nuclear and Allied Sciences, University of Ghana A. K. Anim Ghana Atomic Energy Commission A. Asamoah Ghana Atomic Energy Commission Research Article Keywords: Pediatric, Cereal-based foods, Contamination, Cadmium, Lead Posted Date: January 24th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2493409/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Research Article Keywords: Pediatric, Cereal-based foods, Contamination, Cadmium, Lead Posted Date: January 24th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2493409/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/14 Abstract Dietary intake is a potential route of babies’ exposure to trace metals contamination. Cereal-based pediatric foods on the Ghanaian market can be susceptible to contamination as they are mostly produced on small scales by individuals with little or no knowledge of food safety. This study ascertained trace metals quality of fifty (50) cereal-based pediatric foods composed of: maize, rice, millet, wheat, and mixed cereals from major sales outlets on the Ghanaian market. The samples were acid (HNO3 + H2O2) digested and analyzed using Atomic Absorption Spectrometry (AAS). Concentration (mg/kg) of trace metals were: 0.87–34.74 (Fe), <0.001–1.00 (Zn), <0.003–0.92 (Cu), <0.002–0.82 (Cd), <0.001–0.26 (Cr), <0.005–0.23 (Co), <0.001–0.58 (Pb), and <0.001–0.20 (Ni) across samples. Pb and Cd were respectively measured in 22 % and 44 % of samples that were not registered by the Food and Drug Authority (FDA). Concentration of Cd in 10% of samples exceeded the WHO limit by a factor of 8. Calculated hazard indices > 1.0 indicated potential health risk to baby within the studied age groups with cadmium as the predominate cause. This initial findings show that some locally produced cereal-based pediatric foods on the Ghanaian market can present a route of trace metals contamination and therefore warrant periodic investigations to safeguard public health. 2.1 Sample collection In this study, 50 locally made cereal-based baby foods were sampled from selected major sale outlets (markets, hospitals, and malls) in the Accra Metropolis, Ga East Municipality, La Dadekotopon Municipality, and La Nkwatanang Madina Municipality in the Greater Accra region of Ghana. The samples purchased included; maize (14%), rice (14%), wheat (10%), millet (4%) and mixed cereals (58%). Each sample is made of either one variety of cereal or a combination of different cereals with mostly plant protein such as groundnut and /or soybeans. The samples comprises; seventeen (17) different brands and four (4) sets of unbranded samples (purchased in the same market and by the same producer). Only 5% of the samples were duly registered with Ghana`s Foods and Drugs Authority (FDA). Additional data on the samples are given in Online Resource 1 (Supplementary 1). Each of the samples were labeled with a unique identification code and transported to the laboratory for preparation and analyses. 1. Introduction Cereal-based foods are mostly introduced to babies after the recommended six months exclusive breastfeeding or to compliment breastfeeding (Bonsi, 2014). The cereal-based baby foods are often prepared from single variety of cereals or mixed cereals; rice, maize, millet, sorghum, wheat, barley and oats. The formulation of cereal-based foods are often fortified with plant proteins such as soyabeans or groundnut and are served with animal proteins predominantly milk. One would expect that baby foods should be free from contamination since it is expedience on the manufacturer to use the best of raw materials and the best technology in the most hygienic environment. However, this is often not the case as one of the channels of production may be compromised. Trace metal, aflatoxin, pesticide residues, bacteria overload, polycyclic aromatic hydrocarbons (PAHs) are potential contaminants in cereal-based foods (Korley et al., 2019; Kumar et al., 2019; Fageria et al., 2011; White et al., 2004). The cereals could be contaminated during cultivation via unsafe irrigation water, agrochemicals, proximity to polluted soil, urbanization etc. (Kumar et al., 2019; Fageria et al., 2011). During manufacturing, the food could also be contaminated through milling by wear and tear of the milling plates especially with the use of commercial milling machines. The trace metals iron (Fe), zinc (Zn), copper (Cu), cobalt (Co), chromium (Cr) and nickel (Ni) are essential metals that play various biological roles in the human body in when their correct proportion. However, deficiency-related diseases such as anemia, poor growth and decreased immunity among others were reported to be associated with either limited or excess supply of these essential metals (Codex Alimentarius Commission, 2011). Two notable carcinogenic metals lead (Pb) and cadmium (Cd) have no Page 2/14 Page 2/14 biological function in the human body, hence their presence in food even at trace level have been associated with decline in child intelligence and development, and other organ-related toxicity ( Codex Alimentarius Commission, 2011; Gardener et al., 2019; Kumar et al., 2019; Suomi et al., 2018). biological function in the human body, hence their presence in food even at trace level have been associated with decline in child intelligence and development, and other organ-related toxicity ( Codex Alimentarius Commission, 2011; Gardener et al., 2019; Kumar et al., 2019; Suomi et al., 2018). 1. Introduction In Ghana, majority of the population depends largely on the open market system for most products including baby foods and few of the citizenry depends on the regulated market system. The open market provide grounds for people with little or no knowledge in preparing baby foods to get their unregistered and unwholesome products sold in the market. This may render consumers vulnerable to chemical contaminations including trace metals. A Similar studies has been carried out by Ayivor et al. (2011) in which they determined the concentrations of aluminum (Al), bromine (Br), calcium (Ca), chlorine (Cl), potassium (K), magnesium (Mg), and sodium (Na) in 16 samples of imported cereal-based pediatric foods from various markets in the Greater Accra region. Due to the poor preparation and packaging of the locally produced cereal-based pediatric foods, it is necessary to assess both essential and toxic trace metal contamination in cereal-based baby foods to ensure food safety particularly babies. Accordingly, this work assessed eight (8) trace metals (Fe, Zn, Cr, Co, Cu, Ni, Cd and Pb) and the health risks associated in locally produced cereal-based pediatric foods which are highly patronized in Accra, Ghana. 2.3 Sample Digestion and Metal Determination The concentrations of eight (8) different trace metals were determined in the Inorganic Laboratory of the Nuclear Chemistry and Environmental Research Center (NCERC) of the Ghana Atomic Energy Commission (GAEC), Accra. The samples were subjected to microwave assisted acid digestion using analytical grades of concentrated Trioxonitrate (V) acid (HNO3, 69%), and Hydrogen peroxide (H2O2, 30%). The acid digestion followed was as previously described by Ackah et al. (2014) and Anim et al. (2011). In brief, 2.0 mL of double deionized water was added to approximately 0.50 g of the homogenized sample, followed by 7.0 mL of the concentrated nitric acid for pretreatment. After standing for about 20 minutes, 2.0 mL of the concentrated nitric acid was added, followed by 1.0 mL of the hydrogen peroxide. The complete assembly of the samples in their Teflon bombs were then microwave irradiated using the SINEO JUPITA-A microwave digestion programmed for 50 minutes. The digestates were made up to 20.0 mL using double deionized water and resulting solution analyzed for the metals of interest. The digested samples were analyzed for trace metals using a Varian AA 240FS Fast Sequential Atomic Absorption Spectrometer in an acetylene-air flame. The blanks and reference materials were all analyzed alongside the samples. Quality assurances and quality control measures as such analytical blanks, recoveries, regression curves, repeatability and instrumental protocols were duly followed to ensure accuracy and precision of trace metals concentrations (Supplementary 2). 2.2 Reagents and chemicals All the reagents used were analytical grade with the concentrated trioxonitrate (V) acid (HNO3, 69%) supplied by British Drug House (BDH) and hydrogen peroxide (H2O2, 30%) supplied by MES Equipment Limited. The reference standards materials used for the metal analysis were from FLUKA ANALYTICAL, Page 3/14 Sigma-Aldrich Chemie GmbH - Switzerland. The double deionized water used in this analysis was produced by the Nuclear Chemistry and Environmental Research Centre, GAEC. Sigma-Aldrich Chemie GmbH - Switzerland. The double deionized water used in this analysis was produced by the Nuclear Chemistry and Environmental Research Centre, GAEC. 2.4 Health risk Assessment In assessing the non-carcinogenic health risk associated with the exposure to metals, the Chronic Daily Index (CDI) was calculated using EQ. 1 described by USEPA, (1989) based on parameters specified by Korley et al. (2019). CDI = EQ. 1 Ci⋅IR⋅ED⋅EF BW⋅AT CDI = EQ. 1 Ci⋅IR⋅ED⋅EF BW⋅AT CDI = EQ. 1 Ci⋅IR⋅ED⋅EF BW⋅AT CDI = EQ. 1 Ci⋅IR⋅ED⋅EF BW⋅AT Ci – Concentration of metals in sample in mg/kg IR – Intake Rate in kg/da Ci – Concentration of metals in sample in mg/kg IR – Intake Rate in kg/day EF – Exposure Frequency in day/year ED – Exposure Duration per year EF – Exposure Frequency in day/year ED – Exposure Duration per year EF – Exposure Frequency in day/year ED – Exposure Duration per year Bw – Body weight of babies in Kg AT – Average Life expectancy of the toxicity in day Bw – Body weight of babies in Kg AT – Average Life expectancy of the toxicity in day The potential human risks of babies due to the intake of metals measured in the samples were assessed based on the calculated hazard quotient (HQ) for the individual metals by using EQ. 2 as described by USEPA, (1989). HQ = EQ. 2 CDI RfD Page 4/14 RfD – Oral Reference Dose in mg/kg/day The hazard index (HI) for all the measured metals were calculated using EQ. 3 USEPA, (1989). HI = HQn1 + HQn2 + …+ HQn EQ. 3 HQn(1 – n) is the hazard quotients for the various metals 3. Results The statistical summary of the concentrations of eight (8) metals measured for all the 50 samples was presented in Table 1. The incidence ratios measured were: 100% (Fe), 98% (Zn), 90% (Cu), 44% (Cd), 24% (Ni) 22% (Cr), 22% (Pb), and 8% (Co). There were no significant differences (p > 0.05) in the metal concentrations for the different cereal compositions across all the samples (Supplementary 3.a). Table 1 STATISTICAL SUMMARY OF TOTAL METAL CONCENTRATION (mg/kg), N = 50 Metal Mean St. dev Range Median Percentiles 75th 95th Fe 8.59 9.36 0.87–34.74 4.62 10.03 33.26 Zn 0.13 0.14 < 0.001–1.00 0.12 0.15 0.22 Cu 0.16 0.18 < 0.003–0.92 0.13 0.15 0.81 Cd 0.20 0.34 < 0.002–0.82 < 0.002 0.29 0.81 Cr 0.02 0.04 < 0.001–0.26 < 0.001 < 0.001 0.08 Co 0.01 0.04 < 0.005–0.23 < 0.005 < 0.005 0.10 Pb 0.06 0.12 < 0.001–0.58 < 0.001 0.01 0.35 Ni 0.01 0.03 < 0.001–0.20 < 0.001 0.01 0.07 f th t l t ti i th diff t l th l d h The results of the metal concentrations in the different cereals were then analysed as shown graphically in Fig. 1 (a - f) with box and whiskers plots indicating both minimum and maximum concentrations. The concentrations of eight metals were then analysed across the 17 brands and the 4 sets of unbranded samples as represented graphically with Box and Whiskers plot indicating both the minimum and maximum concentrations in Fig. 2 (a - h). The concentrations of eight metals were then analysed across the 17 brands and the 4 sets of unbranded samples as represented graphically with Box and Whiskers plot indicating both the minimum and maximum concentrations in Fig. 2 (a - h). The results of the metals concentrations of Fe, Cd and Pb were used to calculate for the health risk indices for baby within the ages of 6–8 months, 9–11 months and 12–23 months using Equations 1, 2 The results of the metals concentrations of Fe, Cd and Pb were used to calculate for the health risk indices for baby within the ages of 6–8 months, 9–11 months and 12–23 months using Equations 1, 2 Page 5/14 Page 5/14 and 3. The results of the estimated health risk indices for the calculated ages of babies is represented in Table 2. 3. Results Table 2 ESTIMATED HEALTH RISK INDICES ASSOCIATED WITH METALS CONTAMINATION (n = 50) Age (months) Mean St. dev. Range % above 1.0 HQ (Fe) 6–8 0.41 0.45 0.04–1.68 14 9–11 0.56 0.60 0.06–2.26 14 12–23 0.86 0.92 0.09–3.47 26 HQ (Cd) 6–8 13.61 22.73 <LOD – 55.16 44 9–11 18.30 30.56 <LOD – 74.18 44 12–23 28.12 46.97 <LOD – 114.01 44 HQ (Pb) 6–8 0.53 1.18 <LOD – 5.60 18 9–11 0.71 1.58 <LOD – 7.53 18 12–23 1.09 2.44 <LOD – 11.58 22 HI 6–8 14.55 23.52 0.10–59.84 54 9–11 19.95 32.23 0.13–84.53 54 12–23 30.07 48.61 0.20–123.69 58 Table 2 ESTIMATED HEALTH RISK INDICES ASSOCIATED WITH METALS CONTAMINATION (n = 50) 4. Discussion From Table 1, the mean concentration of all the metal across the samples were relatively low except iron. The high standard deviation could be attributed to the variations of the cereal compositions of the samples. The median concentrations for metals Cr, Co, Ni, Cd and Pb were all below the limit of detection (LOD). The mean iron concentration was higher when compared to similar reports from Lybia and Nigeria (Iwegbue et al., 2010; Elbagermi et al., 2017). However, the mean iron concentration was about 4 folds lower than similar studies done in Suadi Arabia and Turkey (Al Khalifa & Ahmad, 2010; Saracoglu et al., 2007). This was because the products in Suadi Arabia were reported to have been fortified with dairy products (Al Khalifa & Ahmad, 2010). The essential metal Zinc which plays a vital role in baby`s development was found to be very low. This was not surprising since cereals in general have low zinc concentration (Codex Alimentarius Commission, 2011). The Cr, Co, Ni and Cu were very low in concentration. Page 6/14 Page 6/14 The presence of cadmium and lead even at trace level calls for health concern since they do not have biological function in the human body and have the ability to bioaccumulate (Kumar et al., 2019). Though the median cadmium concentration was below LOD, the mean concentration of 0.20 ± 0.34 mg/kg and 95th percentile of 0.81 mg/kg is instructive as it exceeded the Food and Agriculture Organization / World Health Organization (FAO/WHO) and FDA recommended concentration of 0.10 mg/kg ( Codex Alimentarius Commission, 2011). The cadmium concentrations measured were higher when compared to similar studies from other countries (Al Khalifa & Ahmad, 2010; Iwegbue et al., 2010; Edward et al., 2013; Elbagermi et al., 2017; Kazi et al., 2010; Sadeghi et al., 2014). The mean concentration of lead, 0.06 ± 0.12 mg/kg and 75th percentile of 0.01 mg/kg were within recommended limits. However, the 95th percentile concentration of lead, 0.35 mg/kg was of much concern since it exceed the FAO/WHO and FDA recommended concentration of 0.20 mg/kg for cereal-baby foods. The maximum concentration of the lead was in consistent to (Sadeghi et al., 2014), yet were of higher concentration when compared to other studies (Al Khalifa & Ahmad, 2010; Iwegbue et al., 2010; Edward e al., 2013; Elbagermi et al., 2017; Kazi et al., 2010). 4. Discussion Page 7/14 Page 7/14 Page 7/14 Lead concentrations were measured only in BCF (< LOD – 0.26 mg/kg), JWG (0.20 mg/kg), Set 2 (< LOD – 0.35 mg/kg), Set 3 (0.12–0.35 mg/kg) and Set 4 (0.04–0.58 mg/kg), with their maximum concentrations exceeding FAO/WHO and FDA recommended limit of 0.20 mg/kg for cereal-based baby foods (Fig. 2h). Notably, the same brands whose lead concentrations were high also had high cadmium concentrations in the range; <LOD – 0.82 mg/kg (BCF), <LOD-0.81 mg/kg (JWG), <LOD – 0.80 mg/kg (Set 2), 0.80–0.81 mg/kg (Set 3) and < LOD-0.80 mg/kg (Set 4). These brands exceeded the FAO/WHO recommended limit, also exceeded FAO/WHO recommended limit of 0.10 mg/kg for cadmium concentrations (Fig. 2g) (Codex Alimentarius Commission, 2011). However, it is encouraging to note that both Pb and Cd concentrations in the FDA registered samples were below the recommended limits. Lead concentrations were measured only in BCF (< LOD – 0.26 mg/kg), JWG (0.20 mg/kg), Set 2 (< LOD – 0.35 mg/kg), Set 3 (0.12–0.35 mg/kg) and Set 4 (0.04–0.58 mg/kg), with their maximum concentrations exceeding FAO/WHO and FDA recommended limit of 0.20 mg/kg for cereal-based baby foods (Fig. 2h). Notably, the same brands whose lead concentrations were high also had high cadmium concentrations in the range; <LOD – 0.82 mg/kg (BCF), <LOD-0.81 mg/kg (JWG), <LOD – 0.80 mg/kg (Set 2), 0.80–0.81 mg/kg (Set 3) and < LOD-0.80 mg/kg (Set 4). These brands exceeded the FAO/WHO recommended limit, also exceeded FAO/WHO recommended limit of 0.10 mg/kg for cadmium concentrations (Fig. 2g) (Codex Alimentarius Commission, 2011). However, it is encouraging to note that both Pb and Cd concentrations in the FDA registered samples were below the recommended limits. In Table 2. the calculated the hazard indices were all > 1.0 for all the different age groups consuming the locally cereal-based baby`s foods. This means that the studied age groups of babies are likely to experience significant non-cancer risks and the high values indicate increasing the probability of non- carcinogenic health risks (USEPA, 1989). It was observed that though the Fe concentrations were relatively higher than Pb and Cd, yet it generally posed no health risk with mean hazard quotient < 1.0 for all the age groups. The same samples and brands recording both high Pb and Cd concentrations were the key source of the high hazard indices in among the various samples. 4. Discussion Notably, Cd contributed predominately to the high hazard indices with the high hazard quotients > 10 for all the different age groups and hence cause of potential health risk to all the age groups (USEPA, 1989). 5. Conclusion In this study, Eight (8) different metal concentrations were measured in the cereal-based baby foods. Although the mean concentrations of the metals were below the recommended limits, mean concentration and 75th percentile of Cd were notable above the FAO/WHO recommended concentration of 0.10 mg/kg. The presence of Cd and Pb in the samples were assessed to pose non-cancer risk upon exposure to the specified age groups. Generally, metals concentration in all the FDA registered samples were found to be safe while the samples found to have metals concentration above recommended limits were unregistered, specifically; brands codes BCF, JWG, Set 2, Set 3 and Set 4. The high hazard indices > 1.0 indicates that the studied age groups are likely to experience significant non-carcinogenic health risks for consuming the locally produced cereal-based baby`s foods. Such studies are useful to inform the public on the wholesomeness or otherwise of cereal-based baby foods sold on the open market. 4. Discussion Graphical representation of the metal distribution in the samples across the different cereal types was represented in Fig. 1a – f. Apart from Cd which showed a significant difference (p < 0.05) in the concentrations for two varieties; maize only and rice only, there were no significant difference in metal concentrations for the other cereal compositions (Supplementary 3). Fe, Zn, Cu, Cd, and Ni were measured in all the different cereal compositions but Co was measured in only the mixed cereal type. Similarly, Cr was measured in all the other different cereal compositions except in millet only (Fig. 1c). The iron concentrations were highest in millet-only (18.02 ± 15.23 mg/kg) and least in rice-only (2.62 ± 2.67 mg/kg). Copper, chromium, cobalt, and nickel were generally of low concentrations in all the different cereal compositions in consistent with C. Reilly, (2002). The concentrations of cadmium; 0.40 ± 0.60 mg/kg (millet-only), 0.26 ± 0.38 mg/kg (rice-only), 0.17 ± 0.35 (wheat-only), 0.46 ± 0.42 (maize-only) and 0.12 ± 0.28 mg/kg (mixed cereals) were above the FAO/WHO and FDA recommended concentration of 0.10 mg/kg in cereal-based pediatric foods. The mean concentration of lead in the different cereal compositions were generally low concentration, yet with high maximum concentrations above recommended limits (Codex Alimentarius Commission, 2011). The iron and zinc concentrations were the most dominant in all the brands (Fig. 2a and 2b). Aside from Cd and Ni which showed significant differences (p < 0.05) among the various brands, there were no significant differences (p > 0.05) in concentrations between the other metals (Supplementary 3.f). Notably, high iron concentrations were observed for the samples; FSC (3.80–32.47 mg/kg), VFC (2.69 − 28.01 mg/kg), SF (34.22 mg/kg), JF (3.72–34.74 mg/kg), and Set 2 (1.46–28.84 mg/kg) as presented in Fig. 2a. While concentration of Zn across the brands were in the range 0.02–1.0 mg/kg (Fig. 2b). Cobalt was measured in only two brands; FSC (0.02–0.12 mg/kg) and JF (0.08–0.23 mg/kg) as shown Fig. 2e. The level of Copper, chromium, nickel and cobalt in the brands were generally of low concentrations across the brands as shown in Fig. 2c – 2f C. Reilly, (2002). Acknowledgment Our profound gratitude goes to the staffs of School of Nuclear and Allied Sciences (SNAS), the personnel of the NCERC in GAEC, Ghana Young Generation in Nuclear and the Africa Young Generation in Nuclear. Statements and Declarations Page 8/14 Funding No funds, grants or other support was received for conducting this study. No funds, grants or other support was received for conducting this study. Data Availability Data generated and analysed during this current study are included in this published article and its supplementary information files, and any other dataset are available from the corresponding author on reasonable request. Author`s Contribution Authors Contributions A. Domfeh Research idea and plan, project design, sample collection, data acquisition, data analysis, data interpretation and drafting of manuscript A. K. Anim Conceptual design of project, Sample analysis, data processing, supervision and manuscript review A. Asamoah Research idea, approving the final version of data acquisition, analysis and manuscripts review Authors Contributions Page 8/14 The authors declare equivocally that they have no conflict of interest relevant to this article. Funding References 1. Ackah, M., Anim, A. K., Gyamfi, E. T., Zakaria, N., Hanson, J., Tulasi, D., … Osei, J. (2014). Uptake of heavy metals by some edible vegetables irrigated using wastewater: A preliminary study in Accra, Ghana. Environmental Monitoring and Assessment, 186(1), 621–634. https://doi.org/10.1007/s10661-013-3403-0 2. Al Khalifa, A. S., & Ahmad, D. (2010). Determination of key elements by ICP-OES in commercially available infant formulae and baby foods in Saudi Arabia. African Journal of Food Science, 4(7), 464–468. 3. Anim, a K., Ahialey, E. K., Duodu, G. O., Ackah, M., & Bentil, N. O. (2011). Accumulation Profile of Heavy Metals in Fish Samples from Nsawam, Along the Densu River, Ghana. Research Journal of Environmental and Earth Sciences, 3(1), 56–60. 4. Ayivor, J. E., Debrah, S., Forson, A., Nuviadenu, C., Buah Kwofie, A., & Denutsui, D. (2011). Trace elements in some imported commercial infant cereal formulas on the Ghanaian market by INAA. Der Pharma Chemica, 3(5), 94–101. 5. C.M.A Iwegbue, S. O Nwozo, & Nwajei, L. E. O. and G. E. (2010). Survey of trace element composition of commercial infant formulas in the Nigerian market. Food Additives & Contaminants: Part B, 3:3(August 2014), 163–171. https://doi.org/10.1080/19440049.2010.497502 Page 9/14 Page 9/14 6. Committee, C., Additives, F., Cx, D., Committee, F. C., Cccf, F., Commission, C. A., … Session, T. (2011). Joint Fao / Who Food Standards Programme Codex Committee On Contaminants In Foods Fifth Session. In Food and Agriculture organization of the United Nation and World Health Organization. The Hague, The Netherlands. 7. Edward, M., Muntean, N., Creta, C., & Duda, M. (2013). Occurrence of Lead and Cadmium in some Baby Foods and Cereal Products. ProEnvironment/ProMediu, 6(16), 587–590. 8. Elbagermi, M., Alajtal, A., & Edwards, H. (2017). Levels of Major and Minor Elements in Some Commercial Baby Foods Available in Libya. American Journal of Chemistry Application, (June). 9. Eunice Bonsi, P. W. A. and R. Z. (2014). Nutritional enhancement of ghanaian weaning foods using the orange flesh sweetpotato. African Journal of Food, Agriculture, Nutrition and Development, 14 No. 5, 2036–2056. 10. Gardener, H., Bowen, J., & Callan, S. P. (2019). Science of the Total Environment Lead and cadmium contamination in a large sample of United States infant formulas and baby foods. Science of the Total Environment, 651, 822–827. https://doi.org/10.1016/j.scitotenv.2018.09.026 11. Kazi, T. G., Jalbani, N., Baig, J. A., Arain, M. B., Afridi, H. I., Jamali, M. K., … Memon, A. N. (2010). https://doi.org/10.1016/j.foodchem.2006.11.022 https://doi.org/10.1016/j.foodchem.2006.11.022 19. Suomi, J., Tuominen, P., Niinistö, S., & Virtanen, S. M. (2018). Dietary heavy metal exposure of Finnish children of 3 to 6 years. Food Additives & Contaminants: Part A, 35(7), 1305–1315. https://doi.org/10.1080/19440049.2018.1480065 20. US Environmental Protection Agency. (1989). Risk Assessment Guidance for Superfund Volume I Human Health Evaluation Manual (Part A). I(December). 20. US Environmental Protection Agency. (1989). Risk Assessment Guidance for Superfund Volume I Human Health Evaluation Manual (Part A). I(December). 21. White, S., Fernandes, A., Rose, M., & Rose, M. (2004). Infant Formulae And Baby Foods. (December), 1–66. 21. White, S., Fernandes, A., Rose, M., & Rose, M. (2004). Infant Formulae And Baby Foods. (December), 1–66. References Evaluation of toxic elements in baby foods commercially available in Pakistan. Food Chemistry, 119(4), 1313–1317. https://doi.org/10.1016/j.foodchem.2009.09.003 12. Korley Kortei, N., Akomeah Agyekum, A., Akuamoa, F., Baffour, V. K., & Wiisibie Alidu, H. (2019). Risk assessment and exposure to levels of naturally occurring aflatoxins in some packaged cereals and cereal based foods consumed in Accra, Ghana. Toxicology Reports, 6(June 2018), 34–41. https://doi.org/10.1016/j.toxrep.2018.11.012 13. Kumar, S., Prasad, S., Kumar, K., Shrivastava, M., Gupta, N., Nagar, S., … Yadav, S. (2019). Hazardous heavy metals contamination of vegetables and food chain: Role of sustainable remediation approaches - A review. Environmental Research, 179(June), 108792. https://doi.org/10.1016/j.envres.2019.108792 14. Nand Kumar Fageria, Virupax C. Baligar, C. A. J. (2011). Growth and Mineral Nutrition of Field Crops (Third; C. Press, ed.). New York, USA: Taylor & Francis. 15. Rai, P. K., Lee, S. S., Zhang, M., Tsang, Y. F., & Kim, K. H. (2019). Heavy metals in food crops: Health risks, fate, mechanisms, and management. Environment International, 125(February), 365–385. https://doi.org/10.1016/j.envint.2019.01.067 16. Reilly, C. (2002). Metal Contamination of Food Third edition (third; O. B. Unversity, ed.). Oxford, UK: Blackwell PUblishing Company. 17. Sadeghi, N., Oveisi, M. R., Jannat, B., Hajimahmoodi, M., Behfar, A., Behzad, M., … Jannat, B. (2014). Simultaneous measurement of zinc, copper, lead and cadmium in baby weaning food and powder milk by DPASV. Iranian Journal of Pharmaceutical Research, 13(1), 345–349. https://doi.org/10.22037/ijpr.2014.1440 18. Saracoglu, S., Saygi, K. O., Uluozlu, O. D., Tuzen, M., & Soylak, M. (2007). Food Chemistry Determination of trace element contents of baby foods from Turkey. 105, 280–285. Page 10/14 Page 10/14 19. Suomi, J., Tuominen, P., Niinistö, S., & Virtanen, S. M. (2018). Dietary heavy metal exposure of Finnish children of 3 to 6 years. Food Additives & Contaminants: Part A, 35(7), 1305–1315. https://doi.org/10.1080/19440049.2018.1480065 Figures Page 11/14 gure 1 ox and Whiskers plot of metal concentrations: (a) all samples, (b) mixed cereals only, (c) millet on e only, (e) wheat only, and (f) maize only. Whiskers indicate the maximum and minimum ncentrations Figure 1 Box and Whiskers plot of metal concentrations: (a) all samples, (b) mixed cereals only, (c) millet only, (d) rice only, (e) wheat only, and (f) maize only. Whiskers indicate the maximum and minimum concentrations. Page 12/14 Figure 2 Page 13/14 a – d) Box and Whiskers plot of metal concentrations in the samples based on brands: (a) iron, (b) Zinc, c) Copper, and (d) Chromium. Whiskers indicate the maximum and minimum concentrations e – h) Box and Whiskers plot of metal concentrations in the samples based on brands: (e) cobalt, (f) nickel, (g) cadmium, and (h) lead. Whiskers indicate the maximum and minimum concentration (a – d) Box and Whiskers plot of metal concentrations in the samples based on brands: (a) iron, (b) Zinc, (c) Copper, and (d) Chromium. Whiskers indicate the maximum and minimum concentrations (e – h) Box and Whiskers plot of metal concentrations in the samples based on brands: (e) cobalt, (f) nickel, (g) cadmium, and (h) lead. Whiskers indicate the maximum and minimum concentration (e – h) Box and Whiskers plot of metal concentrations in the samples based on brands: (e) cobalt, (f) nickel, (g) cadmium, and (h) lead. Whiskers indicate the maximum and minimum concentration Page 13/14 Page 13/14 Page 13/14 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. ESM1.pdf ESM2.pdf ESM3.pdf ESM1.pdf ESM2.pdf ESM3.pdf Page 14/14
https://openalex.org/W4296458360	https://www.researchsquare.com/article/rs-744483/latest.pdf	English	null	Terahertz Channel Performance in Emulated Falling Rain	Research Square (Research Square)	2,022	cc-by	4,913	Terahertz Channel Performance in Emulated Falling Rain Peian Li Beijing Institute of Technology Jianchen Wang Beijing Institute of Technology Liangbin Zhao Beijing Institute of Technology Jianjun Ma (  jianjun_ma@bit.edu.cn ) Beijing Institute of Technology Houjun Sun Beijing Institute of Technology Xiangyuan Bu Beijing Institute of Technology Jianping An Beijing Institute of Technology Terahertz Channel Performance in Emulated Falling Rain Peian Li Beijing Institute of Technology Jianchen Wang Beijing Institute of Technology Liangbin Zhao Beijing Institute of Technology Jianjun Ma (  jianjun_ma@bit.edu.cn ) Beijing Institute of Technology Houjun Sun Beijing Institute of Technology Xiangyuan Bu Beijing Institute of Technology Jianping An Beijing Institute of Technology Research Article Keywords: Terahertz wireless communication, falling rain, rain chamber, power attenuation, BER, raindrop size distribution Posted Date: September 19th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-744483/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Nano Communication Networks on December 9th, 2022. See the published version at https://doi.org/10.1016/j.nancom.2022.100431. Terahertz Channel Performance in Emulated Falling Rain Peian Li Beijing Institute of Technology Jianchen Wang Beijing Institute of Technology Liangbin Zhao Beijing Institute of Technology Jianjun Ma (  jianjun_ma@bit.edu.cn ) Beijing Institute of Technology Houjun Sun Beijing Institute of Technology Xiangyuan Bu Beijing Institute of Technology Jianping An Beijing Institute of Technology Research Article Keywords: Terahertz wireless communication, falling rain, rain chamber, power attenuation, BER, raindrop size distribution Posted Date: September 19th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-744483/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Nano Communication Networks on December 9th, 2022. See the published version at https://doi.org/10.1016/j.nancom.2022.100431. Abstract The ever-increasing capacity demand (up to Tbps) in wireless connectivity is supposed can be satisfied by using terahertz frequency band ranging from 100 GHz to 10 THz. This has been proved over short channel distances in laboratory with high order modulation schemes (such as QPSK, QAM) employed. However, in outdoor adverse weathers, investigations on channel performance are not enough due to difficulties in outdoor measurements, and then more concentrations are still required. In this article, we report performance of terahertz channels in emulated falling rain in our lab by utilizing a broadband- pulsed terahertz channel and a 16-QAM modulated data stream. We observe that, the variability of raindrop size distribution is a major source of uncertainty in theoretical precipitation. We also find that the channel degradation in falling rain is mainly due to power attenuation, instead of phase dispersion which is negligible in our measurement. Research Article Keywords: Terahertz wireless communication, falling rain, rain chamber, power attenuation, BER, raindrop size distribution Posted Date: September 19th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-744483/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. d ll License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Nano Communication Networks on December 9th, 2022. See the published version at https://doi.org/10.1016/j.nancom.2022.100431. Page 1/10 Page 1/10 2. Terahertz Tds Measurement The schematic diagram of the measurement setup is shown in Fig. 1(a) with a commercial T-Ray 2000™ (Picometrix) terahertz time-domain spectrometer (TDS) employed. This is a conventional TDS system and has been employed in many publications [12-14]. THz pulses are generated by femtosecond optical pulses in the transmitter with a collimated beam diameter of 2 cm and then detected by subsequent optical pulses in the Ti:sapphire laser’s pulse train. There are three gold plated mirrors (1, 2 and 3) used to reflect the THz radiation multiple times to extend the beam path inside the falling rain to be 4 m. When collecting the THz pulses to obtain the spectral attenuation, the system is set to a rapid scan mode with a 1000-pulse average calculation. The whole rain chamber is composed of a rain generator (top p beam path region for rain falling and signal propagation. The b machined with 3261 holes in a cascade distribution. A kind of 3 could be epoxied to generate raindrops with an average diamet radius of the needle. The generator is filled with distilled water t whose dielectric properties could be expressed by a double-deb needles is small enough such that when no air pressure is appl raindrops can be generated. The beam path region could comm conditioned by a central air-conditioning system) to maintain a a channel inside this region, it can be regarded as propagating The whole rain chamber is composed of a rain generator (top part) for controllable rain generation and a beam path region for rain falling and signal propagation. The bottom plate of the rain generator is machined with 3261 holes in a cascade distribution. A kind of 31-gauge needle is sealed to each hole and could be epoxied to generate raindrops with an average diameter of 1.9 mm determined by the inner radius of the needle. The generator is filled with distilled water to emulate the actual material for rain, whose dielectric properties could be expressed by a double-debye model (D3M) [15]. The diameter of the needles is small enough such that when no air pressure is applied on the water inside the generator, no raindrops can be generated. The beam path region could communicate with the laboratory (air- conditioned by a central air-conditioning system) to maintain an identical temperature and humidity. For a channel inside this region, it can be regarded as propagating in free space. 1. Introduction Wireless communication technology operating at terahertz (THz) frequency band provides a new solution for satisfying the demands on large data capacity and high physical layer security [1,2], which could not be achieved by existing radio frequency (RF) band and millimeter wave. For establishing 6G wireless networks, the THz band is regarded as a potential candidate in indoor and outdoor scenarios [3]. However, for outdoor applications, THz wireless channels suffer significant signal losses due to atmospheric weather effects, such as absorption and scattering [4]. Previously, amplitude attenuation and related channel performance degradation caused by water fog, dust cloud, rain and snow have been measured and investigated by employing an on-off keying (OOK) modulated THz links [5-7]. Higher order modulation schemes (such as QPSK, QAM etc.) are necessary to update the data rate with information on phase dispersion required, even though a theoretical investigation is provided in reference [8]. THz time- domain spectroscopy (THz-TDS) technique can be available for measuring and monitoring the variation of amplitude and phase and has been used to sense water vapor contained in air [9]. But there is lack of studies on their degradation in adverse outdoor weathers due to the difficulties for data recording, such as uncontrollable and unrepeatable weather conditions. In this work, we investigate the influence of falling rain on THz channels by employing a commercial T- Ray 2000™ (Picometrix) THz-TDS system and a THz wireless setup with a 16-QAM modulation constellation scheme. A weather chamber was built in our laboratory to emulate stable and controllable falling rain. The lab condition can be reproducible and has been previously demonstrated [10,11]. We investigate and analyze the performance of a wireless terahertz channel in falling rain with a high order modulation scheme, which is usually difficult for actual outdoor measurements. The rest of this article is organized as follows: Section 2 details the schematic diagram of the THz-TDS system with a data processing method mentioned. Section 3 shows measured data and comparison with theoretical calculation results. Section 4 presents experimental performance of a THz wireless channel in falling rain and finally Section 5 concludes this article. Page 2/10 Page 2/10 2. Terahertz Tds Measurement The rainrate of the generated falling rain varies from 50 mm/hr to 500 mm/hr, and is linearly related to the air pressure inside the generator [10]. This means we could generate controllable and reproducible falling rain by controlling the air pressure. Fig. 1(b) shows that the raindrop size follows a log-normal distribution function as with a mean of logarithmic value μ = 0.674 and a standard deviation of logarithmic value σ = 0.157. The parameter D represents the diameter of raindrop particles, which is independent of the pressure and rainrate. It is always true that the rain droplets we generated are spherical. Parameter NRr refers to the total number of raindrop particles in a unit volume [16], which depends on the pressure and rainrate. Inverse fast Fourier transformation is conducted on the measured data, the amplitude spectra of the pulses passing free space (no rain generated) and falling rain are shown in Fig. 1(c) with a rainrate (Rr) of 256 mm/hr. Strong water vapor absorption lines at 0.56 THz, 0.75 THz and 0.98 THz are clearly observable for both curves. Phase spectra is also obtained as shown in Fig. 1(d) without any unwrapping algorithms used. Obvious jumps, corresponding to the strong absorption lines of water vapor [17], are observable for both curves. The phase shift due to falling rain, which could be obtained by comparing both phase spectra curves, is negligible in this work, except at frequencies corresponding to water absorption lines. 3. Experimental Data And Theoretical Modeling Page 3/10 Page 3/10 Mie scattering theory [18] can be used to calculate the signal loss due to absorption and scattering caused by raindrop particles, because the size of the raindrop particles we generated is in the scale of millimeter [19], which is usually comparable to or larger than the THz wavelength. For very small particles, solution given by the Mie theory approaches the Rayleigh approximation [20]. This method systematically describes the scattering mechanism of THz waves by particles of various sizes in the atmosphere. A commonly used theoretical model provided by the ITU Recommendation Sector (ITU-R) [21,22] for gaseous absorption based on the physical model MPM93[23]. ITU-R also provides a prediction model for rain attenuation by equation with the values of a and k determined for a given frequency in the range of 1 GHz to 1 THz, which was given by fitting measurement data. Raindrop size distribution can be affected by various microphysical and dynamic processes inside and below cloud layers. In practical applications, empirical mathematical formulas derived from observed size spectra have been used to approximate natural snow size distributions. Raindrops usually follow exponential [24, 25], Gamma [26] and log-normal distribution [27] in present published investigations. Marshall-Palmer (M-P) distribution [24] is a typical kind of exponential function, and can be proposed for both raindrop and snow description [28]. It is expressed as N(r) = N0 exp(-Λr), where, N is the number of rain droplet radius of r + dr in unit volume. Parameter r is the radius of rain droplet. Parameter N0 = 16×103 [m-3·mm-1] and Λ = 8.2Rr-0.21 [mm-1] are two characteristic parameters and can be retrieved rainrate Rr in mm/hr [29]. The calculation results are shown in Fig. 2 with measured data at 140, 220, 340 and 675 GHz. These frequencies lie in the transparency window and has been used for wireless link setup in several publications [2]. The gaseous absorption, especially due to water vapor under a relative humidity of 37%, is included in the model. An obvious discrepancy between the measurements and the calculated result by ITU-R model or Mie scattering theory with M-P distribution. However, the calculation by Mie scattering together with the log-normal distribution is much better agreed with the measured data. This means Mie scattering is a good theory for the precipitation of signal losses due to falling rain, when a correct raindrop size distribution is ready [30]. 4. Terahertz Data Link Measurement To see the influence of the falling rain on higher order constellation modulation schemes, a 16-QAM THz wireless communication setup is built with its schematic diagram shown in Fig. 3(a). A Xilinx Vertix-7 serial FPGA (XC7VX485T) is used to generate a 16-QAM modulated signal at an intermediate frequency (IF) of 1.25GHz with a data rate of 5 Gbps. The generated digital sequence from FPGA is converted into an analog signal by a DAC (MD662H). After that, it is mixed with a radio frequency (RF) of 162 GHz by a subharmonic mixer (110-170GHz) which is driven by an 81GHz reference signal from an ×6 multiplier. Then, the signal is launched out by a THz antenna. On the receiver side, a low noise amplifier amplifies the D-band signal captured by another identical THz antenna and sends its output to a subharmonic mixer to obtain the IF signal which can be converted to digital sequence by an ADC (EV10AQ190). A Xilinx XC7VX690T FPGA is used for synchronization processing and demodulation operation. The output Page 4/10 Page 4/10 binary sequence would be compared with the transmitting binary sequence, and then the Eb/N0 parameter can be derived by calculating the bit-error-ratio (BER) easily. When the 162 GHz channel propagates through the falling rain with a rainrate of 350 mm/hr, variation of BER value with respect to transmitted THz power is recorded and shown in Fig. 4(b). After through the falling rain, the BER values at the received power below -40.3 dBm could not be recorded due to the scattering and absorption by falling rain droplets. An obvious BER degradation caused by falling rain can be observed when compared with that in free space under the same transmitted power. However, the slopes of both curves are almost identical, which means the BER degradation is mainly due to the power loss by falling rain. In other words, the phase of the channel is more resilience to the influence by falling rain than the amplitude. When we mount the receiver on a movable rail which permits it to translate along an axis perpendicular to the incoming beam’s propagation direction as in Fig. 3(a). By scanning the detector along this line, we map the spatial distribution of the beam arriving at the receiver side. A power loss of 0.5 dB (from -36.4 dBm to -36.9 dBm) due to rain is observed in Fig. 4. Terahertz Data Link Measurement 4(a) along the beam axis (receiver position of 0 m), which is consistent with our prediction in Fig. 2 for a channel distance of 1 m. The corresponding BER degradation is shown in Fig. 4(b), which changes from 7.6×10-11 in free space to 6.5×10-10 in falling rain. An error rate of 10-10 could not be achieved in falling rain due to the signal loss. It should be noted that there is no obvious fluctuation observed in both the power and BER evolution curves, which confirms that the falling rain has negligible influence on the phase of the signal instead of its amplitude. 5. Conclusion In this article, we conduct a study on performance of terahertz wireless channels in emulated falling rain by using a commercial THz time-domain spectroscopy system and a 16-QAM modulated THz wireless channel. The Mie scattering theory is confirmed to be a correct method to predict the attenuation when an accurate raindrop size distribution is chosen or measured. The phase of the wireless channel is more resistive to the falling rain than the amplitude. Further efforts will be conducted to include the influence of rain-lines and wind-induced pole vibration [8], which should be considered in actual outdoor scenarios. References [1] J. Ma et al., "Security and eavesdropping in terahertz wireless links," Nature, vol. 563, p. 89, Oct 15 2018, doi: 10.1038/s41586-018-0609-x. [2] T. Nagatsuma, G. Ducournau, and C. C. Renaud, "Advances in terahertz communications accelerated by photonics," Nature Photonics, vol. 10, no. 6, pp. 371-379, 2016, doi: 10.1038/nphoton.2016.65. [2] T. Nagatsuma, G. Ducournau, and C. C. Renaud, "Advances in terahertz communications accelerated by photonics," Nature Photonics, vol. 10, no. 6, pp. 371-379, 2016, doi: 10.1038/nphoton.2016.65. [3] M. H. Alsharif, A. H. Kelechi, M. A. Albreem, S. A. Chaudhry, M. S. Zia, and S. Kim, "Sixth Generation (6G) Wireless Networks: Vision, Research Activities, Challenges and Potential Solutions," Symmetry, vol. 12, no. 4, p. 676, 2020, doi: 10.3390/sym12040676. Page 5/10 [4] Z. 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References Zimdars, "Fiber-pigtailed terahertz time domain spectroscopy instrumentation for package inspection and security imaging," presented at the Terahertz for Military and Security Applications, Orlando, Florida, United States, 2003. [14] Z. Hossain, S. H. Vedant, C. R. Nicoletti, and J. F. Federici, "Multi-user Interference Modeling and Experimental Characterization for Pulse-based Terahertz Communication," pp. 1-6, 2016, doi: 10.1145/2967446.2967462. [15] F. T. Ulaby and D. G. Long, Microwave Radar and Radiometric Remote Sensing. Ann Arbor, Michigan: University of Michigan Press, 2013. Page 6/10 [16] S. Ishii, S. Sayama, and T. Kamei, "Measurement of Rain Attenuation in Terahertz Wave Range," Wireless Engineering and Technology, vol. 02, no. 03, pp. 119-124, 2011, doi: 10.4236/wet.2011.23017. [17] C. Jördens, S. Wietzke, M. Scheller, and M. Koch, "Investigation of the water absorption in polyamide and wood plastic composite by terahertz time-domain spectroscopy," Polym Test, vol. 29, no. 2, pp. 209- 215, 2010, doi: 10.1016/j.polymertesting.2009.11.003. [18] D. J. Lockwood, "Rayleigh and Mie Scattering," in Encyclopedia of Color Science and Technology, M. R. Luo Ed. New York, NY: Springer, 2016. [18] D. J. Lockwood, "Rayleigh and Mie Scattering," in Encyclopedia of Color Science and Technology, M. R. Luo Ed. New York, NY: Springer, 2016. [19] H. R. Pruppacher and J. D. Klett, Microphysics of clouds and precipitation. Dordrecht: Kluwer Academic Publishers, 1997. [19] H. R. Pruppacher and J. D. Klett, Microphysics of clouds and precipitation. Dordrecht: Kluwer Academic Publishers, 1997. [20] D. Deirmendjian, Electromagnetic scattering on spherical polydispersions. New York: American Elsevier Publishing, 1969. [20] D. Deirmendjian, Electromagnetic scattering on spherical polydispersions. New York: American Elsevier Publishing, 1969. [21] "Recommendation ITU-R P.676-11: Attenuation by Atmospheric Gases." https://www.itu.int/dms_pubrec/itu-r/rec/p/R-REC-P.676-11-201609-I!!PDF-E.pdf (accessed. [21] "Recommendation ITU-R P.676-11: Attenuation by Atmospheric Gases." https://www.itu.int/dms_pubrec/itu-r/rec/p/R-REC-P.676-11-201609-I!!PDF-E.pdf (accessed. [22] " International Telecommunication Union Recommendation (ITU-R) P.676-12: Attenuation by atmospheric gases and related effects." https://www.itu.int/rec/R-REC-P.676-12-201908-I (accessed. [22] " International Telecommunication Union Recommendation (ITU-R) P.676-12: Attenuation by atmospheric gases and related effects." https://www.itu.int/rec/R-REC-P.676-12-201908-I (accessed. [23] H. Liebe, G. Hufford, and M. Cotton, "Propagation modeling of moist air and suspended water/ice particles at frequencies below 1000 GHz," presented at the Proceedings of AGARD, 52nd Specialists Meeting of the Electromagnetic Wave Propagation Panel, 1993. [23] H. Liebe, G. Hufford, and M. Cotton, "Propagation modeling of moist air and suspended water/ice particles at frequencies below 1000 GHz," presented at the Proceedings of AGARD, 52nd Specialists Meeting of the Electromagnetic Wave Propagation Panel, 1993. [24] J. S. Marshall and W. M. References Palmer, "The distribution of raindrops with size," Journal of Meteorology, vol. 5, no. 4, pp. 165-166, 1948. [24] J. S. Marshall and W. M. Palmer, "The distribution of raindrops with size," Journal of Meteorology, vol. 5, no. 4, pp. 165-166, 1948. [25] G. Zhang, M. Xue, Q. Cao, and D. Dawson, "Diagnosing the Intercept Parameter for Exponential Raindrop Size Distribution Based on Video Disdrometer Observations: Model Development," Journal of Applied Meteorology and Climatology, vol. 47, no. 11, pp. 2983-2992, 2008. [26] D. A. de Wolf, "On the Laws‐Parsons distribution of raindrop sizes," Radio Science, vol. 36, pp. 639- 642, 2001. [27] C. Cerro and B. Codina, "Modeling raindrop size distribution and Z(R) relations in the Western Mediterranean Area," Journal of Applied Meteorology, vol. 36, no. 11, pp. 1470-1479, 1997. [28] R. E. Passarelli, "Theoretical and Observational Study of Snow-Size Spectra and Snowflake Aggregation Efficiencies," Journal of the Atmospheric Sciences,, vol. 35, no. 5, pp. 882-889, 1978. [29] Y. Amarasinghe, W. Zhang, R. Zhang, D. M. Mittleman, and J. Ma, "Scattering of Terahertz Waves by Snow," Journal of Infrared, Millimeter, and Terahertz Waves, vol. 41, pp. 215-224, 2019. Page 7/10 Page 7/10 [30] M. Yoseva, H. Hashiguchi, M. Vonnisa, L. Luini, S. Nugroho, and M. A. Shafii., "Characteristics of Rain Attenuation for Microwave-to-terahertz Waveband from Raindrop Size Distribution Observation in Indonesia," presented at the PhotonIcs & Electromagnetics Research Symposium - Spring (PIERS-Spring), Rome, Italy, 2019. [30] M. Yoseva, H. Hashiguchi, M. Vonnisa, L. Luini, S. Nugroho, and M. A. Shafii., "Characteristics of Rain Attenuation for Microwave-to-terahertz Waveband from Raindrop Size Distribution Observation in Indonesia," presented at the PhotonIcs & Electromagnetics Research Symposium - Spring (PIERS-Spring), Rome, Italy, 2019. [30] M. Yoseva, H. Hashiguchi, M. Vonnisa, L. Luini, S. Nugroho, and M. A. Shafii., "Characteristics of Rain Attenuation for Microwave-to-terahertz Waveband from Raindrop Size Distribution Observation in Indonesia," presented at the PhotonIcs & Electromagnetics Research Symposium - Spring (PIERS-Spring), Rome, Italy, 2019. Figures Figure 1 (a) Schematic measurement setup. The rain generator has a dimension of 1.0×0.2×0.2 m3 in L×W×H. It is constructed from aluminum with a transparent Plexiglas window in the center of the side panel in order to monitor the water level while the system is running. The beam path region has a dimension of 1.0×0.2×0.5 m3 in L×W×H. Mirrors (1, 2 and 3) are gold plated and could be used to extend the beam path inside the chamber to be 4 m. (b) Rain drop size distribution. A log-normal distribution fit to measured data is shown with a mean of logarithmic value μ= 0.674 and a standard deviation of logarithmic value σ = 0.157. The measured data was obtained from reference [10]. (c) The broadband- pulsed terahertz source is used to launch a THz pulse into free space and falling rain. The red curve stands for the received signal propagates in free space and the blue one for the signal passing falling rain with a rainrate of 256 mm/hr. (d) Phase performance of THz channels due to falling rain with the same legend as Fig. 1(c). There is no unwrapping algorithms used. Figure 1 (a) Schematic measurement setup. The rain generator has a dimension of 1.0×0.2×0.2 m3 in L×W×H. It i constructed from aluminum with a transparent Plexiglas window in the center of the side panel in order t monitor the water level while the system is running. The beam path region has a dimension of 1.0×0.2×0.5 m3 in L×W×H. Mirrors (1, 2 and 3) are gold plated and could be used to extend the beam Figure 1 (a) Schematic measurement setup. The rain generator has a dimension of 1.0×0.2×0.2 m3 in L×W×H. It is constructed from aluminum with a transparent Plexiglas window in the center of the side panel in order to monitor the water level while the system is running. The beam path region has a dimension of 1.0×0.2×0.5 m3 in L×W×H. Mirrors (1, 2 and 3) are gold plated and could be used to extend the beam path inside the chamber to be 4 m. (b) Rain drop size distribution. A log-normal distribution fit to measured data is shown with a mean of logarithmic value μ= 0.674 and a standard deviation of logarithmic value σ = 0.157. The measured data was obtained from reference [10]. (c) The broadband- pulsed terahertz source is used to launch a THz pulse into free space and falling rain. The red curve stands for the received signal propagates in free space and the blue one for the signal passing falling rain with a rainrate of 256 mm/hr. (d) Phase performance of THz channels due to falling rain with the same legend as Fig. 1(c). There is no unwrapping algorithms used. (a) Schematic measurement setup. The rain generator has a dimension of 1.0×0.2×0.2 m3 in L×W×H. It constructed from aluminum with a transparent Plexiglas window in the center of the side panel in order t monitor the water level while the system is running. The beam path region has a dimension of 1.0×0.2×0.5 m3 in L×W×H. Mirrors (1, 2 and 3) are gold plated and could be used to extend the beam path inside the chamber to be 4 m. (b) Rain drop size distribution. A log-normal distribution fit to measured data is shown with a mean of logarithmic value μ= 0.674 and a standard deviation of logarithmic value σ = 0.157. The measured data was obtained from reference [10]. (c) The broadband- pulsed terahertz source is used to launch a THz pulse into free space and falling rain. The red curve stands for the received signal propagates in free space and the blue one for the signal passing falling rain with a rainrate of 256 mm/hr. (d) Phase performance of THz channels due to falling rain with the same legend as Fig. 1(c). There is no unwrapping algorithms used. Figure 1 Page 8/10 Page 8/10 Figure 2 Power attenuation on wireless channels operating at (a) 140 GHz, (b) 220 GHz, (c) 340 GHz and (d) 675 GHz caused by falling rain with rainrate changing from 50 mm/hr to 450 mm/hr. The black squares represent measured data obtained from the difference between the amplitude spectra in free space and in falling rain. The solid curves stand for calculated results. The red one is for calculation by using Mie scattering theory and the log-normal distribution of raindrop size as in Fig. 1(b). The blue one is for ITU-R model. The black one is by the Mie scattering theory and the Mashall-Palmer (M-P) distribution of raindrop size. Figure 2 Figure 2 Power attenuation on wireless channels operating at (a) 140 GHz, (b) 220 GHz, (c) 340 GHz and (d) 675 GHz caused by falling rain with rainrate changing from 50 mm/hr to 450 mm/hr. The black squares represent measured data obtained from the difference between the amplitude spectra in free space and in falling rain. The solid curves stand for calculated results. The red one is for calculation by using Mie scattering theory and the log-normal distribution of raindrop size as in Fig. 1(b). The blue one is for ITU-R model. The black one is by the Mie scattering theory and the Mashall-Palmer (M-P) distribution of raindrop size. Page 9/10 Page 9/10 Figure 3 Schematic diagram and degradation of a modulated THz channel as a function of transmitted power. (a) Schematic measurement setup, with one transmitter at 162 GHz, and with the receiver mounted on a linear rail to vary the position of the receiver. (b) Measured real-time BER performance of the THz link as a function of the transmitted power at a data rate of 5 Gbps. Values are recorded both after propagating through free space (red) and falling rain (blue) with the receiver fixed along the beam axis. Figure 3 Figure 3 Figure 3 Schematic diagram and degradation of a modulated THz channel as a function of transmitted power. (a) Schematic measurement setup, with one transmitter at 162 GHz, and with the receiver mounted on a linear rail to vary the position of the receiver. (b) Measured real-time BER performance of the THz link as a function of the transmitted power at a data rate of 5 Gbps. Values are recorded both after propagating through free space (red) and falling rain (blue) with the receiver fixed along the beam axis. Schematic diagram and degradation of a modulated THz channel as a function of transmitted power. (a) Schematic measurement setup, with one transmitter at 162 GHz, and with the receiver mounted on a linear rail to vary the position of the receiver. (b) Measured real-time BER performance of the THz link as a function of the transmitted power at a data rate of 5 Gbps. Values are recorded both after propagating through free space (red) and falling rain (blue) with the receiver fixed along the beam axis. Figure 4 Power and BER patterns of the THz channel in falling rain. (a) Power pattern measured when channel propagates through free space (red) at temperature T =17℃, humidity RH 34% and falling rain (blue) at rainrate Rr = 350 mm/hr, temperature T=17℃, humidity RH 34%. (b) Measured BER evolution corresponding to the power patterns in Fig. 4(a). Figure 4 Power and BER patterns of the THz channel in falling rain. (a) Power pattern measured when channel propagates through free space (red) at temperature T =17℃, humidity RH 34% and falling rain (blue) at rainrate Rr = 350 mm/hr, temperature T=17℃, humidity RH 34%. (b) Measured BER evolution corresponding to the power patterns in Fig. 4(a). Page 10/10
https://openalex.org/W2995157772	https://biotechnologyforbiofuels.biomedcentral.com/track/pdf/10.1186/s13068-019-1635-0	English	null	Nitrogen-dependent coordination of cell cycle, quiescence and TAG accumulation in Chlamydomonas	Biotechnology for biofuels	2,019	cc-by	21,933	Abstract Microalgae hold great promises as sustainable cellular factories for the production of alternative fuels, feeds, and biop- harmaceuticals for human health. While the biorefinery approach for fuels along with the coproduction of high-value compounds with industrial, therapeutic, or nutraceutical applications have the potential to make algal biofuels more economically viable, a number of challenges continue to hamper algal production systems at all levels. One such hurdle includes the metabolic trade-off often observed between the increased yields of desired products, such as tria- cylglycerols (TAG), and the growth of an organism. Initial genetic engineering strategies to improve lipid productivity in microalgae, which focused on overproducing the enzymes involved in fatty acid and TAG biosynthesis or inactivat- ing competing carbon (C) metabolism, have seen some successes albeit at the cost of often greatly reduced biomass. Emergent approaches that aim at modifying the dynamics of entire metabolic pathways by engineering of pertinent transcription factors or signaling networks appear to have successfully achieved a balance between growth and neutral lipid accumulation. However, the biological knowledge of key signaling networks and molecular components linking these two processes is still incomplete in photosynthetic eukaryotes, making it difficult to optimize metabolic engineering strategies for microalgae. Here, we focus on nitrogen (N) starvation of the model green microalga, Chla- mydomonas reinhardtii, to present the current understanding of the nutrient-dependent switch between proliferation and quiescence, and the drastic reprogramming of metabolism that results in the storage of C compounds following N starvation. We discuss the potential components mediating the transcriptional repression of cell cycle genes and the establishment of quiescence in Chlamydomonas, and highlight the importance of signaling pathways such as those governed by the target of rapamycin (TOR) and sucrose nonfermenting-related (SnRK) kinases in the coordina- tion of metabolic status with cellular growth. A better understanding of how the cell division cycle is regulated in response to nutrient scarcity and of the signaling pathways linking cellular growth to energy and lipid homeostasis, is essential to improve the prospects of biofuels and biomass production in microalgae. Keywords: Chlamydomonas, Quiescence, Nitrogen deprivation, Triacylglycerols, Cell cycle, DREAM complex, TOR, SnRK/CKIN, Biofuel, Biomass Nitrogen‑dependent coordination of cell cycle, quiescence and TAG accumulation in Chlamydomonas Tomomi Takeuchi1,2 and Christoph Benning1,3,2* © The Author(s) 2019. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco mmons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Biotechnology for Biofuels Biotechnology for Biofuels Takeuchi and Benning Biotechnol Biofuels (2019) 12:292 https://doi.org/10.1186/s13068-019-1635-0 Biotechnology for Biofuels Open Access Backgroundh The use of algae as a potential source of renewable fuel, animal feeds in addition to nutrients and pharmaceuti- cals for human health has been recognized and exploited for decades. Both micro- and macro-algae constitute a diverse group of primarily aquatic photosynthetic organ- isms with varying levels of complexity, and their natural Correspondence: benning@msu.edu 2 Department of Energy‑Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA Full list of author information is available at the end of the article Correspondence: benning@msu.edu 2 Department of Energy‑Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA Full list of author information is available at the end of the article Because algae maintain high productivity in nutrient-rich waters, they can be used to remove excess nutrients from waste water and mitigate fertilizer runoff from farms, while simulta- neously yielding biomass and precursors for the produc- tion of biofuels [13–15]. In addition, the use of industrial flue gas as a source of C, the biorefinery-based approach to biofuel production, and the concurrent cultivation of high-value compounds were also proposed as a potential means to further lower the cost of algal biofuels [8–10, 12, 16]. biochemical compositions (e.g., high contents of oil, carbohydrates, proteins, sugars, vitamins, pigments, or minerals) make them uniquely suitable for different commercial purposes. In addition to their relevance in agriculture as fertilizers, soil conditioners and livestock feeds, algae provide many nutrients essential for human health, including vitamins, minerals, anti-oxidants, and polyunsaturated fatty acids such as docosahexaenoic acids and eicosapentaenoic acids [1–3]. Furthermore, algae-derived products are also used as gelling agents and stabilizers in various food products, cosmetics and pharmaceuticals [1–3]. Over thirty recombinant pro- teins have been successfully produced in microalgae to date, including hormones, enzymes, antibodies, vaccines and immunotoxins, highlighting the biotechnical utili- ties and potentials of these organisms [4–7]. In the past few decades, microalgae have garnered renewed interests as alternative feedstocks for the sustainable production of biofuels, in the forms of biodiesel, bioethanol, biogas and hydrogen. Many microalgae are able to grow rapidly to high cell densities using CO2 or other provided carbon (C) sources, can be cultivated using nonarable land and water sources not suited for agriculture, and accumulate more triacylglycerols (TAG) per dry weight or per unit area than agricultural oil crops [8–12]. Because algae maintain high productivity in nutrient-rich waters, they can be used to remove excess nutrients from waste water and mitigate fertilizer runoff from farms, while simulta- neously yielding biomass and precursors for the produc- tion of biofuels [13–15]. In addition, the use of industrial flue gas as a source of C, the biorefinery-based approach to biofuel production, and the concurrent cultivation of high-value compounds were also proposed as a potential means to further lower the cost of algal biofuels [8–10, 12, 16]. sought-after molecules in algae, including TAG, hydro- gen, and carotenoids like β-carotene and astaxanthin [9, 18–25]. However, the increase in these compounds often comes at the expense of inhibited growth, resulting in the considerable reduction of biomass. A two-stage cultiva- tion strategy, where the algal cells are subjected to stress only after a period of optimal growth and accumulation of biomass, has been proposed and tested to circumvent this problem [26–28], but this production method is still costly due to its extended requirement for time and the inherent complexity in monitoring and optimizing the production process. p p Genetic engineering strategies in algae, which aimed to alter the expression levels of genes encoding individual enzymes involved in lipid metabolism, TAG biosynthesis and catabolism, or other competing C metabolic path- ways, have seen mixed outcomes in achieving the optimal balance between lipid productivity and growth [29, 30]. Recent approaches targeting transcription factors or sign- aling pathways that regulate C and growth metabolisms in algae appear to achieve more consistent successes in increasing TAG content with little or no compromise in cellular growth and proliferation by simultaneously modifying multiple components of a metabolic pathway [31–36]. However, the regulatory components and sign- aling networks coordinating the allocations of C towards storage and growth are still not well characterized in photosynthetic eukaryotes, which continues to hamper the metabolic engineering efforts for algae. Therefore, a better understanding of the molecular mechanisms by which metabolism and growth are regulated and cou- pled is necessary. Here, the nutrient-dependent transi- tions between the cell division and quiescence cycles, the shifts in metabolism towards the synthesis of C storage compounds following nutrient starvation, and the poten- tial molecular components mediating the cessation of growth and entry into quiescence under stress conditions are discussed focusing on nitrogen (N) starvation in the model green microalgae, Chlamydomonas reinhardtii, as a reference. Our current understanding of the signal- ing pathways integrating the changes in metabolism and the cell division cycle in response to nutrient availability in algae is presented, followed by concluding remarks on the potential biotechnological implications of the pre- sented concepts. Although the production of sustainable energy and economically valuable compounds from algae hold great promises, a number of hurdles persist. These include the species-dependent recalcitrance to various genetic manipulations, suboptimal utilization and conversion of light energy and CO2 to biomass due to light satura- tion and or photoinhibition, limited light penetrance in the culture, undesirable contamination, and high costs ultimately associated with sustaining optimal growth and metabolic outputs, as well as high costs of extraction and processing [8, 11, 12, 17, 18]. Another major impedi- ment that prevents algal biofuels from becoming a com- petitive alternative to fossil petroleum on a commercial scale involves the inverse relationship between the yield of cellular products and the growth of the organism. A plethora of abiotic stresses such as nutrient deprivation, extreme light conditions and changes in temperature, salinity or pH is known to induce the accumulation of © The Author(s) 2019. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco mmons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Takeuchi and Benning Biotechnol Biofuels (2019) 12:292 Takeuchi and Benning Biotechnol Biofuels (2019) 12:292 Page 2 of 20 Page 2 of 20 biochemical compositions (e.g., high contents of oil, carbohydrates, proteins, sugars, vitamins, pigments, or minerals) make them uniquely suitable for different commercial purposes. In addition to their relevance in agriculture as fertilizers, soil conditioners and livestock feeds, algae provide many nutrients essential for human health, including vitamins, minerals, anti-oxidants, and polyunsaturated fatty acids such as docosahexaenoic acids and eicosapentaenoic acids [1–3]. Furthermore, algae-derived products are also used as gelling agents and stabilizers in various food products, cosmetics and pharmaceuticals [1–3]. Over thirty recombinant pro- teins have been successfully produced in microalgae to date, including hormones, enzymes, antibodies, vaccines and immunotoxins, highlighting the biotechnical utili- ties and potentials of these organisms [4–7]. In the past few decades, microalgae have garnered renewed interests as alternative feedstocks for the sustainable production of biofuels, in the forms of biodiesel, bioethanol, biogas and hydrogen. Many microalgae are able to grow rapidly to high cell densities using CO2 or other provided carbon (C) sources, can be cultivated using nonarable land and water sources not suited for agriculture, and accumulate more triacylglycerols (TAG) per dry weight or per unit area than agricultural oil crops [8–12]. Chlamydomonas as a model to study key life‑cycle transitions At the cell biological level, many abiotic stresses will induce cells to accumulate storage compounds and exit the normal cell division cycle to enter an alterna- tive reversible state known as quiescence, or G0 [37]. When the conditions are again conducive to growth, Takeuchi and Benning Biotechnol Biofuels (2019) 12:292 Page 3 of 20 cells degrade the accumulated storage compounds, exit quiescence and reenter the cell division cycle [38]. Chla- mydomonas serves as a particularly excellent model sys- tem to study the coordination between metabolism, cell division cycle and quiescence in photosynthetic organ- isms for several reasons. Chlamydomonas can be grown rapidly under heterotrophic, photoautotrophic or mixo- trophic conditions, depending on the research needs [39]. For instance, the growth and division of Chlamydomonas cells can be synchronized with alternating light and dark cycles when they are grown photoautotrophically, ena- bling the facile isolation of cells at different stages of the cell cycle [40] (Fig. 1). In addition, the life-cycle transi- tions between cell division to quiescence and vice versa can be discretely controlled and analyzed by selective removal or resupply of nutrient(s) in the medium (Fig. 1). Furthermore, a great number of -omics-based studies has been conducted using Chlamydomonas under differ- ent stress conditions in the past decade, and a wealth of literature on how Chlamydomonas cells reprogram their metabolism in response to nutrient shortage, such as N starvation at the levels of transcriptome, proteome and metabolome is available [41–49]. While Chlamydomonas is not typically considered a candidate alga for the pro- duction of biofuel feedstocks, Chlamydomonas cells still accumulate a significant amount of TAG just as other oleaginous algae do in response to nutrient starvation [20]. Combined with the availability of well-established molecular genetics and genomic tools (e.g., the anno- tated genome, transformation protocols, reverse genetic engineering tools, and mutant libraries [50–53]) and the haploid genome of Chlamydomonas during vegetative growth [39], there is a solid infrastructure for the further exploration of the regulatory link between metabolism and life-cycle transitions in this alga. The intersection between the cell division cycle and the quiescence cycle in Chlamydomonasfi In the presence of sufficient nutrients, Chlamydomonas and many other green algae grow and divide by a modi- fied cell cycle involving multiple fissions, where the cells go through a prolonged growth or G1 phase followed by a rapid succession of S/M (DNA synthesis and mitosis) cycles [54–56] (Fig. 1). The gap between the S and M phases (known as the G2 phase) is not observed in the cell cycle of Chlamydomonas [57]. In addition, the cell cycle of photoautotrophically grown Chlamydomonas cells synchronizes under diel conditions such that cellular growth, flagella-dependent phototaxis and light-depend- ent reactions of photosynthesis are maximized during the day. Processes such as the replication of DNA and cell division (i.e., S/M phase), which may benefit from the absence of potentially damaging photons and require the resorption of flagella for the basal body-mediated coor- dination of mitosis and cytokinesis, are timed to occur during the night [40, 55, 56, 58]. Early in G1, the newly hatched Chlamydomonas daughter cells are in a stage called pre-commitment, where the cells have not yet reached the critical size necessary to achieve competency for division. When these pre-commitment cells reach a critical volume, they pass a size-regulated checkpoint termed “Commitment”, which is a point of no return similar to “Start” in yeast and “Restriction Point” in mammalian cell cycles [40, 59–61]. Since growing Chla- mydomonas cells may reach more than ten times their initial volume before the start of the S/M phase after a prolonged G1 phase, multiple rounds (n) of S/M cycles are necessary to produce 2n daughters of equal size [61]. Thus, the number of S/M cycles that each mother cell undergoes is determined by its cell size such that daugh- ters of uniform size distributions are always achieved [59, 60] (Fig. 1). O th th h d h f d ith t i t li it ti G1 G0 N- NR G1 CP S M N+ Cell division cycle Quiescence cycle n=1 n divisions n=2 ... Fig. 1 The intersection between the cell division and the quiescence cycles in Chlamydomonas. The intersection between the cell division cycle and the quiescence cycle in Chlamydomonasfi The circles to the right represent the cell division cycle of Chlamydomonas characterized by multiple fissions, where the cells increase in volume during a prolonged growth (G1) phase during the light phase (white left half), followed by rapid rounds of S/M (DNA synthesis and mitosis) cycles during the dark phase (shaded right half) to give rise to 2n daughter cells of equal size. The commitment point (CP) represents the size-dependent checkpoint. Upon the passage of CP, the cells commit to completing at least one round of division even when the light or nutrients are subsequently withdrawn. The left circle represents the quiescence cycle, where the cells cease further growth and division with 1C (one copy) chromatin content. The entry into and exit from the quiescence (G0) are controlled by the availability of nutrients, such as nitrogen (N), and the respective changes in chlorophyll content of Chlamydomonas cells are depicted by different shades of green. The coupling of these two opposing cycles occurs during the post-mitotic resting stage or G1 phase prior to the passage of CP. Cell cycle-dependent steps are represented by the black arrow heads, while the nutrient-dependent steps are represented by the white arrow heads. N+: N-replete growth; N−: N deprivation; NR: N refeeding G1 G0 N- NR G1 CP S M N+ Cell division cycle Quiescence cycle n=1 n divisions n=2 ... G1 G1 CP S M N+ Cell division cycle cycle n=1 n divisions n=2 ... Fig. 1 The intersection between the cell division and the quiescence cycles in Chlamydomonas. The circles to the right represent the cell division cycle of Chlamydomonas characterized by multiple fissions, where the cells increase in volume during a prolonged growth (G1) phase during the light phase (white left half), followed by rapid rounds of S/M (DNA synthesis and mitosis) cycles during the dark phase (shaded right half) to give rise to 2n daughter cells of equal size. The commitment point (CP) represents the size-dependent checkpoint. Upon the passage of CP, the cells commit to completing at least one round of division even when the light or nutrients are subsequently withdrawn. The left circle represents the quiescence cycle, where the cells cease further growth and division with 1C (one copy) chromatin content. The intersection between the cell division cycle and the quiescence cycle in Chlamydomonasfi In Chlamydomonas, the cell density of mixotrophically grown cells will approximately double using the finite reservoir of intracellular N within the first 24 h of N starvation [43, 51]. Following this increase in cell number, the expression of genes involved in cell cycle progression, DNA synthesis, and replication is substan- tially reduced [42], and by day 2 of N deprivation, greater than 70% of the cellular population arrests growth with 1C (one copy) chromatin content [72]. Therefore, in the face of starvation, the arrest of further growth and divi- sion prior to DNA replication during the pre-commit- ment phase is also likely an important factor enabling successful life-cycle transitions of Chlamydomonas. Cellular changes that accompany N starvation d h i i i Chl d cycle and forego the anabolic, energy-consuming metab- olism that is required for growth and division in favor of energy-saving metabolism that defines quiescence [62]. This is also the case for microalgae such as Chla- mydomonas (Fig. 1). The ability of ancestral eukaryotic cells to enter a state of quiescence, maintain viability, and subsequently resume growth when the conditions improve, was likely critical, not only for their immedi- ate survival but the subsequent evolution of species. The molecular mechanisms by which cells transition from active cell division to quiescence cycles and vice versa in response to nutrient availability are best characterized in yeast [38]. However, the capacity to orchestrate these life- cycle changes is evolutionarily conserved in many organ- isms. For instance, the entry into quiescence in cultured mammalian cells can be induced by serum and amino acid starvation, high cell density, and anchorage depriva- tion [63–67], although their proliferation is typically con- trolled by the developmental and contextual cues within the organism. Thus, some features of quiescent cells appear to be more universal. Although exceptions exist, these include the arrest of growth and cell division before the genome replicates, chromosome compaction, induc- tion of autophagy, reduced rates of transcription and translation, and increased content of C storage molecules [37, 38, 62, 68]. An increasing body of work in opisthokonts as well as in Chlamydomonas suggests that quiescence is a poised and actively maintained state, where the entry into and exit from such a state represent distinct processes gov- erned by unique signaling and gene-regulatory networks, rather than a phase of the cell division cycle or a passive inactive state [38, 67–72] (Fig. 1). The intersection between the cell division cycle and the quiescence cycle in Chlamydomonasfi The entry into and exit from the quiescence (G0) are controlled by the availability of nutrients, such as nitrogen (N), and the respective changes in chlorophyll content of Chlamydomonas cells are depicted by different shades of green. The coupling of these two opposing cycles occurs during the post-mitotic resting stage or G1 phase prior to the passage of CP. Cell cycle-dependent steps are represented by the black arrow heads, while the nutrient-dependent steps are represented by the white arrow heads. N+: N-replete growth; N−: N deprivation; NR: N refeeding On the other hand, when faced with nutrient limitation, single-celled organisms exit from the active proliferative Takeuchi and Benning Biotechnol Biofuels (2019) 12:292 Takeuchi and Benning Biotechnol Biofuels (2019) 12:292 Takeuchi and Benning Biotechnol Biofuels (2019) 12:292 Page 4 of 20 Page 4 of 20 The entry into the quiescence cycle in the early G1 phase before genome replication is likely important for the maintenance of viability during quiescence and the successful reentry into the cell division cycle in response to growth-promoting cues. Because quiescent cells can- not effectively dilute out molecules such as DNA dam- aged by reactive oxygen species (ROS) through growth and cell division, replace them through active synthesis, or repair them by energy-costly mechanisms, the con- densation of chromosomes facilitates the preservation of genomic integrity and promotes survival [37, 38, 62]. Although the transcripts and protein products of most cell cycle genes are not essential for the maintenance of quiescence and survival in mammalian quiescent cells, the repression of genes that promote cell cycle progres- sion, including genes encoding mitotic CDKs and their associated cyclins, is critical for the appropriate exit from the cell division cycle, the establishment of quies- cence and the subsequent resumption of growth [62, 73, 74]. In response to quiescence-inducing cues, the inhibitors of G1 CDKs become upregulated in various quiescent mammalian cell-lineages [62]. For instance, these inhibitors act to maintain the hematopoietic stem cells in quiescence and prevent them from inappropri- ately or precociously entering the cell cycle [62, 75, 76]. These functions of CDK inhibitors appear conserved also in yeast [77]. Yeast mutant cells that have lost the abil- ity to repress certain growth and cell cycle-related genes following glucose exhaustion also have shortened lifes- pan and fail to successfully exit quiescence upon glucose refeeding [78]. The intersection between the cell division cycle and the quiescence cycle in Chlamydomonasfi In the opisthokont models of quiescence, the intersection between the cell division cycle and the so called quiescence cycle is thought to occur early in the G1 phase or during the “restrictive window” following the completion of a pre- vious cell cycle before the cells pass their respective G1 checkpoints [38, 68]. In Chlamydomonas, it is also dur- ing the G1 or the post-mitotic resting phase prior to the passage of the commitment point that the cells are faced with a decision whether to proceed with the subsequent steps of the cell division cycle or to enter an alternative quiescence cycle (Fig. 1). After passing the commitment point, Chlamydomonas cells will undergo at least one round of division even after nutrients or light are with- drawn [40, 60], likely because the completion of the cell division cycle is under the control of the intrinsic oscil- lation of cell cycle regulators such as cyclin-dependent kinases (CDKs) [56]. Thus, it is only when the cyclical transcriptional waves during the cell cycle cease and the cells arrive at the pre-commitment stage that they are able to enter into the quiescence cycle. Cellular changes that accompany N starvation and the entry into quiescence in Chlamydomonas As the universal features of quiescence are further refined, it is becoming evident that the transition from the cell division cycle to the quiescence cycle and its reversal require the genome-wide adjustment of regula- tory networks, metabolism, and intracellular structures (discussed in detail below and summarized in Fig. 2), and where applicable, necessitate the repression of alternative Cellular changes that accompany N starvation and the entry into quiescence in Chlamydomonas Although ammonium is the preferred source of N, Chlamydomonas cells can also assimilate other inorganic N-containing compounds [85]. Thus, following N deprivation, the abundance of transcripts and proteins involved in the transport and the metabolism of alternative, less favorable N sources increases almost immediately (within 0.5–1 h) [43, 85]. The cellular-wide reprogramming of metabolism occurs at the levels of transcripts and proteins to conserve energy and minimize N consumption. The levels of both cytoplasmic and chloroplast ribosomes decrease sub- stantially [86, 87], and the total cellular contents of RNA [88] and protein [43] per cell become reduced by approx- imately 60% and 50%, respectively. It has been reported that the proteins whose abundance increases upon trans- fer of cells to N-free medium, such as those needed for N acquisition and metabolism, contain less N on average than those that decrease in abundance, highlighting the evolutionarily selected N sparing strategy to reduce the cellular demand for N when it is not readily available in the environment [43]. Chlamydomonas cells also utilize a similar conservation mechanism during sulfur (S) short- age, such that the abundant proteins under S-deficient conditions contain less S in their amino acid side chains [89]. While the genes encoding the key enzymes of gly- oxylate cycle and gluconeogenesis are downregulated [42], those involved in the biosynthesis and branching of starch peak shortly after the transfer of cells to N-free medium, followed by a steady decrease in their transcript levels until the new basal level is achieved [46]. This is in contrast to genes encoding enzymes of TAG biosynthesis, Exit out of G0: Recover and resume growth D Exit out of G0: Recover and resume growth D Up Photosynthesis Tetrapyrrole synthesis Membrane lipids Transcription Protein synthesis Fig. 2 Cellular changes accompanying the entry into and exit out of quiescence in Chlamydomonas. The quiescence cycle of Chlamydomonas cells is depicted, where the cells are colored in different shades of green according to the respective changes in chlorophyll content. The summary of characteristics that Chlamydomonas cells must acquire during the entry into (following N deprivation, N−) and exit of quiescence (G0) (following N refeeding, NR) are shown. The maintenance of a quiescent state is an active process. Cellular changes that accompany N starvation and the entry into quiescence in Chlamydomonas Takeuchi and Benning Biotechnol Biofuels (2019) 12:292 Page 5 of 20 G1 G0 N- NR Entry into G0: Scavenge, save, repurpose Arrest of growth and division Non-replicated genome Redox homeostasis Exit out of G0: Recover and resume growth Up Down Respiration Photosynthesis Autophagy Chlorophyll Carbon storage Membrane lipids Nutrient uptake Translation Gametogenesis Transcription Up Down Photosynthesis Carbon storage (TAG degradation) Tetrapyrrole synthesis Membrane lipids Transcription Protein synthesis Maintenance of G0 Fig. 2 Cellular changes accompanying the entry into and exit out of quiescence in Chlamydomonas. The quiescence cycle of Chlamydomonas cells is depicted, where the cells are colored in different shades of green according to the respective changes in chlorophyll content. The summary of characteristics that Chlamydomonas cells must acquire during the entry into (following induces the transcriptional program necessary for game- togenesis, during which the cells of opposite mating types differentiate into gametes capable of mating [80]. The fusion of these gametes allows for the formation of diploid zygospores, which are markedly more resilient to environmental insults than Chlamydomonas cells during vegetative growth [81, 82]. In more recent years, multiple -omics-based approaches have been successfully applied to study the systems-level responses of this alga to N dep- rivation, revealing the wholesale cellular reprogramming of transcriptome, proteome and metabolome that results in the accumulation of C storage and a reversible quies- cent state [41–49, 71, 72, 83] (Fig. 2). G1 G0 N- NR Entry into G0: Scavenge, save, repurpose Arrest of growth and division Non-replicated genome Redox homeostasis Exit out of G0: Recover and resume growth Up Down Respiration Photosynthesis Autophagy Chlorophyll Carbon storage Membrane lipids Nutrient uptake Translation Gametogenesis Transcription Up Down Photosynthesis Carbon storage (TAG degradation) Maintenance of G0 G1 G0 N- NR Entry into G0: Scavenge, save, repurpose Arrest of growth and division Non-replicated genome Redox homeostasis Up Down Respiration Photosynthesis Autophagy Chlorophyll Carbon storage Membrane lipids Nutrient uptake Translation Gametogenesis Transcription Maintenance of G0 Arrest of growth and division Non-replicated genome Redox homeostasis Maintenance of G0 Although some responses of the starved cells are nutrient-specific, the underlying fundamental principles governing microbes under starvation can be summa- rized in three words—scavenge, conserve, and recycle [84]. In general, nutrient-deprived Chlamydomonas cells actively increase the scavenging and uptake of the limiting nutrient(s), curtail anabolic energy-consuming metabolism associated with growth and proliferation, and strategically repurpose nonessential macromolecules to maximize survival (Fig. 2). Cellular changes that accompany N starvation and the entry into quiescence in Chlamydomonas A similar loss of viability was recently shown for autophagy-defective mutants of Chlamydomonas in response to deprivation of N, P (phosphorus) and S [98], suggesting the impor- tance of this catabolic pathway for stress acclimation and cellular homeostasis of this alga [94]. oxygen consumption increases on a protein basis, further corroborating the bioenergetic preference for respiration over photosynthesis during N deprivation [43]. The recent interest in regulatory and metabolic path- ways governing TAG accumulation in microalgae has led to the identification of key enzymes responsible for the biosynthesis of TAG in Chlamydomonas [20]. Although the expression changes of many fatty acid and lipid metabolism genes are modest, notable changes in the transcript levels of genes involved in TAG biosynthesis and a number of lipases are observed following N depri- vation [42, 43, 90]. The Chlamydomonas genome encodes one type I (DGAT1) and five type II (DGTT1–5) diacylg- lycerol acyltransferases, which catalyze the transfer of an acyl-moiety from acyl-CoA to the sn-3 position of diacyl- glycerols (DAG), and one phospholipid: DAG acyltrans- ferase (PDAT), which catalyzes the transfer of an acyl chain at the sn-2 position of membrane lipids to the sn-3 position of DAG, resulting in the synthesis of TAG [20, 106, 107]. Among them, the transcript levels of DGAT1, DGTT1, and PDAT show the most significant upregu- lation following N deprivation [42, 43, 90]. The Chla- mydomonas PDAT catalyzes the biosynthesis of TAG through its acyltransferase and acylhydrolase activities toward a broad range of acyl-lipid substrates, including galactolipids, phospholipids, cholesteryl esters and TAG [104]. The genes encoding proteins with potential roles in TAG lipolysis, such as acylglycerol lipase, LIP1 (Lipase 1) with a likely role in DAG turnover [108] and those encoding the putative peroxisomal β-oxidation enzymes are concurrently downregulated [42]. Chlamydomonas cells can directly funnel exogenous acetate towards the synthesis of fatty acids and TAG following N deprivation, and the presence of acetate increases the TAG yield [42, 92, 103, 109]. Under mixotrophic conditions, over 80% of starch molecules are produced from the assimilated pho- tosynthates or CO2. However, under these conditions, approximately 75% of the C used for the de novo-synthe- sis of fatty acids and 70% of the subsequently assembled TAG or other lipid species are derived from acetate fol- lowing N starvation [105], supporting the previous 30% estimate of the contribution of membrane lipid turnover to TAG synthesis [103]. Cellular changes that accompany N starvation and the entry into quiescence in Chlamydomonas The repression of genes associated with cell cycle progression, DNA synthesis and replication must be maintained in order to prevent the premature entry into the cell division cycle in the absence of nutrient(s), such as N. The effective management of damaging reactive oxygen species (ROS) and the achievement of redox homeostasis are necessary to promote cellular survival during the non-dividing, energy-limited state. When N becomes available, the cells that remain viable and metabolically active are able to remobilize the accumulated carbon storage, such as triacylglycerols (TAG), remodel photosynthetic membranes, and resume the synthesis of macromolecules in order to reenter the growth (G1) phase. The white arrow heads depict the nutrient-dependent nature of these steps non-dividing cell fates such as apoptosis, senescence and differentiation [67, 68, 79]. Despite the conservation of many quiescence-associated components and processes, the generation of chemical energy from light and CO2 imposes another layer of complexity on the mainte- nance of a non-replicating, viable, and reversible state in photosynthetic organisms. While many abiotic stresses trigger algal cells to enter a quiescent state and to form lipid droplets rich in TAG, the consequences of nutrient deprivation, especially that of N, is the best studied pro- cess [26, 71]. It has been long known that N starvation Takeuchi and Benning Biotechnol Biofuels (2019) 12:292 Page 6 of 20 Page 6 of 20 whose transcript abundance gradually increases over the 2 day time course [46, 90], consistent with the observa- tions that starch accumulation precedes the increase in TAG [45, 91, 92]. To recycle and repurpose intracellular reserves of N, the nonessential or damaged macromol- ecules are engulfed within a specialized double-mem- brane vesicle called autophagosome and are trafficked for degradation into the vacuole [93]. The induction of autophagy is one of the hallmarks of quiescent cells. Although many growth-inhibiting stresses lead to the activation of autophagy in eukaryotes [38, 93, 94], this catabolic process is also necessary for the survival and maintenance of quiescent lymphocytes and hematopoi- etic stem cells in mammals [62]. Yeast mutants defective in autophagy accumulate higher levels of ROS and rapidly lose viability during nutrient starvation due to their ina- bility to remobilize amino acids and synthesize proteins necessary for stress adaptation [95–97]. Cellular changes that accompany N starvation and the entry into quiescence in Chlamydomonas Additional fatty acids are derived from the remodeling of plastid membranes by enzymes such as PGD1 (Plastid Galactoglycerolipid Degradation 1), a lipase responsible for cleaving the acyl chains from MGDG for the synthesis of TAG [110]. Many studies have historically observed how N dep- g [ ] Following N deprivation, Chlamydomonas cells increasingly rely on respiratory metabolism to produce energy instead of photosynthesis [43]. The cessation of chlorophyll synthesis, which is regulated both tran- scriptionally and post-translationally, leads to a drastic decrease in cellular chlorophyll content [43, 46, 99]. A marked multi-level downregulation of photosynthesis takes place. The abundances of many transcripts and proteins encoding the subunits of light-harvesting com- plexes, the cytochrome b6f complex, photosystems I and II, and the plastid ATP synthase complex decrease with different kinetics, ultimately leading to reduced photosynthetic capacity, efficiency and flux [42, 43, 88, 99–102]. After 2 days of N deprivation, the cellular lev- els of plastid membrane lipids, especially of monogalac- tosyldiacylglycerol (MGDG), are reduced while the TAG content increases, likely to sequester acyl groups inertly in lipid droplets as the extent of the photosynthetic mem- brane decreases [91, 99, 103, 104]. Most transcripts and enzymes of the Calvin–Benson cycle, especially rubisco, are reduced in abundance following 2 days of N depriva- tion, resulting in the increased levels of its intermediates [42, 43]. In agreement with these observations, the rates of carbon assimilation and consumption decrease sig- nificantly during N starvation [105]. Although the mRNA levels of mitochondrial respiratory complexes remain relatively stable during the 2 days of N starvation, their protein levels, along with the corresponding mitochon- drial ATP synthase and cytochrome bc1 complex com- ponents, become more abundant [43]. Consistently, the Many studies have historically observed how N dep- rivation results in the diversion of C towards storage compounds, namely TAG and starch, at the expense of decreased growth in a number of microalgae [9, 80, 111– 113]. The tight coupling and inverse relationship between TAG accumulation and proliferation have also been Takeuchi and Benning Biotechnol Biofuels (2019) 12:292 Page 7 of 20 (See figure on next page.) Fig. 3 Proposed role of putative DREAM-like complexes in the nutrient-dependent life-cycle transitions of Chlamydomonas. The DREAM complex: a master transcriptional regulator of cell division cycle also in algae? demonstrated in yeast, where the inhibition of cell cycle progression leads to the increased formation of lipid droplets, regardless of whether the delay is caused by drugs or mutations in genes encoding cell cycle regula- tors [114]. It was also recently reported that the mRNAs encoding early fatty acid synthesis enzymes (e.g., acetyl- CoA carboxylase 1, ACC1 and fatty acid synthase 1 and 2, FAS1 and 2) are translated in a cell cycle and nutrient dependent manner in yeast [115]. However, the analo- gous proteins in Chlamydomonas are less abundant fol- lowing N deprivation than in yeast [43]. Nevertheless, different hypotheses and theories were put forward to answer the question of why algae accumulate TAG in response to growth-inhibiting stresses following entry into quiescence. The potential physiological roles of lipid droplets and TAG during stress include fatty acid stor- age for survival and future membrane biosynthesis, a transient reservoir of acyl groups for the remodeling of the lipids in the photosynthetic membrane, a reservoir of carotenoids for photoprotection, and a sink for excess and unused photosynthetic energy and reductants to pre- vent photo-oxidative damage [9, 24, 105, 110, 116–121]. The studies in yeast suggest that the availability of acetyl- CoA, a central carbon metabolite that is derived from the breakdown of C storage, is a crucial factor for the cel- lular exit from quiescence and reentry into the cell divi- sion cycle. These studies suggest that the rapid increase in acetyl-CoA that results upon a suitable metabolic stimulation is necessary for driving the acetylation of his- tones at growth regulatory genes, their activation, and consequently enable cells to exit from quiescence [122, 123]. Whether these regulatory principles apply to algae remain to be explored. Despite the recent advances in understanding the impact of nutrient availability on gene expression and metabo- lism of Chlamydomonas, the signaling pathways and molecular components enabling the entry into, main- tenance of, and exit from quiescence remain largely unknown in photosynthetic eukaryotes. One potential regulatory component that may play a role in the nutri- ent-dependent life-cycle transitions of Chlamydomonas is the evolutionarily conserved multi-protein transcrip- tional regulatory complex known as DREAM (DP, RB, E2F and Myb-MuvB) (Fig. 3), although its presence in algae has yet to be confirmed. Cellular changes that accompany N starvation and the entry into quiescence in Chlamydomonas Although the existence of DREAM-like (DP, RB, E2F and Myb-MuvB) complexes has not been confirmed for the algal lineage, the repression of genes related to the cell cycle and the cessation of growth and division with 1C (one copy) DNA content in the absence of N have been previously observed. Furthermore, some components of DREAM-like complexes are conserved in Chlamydomonas, including the RB pathway proteins (MAT3/RB, Cre06. g255450; E2F1, Cre01.g052300; DP1, Cre07.g323000), three CXC domain-containing proteins (CHT7, Cre11.g481800; CXC2, Cre08.g361400 and CXC3, Cre12.g550250; potential orthologs of mammalian LIN54, fly Mip120, worm lin-54, and Arabidopsis TCX5), and one Myb protein with three Myb-repeats (Myb3R, Cre12.g522400). The model of their hypothetical functions within the putative DREAM-like complexes in mediating the nutrient-dependent entry into and exit from quiescence (G0) is illustrated. The grey dotted lines are used to denote the hypothesized interactions. In line with the literature demonstrating their importance in the transcriptional regulation of cell cycle-dependent gene expression in other model organisms, the putative Chlamydomonas DREAM-like repressor complex is postulated to repress the genes associated with cell cycle progression during the post-mitotic or G1 phase prior to the passage of commitment point (CP) in response to N deprivation (N−), allowing the exit from active proliferation and entry into quiescence. Conversely, upon sensing the replenishment of N, the cells need to reinstate their capacity for energy capture and macromolecular synthesis. Once their metabolism is sufficiently restored to sustain further growth, the cell cycle-related genes are postulated to become activated by the dissociation of a DREAM-like repressor complex and or the formation of its activator counterpart, allowing the cells to fully exit from quiescence to reenter the cell division cycle. Although these complexes may also play a role in the progression of the cell division cycle itself, they are omitted from the model for the sake of simplicity. The plus and the minus signs next to the energy status represent energy sufficient and deficient states, respectively. Cell cycle-dependent steps are represented by black arrow heads, while the nutrient-dependent steps are represented by white arrow heads. N+: N-replete growth; N−: N deprivation; NR: N refeeding The DREAM complex: a master transcriptional regulator of cell division cycle also in algae? It is notably absent from yeast, but organisms from many evolutionary lineages including mammals [124–126], fruit flies [127, 128], worms [129, 130] and plants [131] utilize this structur- ally conserved module to coordinate the expression of the cell division cycle-dependent and development- specific genes in response to different cues present dur- ing quiescence, cell proliferation and differentiation, and organismal or sexual organ development [73, 74]. These complexes, whose activities are determined by the com- binatorial presence of distinct components, are impor- tant for the context-dependent transcriptional regulation of cell cycle genes, whose protein abundance is largely determined at the transcriptional level [74]. The core components of DREAM complexes are conserved among species, which include retinoblastoma (RB) tumor sup- pressor proteins, adenovirus early gene 2 binding factor (E2F) family of transcription factors and their dimeriza- tion partners (DP), and the members of the multi-vulva class B (MuvB) complexes, which were initially identified Takeuchi and Benning Biotechnol Biofuels (2019) 12:292 Page 8 of 20 N- MAT3 DP E2F Myb CHT7 CXC2 CXC3 RB complex ∆Energy status (-) Signaling (N-) Metabolic reprograming (N-) Cell cycle-related genes Cell division cycle exit ∆Energy Status (+) Signaling (NR) Recovery/ re-constitution of macromolecules Cell division cycle reentry Cell cycle-related genes and or ? Repressor dissociation/ Activator formation Metabolic reprograming (NR) G0 N- NR G1 CP S M N+ n=1 n divisions n=2 ... DREAM-like repressor complex DREAM-like Activator complex DREAM-like repressor complex Repressor formation N refeeding (NR) ∆Energy status ( ) Signaling (N-) ∆Energy Status (+) Repressor formation M Metabolic reprograming (NR) n=1 n division n=2 ... n=1 n division n=2 ... Recovery/ re-constitution of macromolecules Cell division cycle reentry Cell division cycle reentry Cell cycle-related genes n divisions . Repressor dissociation/ Activator formation collectively referred hereafter as the CXC domain. These CXC domain proteins include the mammalian LIN54 [124, 126], Drosophila Mip120 [127, 132], and C. ele- gans Lin-54 [129], and their CXC domains are necessary for the sequence-specific binding of DNA [132–135]. through mutations that cause synthetic multi-vulva phe- notypes in Caenorhabditis elegans [73, 74]. through mutations that cause synthetic multi-vulva phe- notypes in Caenorhabditis elegans [73, 74]. collectively referred hereafter as the CXC domain. These CXC domain proteins include the mammalian LIN54 [124, 126], Drosophila Mip120 [127, 132], and C. ele- gans Lin-54 [129], and their CXC domains are necessary for the sequence-specific binding of DNA [132–135]. The DREAM complex: a master transcriptional regulator of cell division cycle also in algae? Chlamydomonas utilizes a homolog of the mammalian RB protein, MAT3, in coordination with the E2F1 transcriptional activator and its dimeriza- tion partner, DP1, to regulate cell size and cell cycle pro- gression [61, 155, 156]. However, unlike its mammalian counterpart, the Chlamydomonas MAT3/RB complex is stably associated with chromatin, and the progression through the cell division cycle is thought to involve dif- ferential phosphorylation of the RB protein or the partic- ipation of additional activator or repressor proteins [156]. The mat3–4 mutant is characterized by a misregulation of cell size homeostasis. Its cells are smaller in size than wild-type cells, because they pass the commitment point at a smaller volume and also undergo more rounds of the S/M cycle than the wild type [61, 155]. A novel class of cyclin dependent kinase, CDKG1, is one of the regulators responsible for coupling the mother cell size to the num- ber of subsequent divisions, by phosphorylating MAT3 in a cyclin D-dependent manner [157]. A single Myb3R gene is encoded within the genome of Chlamydomonas, whose expression is upregulated when the light–dark synchronized Chlamydomonas cells go through divi- sion [158]. The coexpression network generated for Chlamydomonas using genome-wide transcriptom- ics conducted under a number of conditions, including nutrient deprivation, has also found this gene to coex- press or cluster closely with other cell cycle genes [159]. Despite the intriguing observation that the five repeats of the Arabidopsis MSA elements are found within the 600 bp upstream of the translational start site of Myb3R gene itself, no obvious enrichment of this motif has been observed in the promoter regions of cell cycle genes when the entire genome was used as a Ref. [158]. Fur- thermore, since none of the previously identified candi- date cis-regulatory elements with a potential to regulate the diurnal transcription programs in Chlamydomonas appear to resemble the MSA motif [158, 160], further studies are needed to implicate Myb3R in cell cycle regu- lation or cell division. dd h f d h hl Other transcription factors are also known to associate with the core complexes, but their presence is less con- served among different organisms. While recruitment of the forkhead box M1 (FOXM1) transcription factor to the MuvB complex is necessary for the full activation of G2/M phase genes in mammals, no forkhead transcrip- tion factors have been found in the orthologous com- plexes of flies, worms, and plants [74]. The DREAM complex: a master transcriptional regulator of cell division cycle also in algae? One of the best characterized core constituents of the metazoan MuvB complexes are the proteins that con- tain two tandem cysteine-rich motifs or domains that are Takeuchi and Benning Biotechnol Biofuels (2019) 12:292 Page 9 of 20 For instance, the CXC domain of mammalian LIN54 is known to directly bind the cis-regulatory element known as the cell cycle genes homology region (CHR) [133, 135], which is a primary promoter element involved in the regulation of G2/M phase genes [136–139]. Conse- quently, LIN54 is essential for the recruitment of both activator and repressor DREAM complexes to these sites [74]. The CHR consensus sequences, defined by TTY RAA, where Y and R represent pyrimidine and purine bases, respectively [135], are enriched in the promoter regions of MuvB-target genes, not only in humans [137– 139] but also in flies [140] and worms [134]. Although a CXC domain protein, TCX5, is present in both the acti- vator and repressor forms of Arabidopsis DREAM-like complexes, which play a crucial role in the regulation of G2/M phase-specific gene expression, its functional contributions within these complexes remain unknown, and the CHR elements are yet to be identified in plants [74, 131, 141]. Despite the ubiquitous presence of CXC domain-containing proteins in plants, the soybean CPP1 and maize CBBP remain some of the few cases where their CXC domains have been implicated in the bind- ing of DNA [142, 143]. Regardless, in addition to TCX5, increasing numbers of studies are revealing the functions of CXC domain proteins in the regulation of the cell cycle and cell division of Arabidopsis. One such protein, TSO1, is involved in the control of the cell division cycle in meristems, shoots, and roots during plant development [144–149], and its closest paralogs, SOL1 and 2 have recently been implicated in the regulation of stomatal cell division and fate transition [150]. corresponding E2Fs, and despite the seeming absence of the CHR elements in plants, the target promoter regions of Myb3R-containing DREAM-like complexes of Arabi- dopsis are found to be enriched in MSA and or E2F ele- ments [131]. The Chlamydomonas genome also encodes some of the conserved components of DREAM complexes (Fig. 3), and their protein products have documented roles in the control of cell-size homeostasis, the cell division cycle, and quiescence. The DREAM complex: a master transcriptional regulator of cell division cycle also in algae? Thus, further studies are needed to explore the molecular mechanisms by which the CHT7 complex affects quiescence, whether the CHT7 protein plays a direct or indirect role in the transcriptional reg- ulation of cell cycle genes, or whether or not the Chla- mydomonas CHT7 complex is functionally analogous to DREAM complexes in other organisms. Translation Fig. 4 Working model of TOR and SnRK/CKIN family of kinases and their major functions in Chlamydomonas stress biology. Chlamydomonas rapamycin-sensitive TOR complex 1 (TORC1) consists of TOR, LST8 and RAPTOR. The inhibition of TORC1 by pharmacological means (rapamycin, AZD-8055 or Torin1) has enabled the studies of TOR pathway functions in Chlamydomonas, whereas the components of TORC2 have not been identified in photosynthetic organisms. When the conditions are conducive to growth (i.e., in the absence of abiotic stresses), the TORC1 complex promotes anabolic processes such as protein synthesis and therefore growth and represses stress-induced responses such as TAG synthesis and the induction of autophagy. TORC1 has also been shown to positively regulate the levels of inositol polyphosphates (InsPs), InsP7 and InsP8, to promote growth and inhibit TAG accumulation. Although the members of the SnRK and CKIN family of kinases in Chlamydomonas and microalgae are not well characterized to date, several studies have shown the functions of some members in the acclimation of cells to abiotic stresses, including the adjustment of reactive oxygen species (ROS), accumulation of TAG and sulfur (S) assimilation during sulfur deprivation. The relationships that are supported by experiments are shown in solid black lines, whereas the dotted grey lines with question marks represent the hypothesized regulatory links based on previous findings in other model organisms that require further studies in Chlamydomonas. The positive and negative regulatory relationships are represented by arrows and T-bars, respectively The DREAM complex: a master transcriptional regulator of cell division cycle also in algae? The relationships that are supported by experiments are shown in solid black lines, whereas the dotted grey lines with question marks represent the hypothesized regulatory links based on previous findings in other model organisms that require further studies in Chlamydomonas. The positive and negative regulatory relationships are represented by arrows and T-bars, respectively transcriptional regulation of quiescence-associated pro- grams is the Compromised Hydrolysis of TAG 7 (CHT7) protein. The mutant ablated in CHT7 is impaired in its ability to remobilize TAG and shows delayed growth upon N or P resupply and rapamycin removal (follow- ing a period of N or P starvation or rapamycin treat- ment, respectively [71]). Decades ago, a similar delay in the resumption of growth was also observed for the mat3 mutant during N refeeding [161]. Similarly to RB and related proteins, CHT7 proteins are located in the nucleus, although some are also observed in the cytosol [71]. No obvious defects in growth are detected in the cht7 mutant during N-replete growth despite the large number of misregulated genes in this mutant under this condition. Of these genes, nearly 50%, including those involved in photosynthesis, flagellum assembly and autophagy, are expressed in cht7 cells under N-replete conditions in a similar manner as in wild-type cells, which are subjected to N deprivation [71]. In addition, many genes involved in chloroplast-related functions, including photosynthesis, tetrapyrrole synthesis and plastid ribosomal protein synthesis, fail to reverse their expression upon N refeeding in cht7 as would be typi- cal for genes in the wild type [72]. Thus, CHT7 has been hypothesized as an apparent repressor, during N-replete growth and N refeeding of a subset of transcriptomic programs associated with N deprivation-induced quies- cence. Furthermore, it was hypothesized that the com- plete exit from quiescence during N refeeding requires the repression of these programs by CHT7 [71, 72]. How- ever, the mechanisms governing CHT7 activity remain unclear. Although the majority of CHT7 proteins of Chlamydomonas exist as part of a protein complex, the levels of CHT7 (per total protein) do not fluctuate with changes in N abundance, and the apparent size and abun- dance of the observed complex stay constant regardless of N availability [71]. The DREAM complex: a master transcriptional regulator of cell division cycle also in algae? Thus, further studies are needed to explore the molecular mechanisms by which the CHT7 complex affects quiescence, whether the CHT7 protein plays a direct or indirect role in the transcriptional reg- ulation of cell cycle genes, or whether or not the Chla- mydomonas CHT7 complex is functionally analogous to DREAM complexes in other organisms. V Autophagy Translation TAG synthesis Growth Abiotic stresses (e.g., nutrient deficiency) SnRK/ CKIN TOR RAPTOR TORC1 Growth conducive (e.g., sufficient nutrient) Rapamycin- FBK12 AZD-8055 Torin1 ? ROS homeostasis Acetate InsPs ? ? ? LST8 transcriptional regulation of quiescence associated pro grams is the Compromised Hydrolysis of TAG 7 (CHT7) protein. The mutant ablated in CHT7 is impaired in its ability to remobilize TAG and shows delayed growth upon N or P resupply and rapamycin removal (follow- ing a period of N or P starvation or rapamycin treat- ment, respectively [71]). Decades ago, a similar delay in the resumption of growth was also observed for the mat3 mutant during N refeeding [161]. Similarly to RB and related proteins, CHT7 proteins are located in the nucleus, although some are also observed in the cytosol [71]. No obvious defects in growth are detected in the cht7 mutant during N-replete growth despite the large number of misregulated genes in this mutant under this condition. Of these genes, nearly 50%, including those involved in photosynthesis, flagellum assembly and autophagy, are expressed in cht7 cells under N-replete conditions in a similar manner as in wild-type cells, which are subjected to N deprivation [71]. In addition, many genes involved in chloroplast-related functions, including photosynthesis, tetrapyrrole synthesis and plastid ribosomal protein synthesis, fail to reverse their expression upon N refeeding in cht7 as would be typi- cal for genes in the wild type [72]. Thus, CHT7 has been hypothesized as an apparent repressor, during N-replete growth and N refeeding of a subset of transcriptomic programs associated with N deprivation-induced quies- cence. Furthermore, it was hypothesized that the com- plete exit from quiescence during N refeeding requires the repression of these programs by CHT7 [71, 72]. How- ever, the mechanisms governing CHT7 activity remain unclear. Although the majority of CHT7 proteins of Chlamydomonas exist as part of a protein complex, the levels of CHT7 (per total protein) do not fluctuate with changes in N abundance, and the apparent size and abun- dance of the observed complex stay constant regardless of N availability [71]. The DREAM complex: a master transcriptional regulator of cell division cycle also in algae? Furthermore, Myb-type transcription factors, which function as acti- vators of gene expression both in mammals and flies, are apparently missing from the DRM complex of C. elegans, and the C. elegans DRM is thought to act primarily as a transcriptional repressor [74, 129]. This is in contrast to DREAM-like complexes of Arabidopsis, where a small family of Myb3R transcription factors with three Myb- repeats with activator or repressor function(s) regulate the expression of many G2/M phase-specific genes by interacting with the promoter sequence known as the mitosis-specific activator (MSA) element [131, 151–154]. Therefore, Myb3Rs play an important role in determin- ing the direction of transcriptional regulation mediated by Arabidopsis DREAM-like complexes along with the In addition to the aforementioned genes, the Chla- mydomonas genome encodes at least three proteins with annotated CXC domains. Although the literature on these proteins is scarce, one CXC domain protein in Chlamydomonas with characterized functions in the Takeuchi and Benning Biotechnol Biofuels (2019) 12:292 Page 10 of 20 V Autophagy Translation TAG synthesis Growth Abiotic stresses (e.g., nutrient deficiency) SnRK/ CKIN TOR RAPTOR TORC1 Growth conducive (e.g., sufficient nutrient) Rapamycin- FBK12 AZD-8055 Torin1 ? ROS homeostasis Acetate InsPs ? ? ? LST8 Fig. 4 Working model of TOR and SnRK/CKIN family of kinases and their major functions in Chlamydomonas stress biology. Chlamydomonas rapamycin-sensitive TOR complex 1 (TORC1) consists of TOR, LST8 and RAPTOR. The inhibition of TORC1 by pharmacological means (rapamycin, AZD-8055 or Torin1) has enabled the studies of TOR pathway functions in Chlamydomonas, whereas the components of TORC2 have not been identified in photosynthetic organisms. When the conditions are conducive to growth (i.e., in the absence of abiotic stresses), the TORC1 complex promotes anabolic processes such as protein synthesis and therefore growth and represses stress-induced responses such as TAG synthesis and the induction of autophagy. TORC1 has also been shown to positively regulate the levels of inositol polyphosphates (InsPs), InsP7 and InsP8, to promote growth and inhibit TAG accumulation. Although the members of the SnRK and CKIN family of kinases in Chlamydomonas and microalgae are not well characterized to date, several studies have shown the functions of some members in the acclimation of cells to abiotic stresses, including the adjustment of reactive oxygen species (ROS), accumulation of TAG and sulfur (S) assimilation during sulfur deprivation. Signaling networks linking the metabolic status to growth in Chlamydomonas: TOR and SnRK/CKIN pathways Despite the identification of TOR and the conservation of TORC1 components, such as RAPTOR (regulatory-associated protein of TOR) and LST8 (lethal with SEC13 protein 8) in plants and algae [172–177], no obvious homologs of TORC2 components have been identified in organisms of the green-lineage, although its functional equivalent is postulated to exist [177–181]. Nevertheless, the primary functions of the TOR pathways and the mechanisms of TOR inhibition by rapamycin appear conserved in nearly all groups of organisms. When nutrients are ample, TOR complexes act as positive regulators of cellular growth by promoting anabolic processes such as nucleotide synthe- sis, transcription, ribosome biogenesis and translation, while inhibiting the opposing catabolic processes includ- ing mRNA degradation and autophagy [170, 179–183] (Fig. 4). plays a functionally analogous role to those of yeast and mammals, where the associations of the LST8 proteins with the kinase domains of respective TORs are neces- sary for their full catalytic activities [194, 195], and the seven WD-40 domains present within Chlamydomonas LST8 may have an additional function in facilitating the association of TORC1 with its various protein substrates [175]. Furthermore, some fractions of both TOR and LST8 appear to be peripherally associated with mem- branes of the endoplasmic reticulum (ER) system, par- ticularly near the peri-basal body regions at the base of flagella, where the demand for protein synthesis is likely high [175, 196]. In animals and yeast, the network governed by TORC1 constitutes one of the major signaling pathways link- ing nutrient availability to the autophagic machin- ery, by the phosphorylation-mediated regulation of ATG (autophagy-related) proteins that orchestrate autophagy [197–199]. As discussed earlier, the activa- tion of autophagy is a necessary cellular response to promote survival during starvation and the consequent establishment of a reversible state of quiescence. The ATG proteins are also conserved in Chlamydomonas [183], and the FKBP12–rapamycin mediated inhibition of TORC1 leads to an increased bleaching and vacuoli- zation [173]. One such conserved ATG protein, ATG8 has also been demonstrated as an autophagy-specific marker in Chlamydomonas [93, 200]. In many organ- isms, the covalent attachment of phosphatidylethanola- mine (PE) to ATG8 (known as lipidation) allows for the association of ATG8 proteins with the autophagosome vesicle until the fusion of the ATG8–autophagosome with the vacuole takes place [201]. Signaling networks linking the metabolic status to growth in Chlamydomonas: TOR and SnRK/CKIN pathways Evolutionarily conserved signaling pathways playing a central role in the coordination of nutrient status with metabolism and cellular growth in eukaryotes, includ- ing photosynthetic organisms, are those involving the target of rapamycin (TOR) kinases and their antagonists, AMPK/Snf1/SnRK/CKIN kinases (Fig. 4). As suggested Takeuchi and Benning Biotechnol Biofuels (2019) 12:292 Page 11 of 20 Page 11 of 20 by their names, TOR1 and TOR2 kinases were first iden- tified by a genetic mutant screen in yeast as targets of rapamycin [162], an antifungal and immunosuppressant compound isolated from the soil bacterium Streptomy- ces hygroscopicus [163, 164]. Whereas the treatment of wild-type yeast cells leads to the arrest of the cell cycle in the G1 phase, those with a mutation in the TOR1 or 2 are resistant to the drug [162]. Although most organisms contain only a single TOR [165], the functional equiva- lents of two distinct multi-protein complexes discovered in yeast, TOR complex 1 (TORC1) and 2 (TORC2) [166– 168], are present in many organisms [169–171]. Despite the identification of TOR and the conservation of TORC1 components, such as RAPTOR (regulatory-associated protein of TOR) and LST8 (lethal with SEC13 protein 8) in plants and algae [172–177], no obvious homologs of TORC2 components have been identified in organisms of the green-lineage, although its functional equivalent is postulated to exist [177–181]. Nevertheless, the primary functions of the TOR pathways and the mechanisms of TOR inhibition by rapamycin appear conserved in nearly all groups of organisms. When nutrients are ample, TOR complexes act as positive regulators of cellular growth by promoting anabolic processes such as nucleotide synthe- sis, transcription, ribosome biogenesis and translation, while inhibiting the opposing catabolic processes includ- ing mRNA degradation and autophagy [170, 179–183] (Fig. 4). by their names, TOR1 and TOR2 kinases were first iden- tified by a genetic mutant screen in yeast as targets of rapamycin [162], an antifungal and immunosuppressant compound isolated from the soil bacterium Streptomy- ces hygroscopicus [163, 164]. Whereas the treatment of wild-type yeast cells leads to the arrest of the cell cycle in the G1 phase, those with a mutation in the TOR1 or 2 are resistant to the drug [162]. Although most organisms contain only a single TOR [165], the functional equiva- lents of two distinct multi-protein complexes discovered in yeast, TOR complex 1 (TORC1) and 2 (TORC2) [166– 168], are present in many organisms [169–171]. Signaling networks linking the metabolic status to growth in Chlamydomonas: TOR and SnRK/CKIN pathways Although the molecular mechanisms by which TOR sensitivity to rapamycin has identified the VIP1 locus, suggesting a relationship between inositol polyphos- phates (InsPs), TAG accumulation, and TOR [229]. The VIP1 gene encodes a kinase responsible for the pyroph- osphorylation of InsP6 to yield InsP7 and InsP8, which are important signaling molecules [229]. The vip1-1 mutant has decreased levels of InsP7 and InsP8, slower growth and increased levels of TAG during mixotrophic growth in the presence of acetate [229]. A similar reduction in the InsP7 and InsP8 content was observed for rapamycin- treated wild-type cells, further suggesting a link between InsPs, TAG, and the TOR pathway [229]. In addition, the expression profiles of thousands of genes are reported to change in response to the rapamycin treatment of Chla- mydomonas cells [230], and they appear to at least par- tially resemble the transcriptional program associated with nutrient starvation. Following rapamycin treatment, where genes involved in autophagy, vacuolar function, amino acid metabolism and transport tend to be upregu- lated, genes involved in processes that require a robust anabolic metabolism, e.g., nucleotide synthesis to sustain DNA replication and the cell cycle become downregu- lated [230]. The decrease in the transcript levels of cell cycle-related genes in response to rapamycin is not only consistent with the observed inhibition of growth follow- ing TORC1 inactivation in Chlamydomonas [173, 211], but also with studies in Arabidopsis, where the expression of E2Fa and E2Fb targets with central roles in the regu- lation of cell cycle is activated by the TORC1-mediated phosphorylation and repressed upon TORC1 inhibition [231, 232]. Arabidopsis TORC1 was also recently shown to phosphorylate and inhibit a member of the dual-spec- ificity tyrosine phosphorylation-regulated kinase (DRYK) family, AtYAK1 an orthologue of Yet Another Kinase 1 in yeast), which acts as a negative regulator of plant growth [233–235]. Under conditions where TORC1 is inactive, the repression on AtYAK1 is lifted, and AtYAK1 activates plant-specific CDK inhibitors, SMR (Siamese-related) proteins, to negatively regulate cell cycle progression [235]. Yeast YAK1 and its metazoan orthologs, mam- malian DYRK1A and fly Minibrain kinases, also have known roles in inhibiting proliferation [236–242]. The mammalian DYRK1A upregulates the expression of the gene encoding CDK inhibitor, CDKN1B (also known as p27KIP1) [239]. The DYRK1A-mediated phosphoryla- tion of CDKN1B, in addition to cyclin D1 and D3, pro- motes CDKN1B stabilization, cyclin D degradation and consequently cell cycle exit [237, 240]. Signaling networks linking the metabolic status to growth in Chlamydomonas: TOR and SnRK/CKIN pathways Furthermore, the mammalian DYRK1A also phosphorylates LIN52, a component of the MuvB core, to facilitate DREAM com- plex formation and to promote entry into quiescence or senescence [241]. In these contexts, it may be worthy to translation initiation [204, 207]. The TOR–S6K pathway is also conserved in plants, and the translation initia- tion of cytosolic S6 ribosomal protein in Arabidopsis is likewise regulated by this pathway [192, 208–210]. The Arabidopsis TOR kinase also promotes plastid ribo- somal biogenesis by upregulating the transcription and translation of genes and mRNAs, respectively, for nuclear encoded plastid ribosomal proteins [208]. Although Chlamydomonas TOR kinase is implicated in the regulation of de novo amino acid synthesis [211, 212], and rapamycin treatment is also known to inhibit protein synthesis in this alga [213], the signaling path- ways downstream of TOR controlling protein synthesis are generally less well characterized in algae. However, TOR-dependent phosphorylation sites were also recently identified in S6K and ribosomal S6 protein of Chla- mydomonas through phosphoproteomic studies of cells treated with rapamycin, AZD-8055, or Torin1 [214–217], and TORC1-mediated phosphorylation of the riboso- mal S6 protein at serine-245 was shown to be regulated by N as well as P availability in Chlamydomonas [218, 219]. Furthermore, a recent study has begun to establish the regulatory link between P availability and TORC1- signaling Chlamydomonas [219]. Using the phospho- rylation of ribosomal S6 protein as a marker of TORC1 activity, it was shown that TORC1 becomes inhibited following P starvation likely through a drastic reduction in the abundance of LST8 proteins, which are necessary for the activity of TOR complexes [166, 194, 195, 219]. Therefore, it is increasingly evident that the inhibition of TORC1 and the deprivation of nutrients both trigger similar cellular processes and stress responses, not only in yeast where the role of TOR pathways in the coor- dination of nutrient status to cellular growth is firmly established [220–223], but also in algae [35, 224–227]. In addition to the cessation of growth, the activation of autophagy and the reduction in protein synthesis that occur upon TORC1 inhibition and nutrient starvation, the repression of TORC1 and N shortage both induce the formation of TAG-rich lipid droplets in various species of algae [35, 224–226]. Signaling networks linking the metabolic status to growth in Chlamydomonas: TOR and SnRK/CKIN pathways Because the amount of ATG8 proteins is directly related to the number and size of autophagosomes, the levels of lipidated ATG8 and their altered cellular localization can be used as mark- ers of active autophagy [202], which holds true also for Chlamydomonas [93, 200]. The treatment of Chla- mydomonas cells with rapamycin leads to the accumula- tion of ATG8 and its lipidated forms, followed by their relocation to large punctate structures in the cytoplasm, indicating the inhibition of TORC1 as an important step in the activation of autophagy [93]. Moreover, the same ATG8-marker responses are induced upon subjecting the cells to nutrient starvation and oxidative or ER stresses, illuminating the role of the TORC1 pathway in regulating stress-induced autophagy of this alga [93, 203]. Rapamycin acts to inhibit TOR by interacting with a 12-kDa proline isomerase immunophilin known as FK506-binding protein (FKBP12) [162]. The FKBP12– rapamycin complex subsequently binds to the FRB (FKBP12–rapamycin binding) domain of TOR, lead- ing to its inactivation by limiting the accessibility of its kinase domain to the substrate [184]. In opisthokonts, TORC1 is sensitive to rapamycin whereas TORC2 is not [166, 167, 185–187], largely owing to the presence of RICTOR (rapamycin-insensitive companion of TOR) in TORC2 which renders the FRB domain inaccessible to the FKBP12–rapamycin [188, 189]. In addition, since the FKBP12 proteins of many plant species are unable to stably associate with rapamycin, land plants are resist- ant or highly tolerant to the growth-inhibitory effects of rapamycin [172, 190–193]. In contrast, growth and cell cycle progression of Chlamydomonas are sensitive to rapamycin treatment, although to a lesser extent when compared to yeast or mammals due to the lower affinity of its FKBP12 protein to rapamycin [173]. As observed for other organisms, the Chlamydomonas TOR protein exists as part of a large molecular weight complex, and its single copy LST8 co-purifies with TOR and FKB12 in the presence of rapamycin, confirming the existence of Chla- mydomonas TORC1 [175]. The Chlamydomonas LST8 In opisthokonts as well as plants, the mechanisms by which TORC1 promotes protein synthesis are known [204–208]. For instance, in mammals, the direct phos- phorylation and activation of S6 kinase (S6K) by TORC1 leads to the S6K-dependent phosphorylation of the ribosomal protein S6, resulting in increased rates of Takeuchi and Benning Biotechnol Biofuels (2019) 12:292 Page 12 of 20 Page 12 of 20 translation initiation [204, 207]. Signaling networks linking the metabolic status to growth in Chlamydomonas: TOR and SnRK/CKIN pathways The TOR–S6K pathway is also conserved in plants, and the translation initia- tion of cytosolic S6 ribosomal protein in Arabidopsis is likewise regulated by this pathway [192, 208–210]. The Arabidopsis TOR kinase also promotes plastid ribo- somal biogenesis by upregulating the transcription and translation of genes and mRNAs, respectively, for nuclear encoded plastid ribosomal proteins [208]. Although Chlamydomonas TOR kinase is implicated in the regulation of de novo amino acid synthesis [211, 212], and rapamycin treatment is also known to inhibit protein synthesis in this alga [213], the signaling path- ways downstream of TOR controlling protein synthesis are generally less well characterized in algae. However, TOR-dependent phosphorylation sites were also recently identified in S6K and ribosomal S6 protein of Chla- mydomonas through phosphoproteomic studies of cells treated with rapamycin, AZD-8055, or Torin1 [214–217], and TORC1-mediated phosphorylation of the riboso- mal S6 protein at serine-245 was shown to be regulated by N as well as P availability in Chlamydomonas [218, 219]. Furthermore, a recent study has begun to establish the regulatory link between P availability and TORC1- signaling Chlamydomonas [219]. Using the phospho- rylation of ribosomal S6 protein as a marker of TORC1 activity, it was shown that TORC1 becomes inhibited following P starvation likely through a drastic reduction in the abundance of LST8 proteins, which are necessary for the activity of TOR complexes [166, 194, 195, 219]. Therefore, it is increasingly evident that the inhibition of TORC1 and the deprivation of nutrients both trigger similar cellular processes and stress responses, not only in yeast where the role of TOR pathways in the coor- dination of nutrient status to cellular growth is firmly established [220–223], but also in algae [35, 224–227]. In addition to the cessation of growth, the activation of autophagy and the reduction in protein synthesis that occur upon TORC1 inhibition and nutrient starvation, the repression of TORC1 and N shortage both induce the formation of TAG-rich lipid droplets in various species of algae [35, 224–226]. In Chlamydomonas and the red alga Cyanidioschyzon merolae, the repression of TORC1 path- ways by pharmacological means (rapamycin, AZD8055, or Torin1) has been shown to result in the upregulation of key enzymes involved in TAG biosynthesis, such as glycerol-3-phosphate acyltransferase (GPAT) and DGAT [224, 225]. Consistent with these observations, the accu- mulation of TAG and starch is also reported for Arabi- dopsis seedlings with inducible repression of TOR [228]. Signaling networks linking the metabolic status to growth in Chlamydomonas: TOR and SnRK/CKIN pathways However, these studies are starting to shed light on the significance of signaling pathways involving TOR and SnRK/CKIN in the coordination of nutrient availability, energy metabolism, and cellular growth in photosyn- thetic organisms. This emerging knowledge provides an essential basis for the further exploration of these sign- aling networks and assessment of their bioengineering potential in microalgae. named TAR1 (TAG accumulation regulator 1), another green-lineage-specific DRYK kinase, DYRKP, and other DRYK-related kinases [243, 244]. While both TAR1 and DYRKP have been shown to regulate the accumulation of TAG and for DYRKP, of starch during N and S dep- rivation [243, 244], their potential role in the nutrient- dependent regulation of the Chlamydomonas cell cycle is not yet clear. proliferation, while activating stress-induced responses such as gluconeogenesis and starch synthesis in plants [179]. The antagonistic activities of SnRK1α1 towards TORC1 have also been demonstrated in Arabidopsis by its ability to interact with and phosphorylate RAP- TOR1B [262]. Although a complete knockout of SnRK1 genes results in embryonic lethality in Arabidopsis [262], similarly to the knockout mutants of TOR [172], induc- ible amiRNA::SnRK1α2 transgenic plants in the snrk1α1 mutant background show a hyper-phosphorylation of ribosomal protein S6, indicating their crucial role in the suppression of TORC1 and the downregulation of trans- lation [262]. The SnRK2 and 3 subfamilies in Arabidopsis are also reported to function in the adaptation of plants to a wide range of abiotic stresses, including drought, flood, cold, salinity, and nutrient scarcity [263]. Although the characterization of the SnRK family in algae lags behind, a number of studies in Chlamydomonas have suggested the role of SnRKs/CKINs in the cellular response to abiotic stresses, including cold [264] and shortages of S [89, 265, 266] and N [47]. In Chlamydomonas SAC1 and SnRK2.2 have been implicated in the regulation of TAG synthesis during S deprivation by modulating the expres- sion of DGTT1–4 [267]. Indeed, the biotechnical impli- cations of this signaling pathway have recently prompted the genome-wide identification of 22 CKIN proteins in Chlamydomonas as orthologs of plant SnRKs [250]. Whether the orthologous DREAM complex components in Chlamydomonas are targeted by members of a TOR or SnRK/CKIN signaling cascade to relay cellular nutri- ent status and to regulate the transitions between cell division and quiescence cycles, remain to be elucidated. Signaling networks linking the metabolic status to growth in Chlamydomonas: TOR and SnRK/CKIN pathways However, these studies are starting to shed light on the significance of signaling pathways involving TOR and SnRK/CKIN in the coordination of nutrient availability, energy metabolism, and cellular growth in photosyn- thetic organisms. This emerging knowledge provides an essential basis for the further exploration of these sign- aling networks and assessment of their bioengineering potential in microalgae. In animals and yeast, AMP-activated kinase (AMPK) and sucrose nonfermenting 1 (Snf1) kinase, respectively, are central signaling components that are activated by nutrient limitation and other stresses, and act antago- nistically to TORC1 [245–249]. The orthologs of AMPK/ Snf1 are also conserved in plants and algae, and they are known as Snf-related kinases (SnRKs) in Arabidopsis and sometimes referred to as CKINs (Chlamydomonas kinases) in Chlamydomonas [179, 250]. In general, the activated AMPK/Snf1/SnRK/CKIN signaling pathway promotes cellular survival and cessation of growth dur- ing stress by upregulating catabolic processes to gener- ate more energy and downregulating growth-promoting processes to consume less energy [179, 247, 249–253] (Fig. 4). In mammalian cells, AMPK is known to inacti- vate TORC1 in response to energy and nutrient stresses by phosphorylating one of its constituents, RAPTOR, and by the subsequent recruitment of 14-3-3 proteins [254]. In addition to its inhibitory effect on TORC1, AMPK facilitates the arrest of the cell cycle during the G1 phase prior to the replication of DNA by upregulating and stabilizing the levels of CDK inhibitors, CDKN1A (also known as p21WAF1) and CDKN1B (also known as p27KIP1), respectively [255–257]. The AMPK also pro- motes the initiation of autophagy, upregulates the uptake of glucose and fatty acids, and facilitates the breakdown of these molecules by the activation of glycolysis and fatty acid oxidation, respectively [249, 252]. In a similarly opposing manner to TORC1, AMPK acts to inhibit the biosynthesis of nearly all macromolecules, including pro- teins, ribosomal RNA, lipids, and carbohydrates by the direct phosphorylation of various key components and regulatory factors of these anabolic pathways [249].hi Signaling networks linking the metabolic status to growth in Chlamydomonas: TOR and SnRK/CKIN pathways In Chlamydomonas and the red alga Cyanidioschyzon merolae, the repression of TORC1 path- ways by pharmacological means (rapamycin, AZD8055, or Torin1) has been shown to result in the upregulation of key enzymes involved in TAG biosynthesis, such as glycerol-3-phosphate acyltransferase (GPAT) and DGAT [224, 225]. Consistent with these observations, the accu- mulation of TAG and starch is also reported for Arabi- dopsis seedlings with inducible repression of TOR [228]. Although the molecular mechanisms by which TOR pathways regulate lipid metabolism or TAG accumulation Although the molecular mechanisms by which TOR pathways regulate lipid metabolism or TAG accumulation are currently not well known in algae, a recent genetic screen for Chlamydomonas mutants with increased Takeuchi and Benning Biotechnol Biofuels (2019) 12:292 Page 13 of 20 Page 13 of 20 proliferation, while activating stress-induced responses such as gluconeogenesis and starch synthesis in plants [179]. The antagonistic activities of SnRK1α1 towards TORC1 have also been demonstrated in Arabidopsis by its ability to interact with and phosphorylate RAP- TOR1B [262]. Although a complete knockout of SnRK1 genes results in embryonic lethality in Arabidopsis [262], similarly to the knockout mutants of TOR [172], induc- ible amiRNA::SnRK1α2 transgenic plants in the snrk1α1 mutant background show a hyper-phosphorylation of ribosomal protein S6, indicating their crucial role in the suppression of TORC1 and the downregulation of trans- lation [262]. The SnRK2 and 3 subfamilies in Arabidopsis are also reported to function in the adaptation of plants to a wide range of abiotic stresses, including drought, flood, cold, salinity, and nutrient scarcity [263]. Although the characterization of the SnRK family in algae lags behind, a number of studies in Chlamydomonas have suggested the role of SnRKs/CKINs in the cellular response to abiotic stresses, including cold [264] and shortages of S [89, 265, 266] and N [47]. In Chlamydomonas SAC1 and SnRK2.2 have been implicated in the regulation of TAG synthesis during S deprivation by modulating the expres- sion of DGTT1–4 [267]. Indeed, the biotechnical impli- cations of this signaling pathway have recently prompted the genome-wide identification of 22 CKIN proteins in Chlamydomonas as orthologs of plant SnRKs [250]. Whether the orthologous DREAM complex components in Chlamydomonas are targeted by members of a TOR or SnRK/CKIN signaling cascade to relay cellular nutri- ent status and to regulate the transitions between cell division and quiescence cycles, remain to be elucidated. Nothing to report. Only the authors contributed to this review. Nothing to report. Only the authors contributed to this review. Abbreviations ACC1 l C A Chisti Y. Biodiesel from microalgae. Biotechnol Adv. 2007;25(3):294–306 8. Chisti Y. Biodiesel from microalgae. Biotechnol Adv. 2007;25(3):294–306 9. Hu Q, Sommerfeld M, Jarvis E, Ghirardi M, Posewitz M, Seibert M, Dar- zins A. Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J. 2008;54(4):621–39. 9. Hu Q, Sommerfeld M, Jarvis E, Ghirardi M, Posewitz M, Seibert M, Dar- zins A. Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J. 2008;54(4):621–39. 10. Li Y, Horsman M, Wu N, Lan CQ, Dubois-Calero N. Biofuels from microal- gae. Biotechnol Prog. 2008;24(4):815–20. 11. Scott SA, Davey MP, Dennis JS, Horst I, Howe CJ, Lea-Smith DJ, Smith AG. Biodiesel from algae: challenges and prospects. Curr Opin Biotech- nol. 2010;21(3):277–86. 12. Jones CS, Mayfield SP. Algae biofuels: versatility for the future of bioen- ergy. Curr Opin Biotechnol. 2012;23(3):346–51.f Author details 1 Department of Biochemistry and Molecular Biology, Michigan State Uni- versity, East Lansing, MI 48824, USA. 2 Department of Energy‑Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA. 3 Depart- ment of Plant Biology, Michigan State University, East Lansing, MI 48824, USA. Received: 15 October 2019 Accepted: 12 December 2019 Consent for publication Not applicable. Consent for publication Not applicable. Authors’ contributions 14. Park JBK, Craggs RJ, Shilton AN. Wastewater treatment high rate algal ponds for biofuel production. Bioresour Technol. 2011;102(1):35–42. TT conceptualized the manuscript and prepared the draft of all sections. CB provided critical review and contributed to the writing and editing of all sec- tions. Both authors read and approved the final manuscript. 15. Pittman JK, Dean AP, Osundeko O. The potential of sustainable algal biofuel production using wastewater resources. Bioresour Technol. 2011;102(1):17–25. References l 1. Pulz O, Gross W. Valuable products from biotechnology of microalgae. Appl Microbiol Biotechnol. 2004;65(6):635–48. 2. Mata TM, Martins AA, Caetano NS. Microalgae for biodiesel produc- tion and other applications: a review. Renew Sustain Energy Rev. 2010;14(1):217–32. 3. Yen H-W, Hu IC, Chen C-Y, Ho S-H, Lee D-J, Chang J-S. Microalgae-based biorefinery—from biofuels to natural products. Bioresour Technol. 2013;135:166–74. 3. Yen H-W, Hu IC, Chen C-Y, Ho S-H, Lee D-J, Chang J-S. Microalgae-based biorefinery—from biofuels to natural products. Bioresour Technol. 2013;135:166–74. Funding 16. Wang B, Li Y, Wu N, Lan CQ. CO2 bio-mitigation using microalgae. Appl Microbiol Biotechnol. 2008;79(5):707–18. T.T. was supported by a Grant from the National Science Foundation MCB- 1515169 to C.B. and partly by the Plant Biotechnology for Health and Sustain- ability Training Program at MSU (NIH T32 GM110523). C.B. was supported in part by a Grant from the Chemical Sciences, Geosciences, and Biosciences Division, Office of Basic Energy Sciences, Office of Science, U.S. Department of Energy (DE–FG02–91ER20021) and MSU AgBioResearch (MICL02357). 17. Hannon M, Gimpel J, Tran M, Rasala B, Mayfield S. Biofuels from algae: challenges and potential. Biofuels. 2010;1(5):763–84. 18. Radakovits R, Jinkerson RE, Darzins A, Posewitz MC. Genetic engi- neering of algae for enhanced biofuel production. Eukaryot Cell. 2010;9(4):486–501. 19. Sharma KKS, Schuhmann H, Schenk PM. High lipid induction in microal- gae for biodiesel production. Energies. 2012;5:1532–53. Concluding remarks The SnRK family of kinases in Arabidopsis is classified into three subfamilies. The SnRK1 subfamily represents the smallest group with three genes (SnRK1α1–3), and they have the greatest similarity to the yeast Snf1 [258]. The SnRK1 family of genes coordinates the energy and redox homeostasis of plants in response to a plethora of growth-inhibiting stresses and regulates a broad range of metabolic pathways through the phosphorylation of the key enzymes or transcription factors to improve stress tolerance and promote survival [259–261]. In this context, the members of the SnRK1 family act to inhibit highly anabolic processes such as protein synthesis and Given the ongoing biotechnological interests in algae, increasing numbers of studies are giving rise to a sys- tems-level understanding of how various algal species respond to nutrient starvation, and how the metabolic pathways leading to the accumulation of TAG are regu- lated. Although the knowledge of transcriptomic and metabolic changes accompanying nutrient short- age and the entry into quiescence in algae continues to improve and evolve, the signaling and molecular com- ponents coordinating metabolism, energy status and cell division cycle are still not well-understood. The Takeuchi and Benning Biotechnol Biofuels (2019) 12:292 Page 14 of 20 trade-offs between growth and the accumulation of eco- nomically valuable compounds thus continue to hinder the directed metabolic engineering of algae for biofuels and the commercial viability of utilizing algae as a chas- sis for the synthesis of high-value products. However, a better understanding of the controls of the cell division cycle in response to nutrient shortage and the signal- ing pathways coupling the cellular growth to energy and lipid homeostasis has the potential to improve the future metabolic engineering strategies of algae. Indeed, emerg- ing evidence suggests that the manipulation of signaling pathways, such as TOR, represents a viable approach to increasing the lipid productivity in algae with little to no growth penalties [35, 268]. Thus, further studies of the signaling networks and the downstream components mediating and linking these biological processes are cru- cial in bridging a critical knowledge gap, which currently prevents us from achieving the optimal balance between the production of biofuels and biomass in algae employ- ing simple and robust culturing conditions. Ethics approval and consent to participate Not applicable. Ethics approval and consent to participate Not applicable. Concluding remarks trade-offs between growth and the accumulation of eco- nomically valuable compounds thus continue to hinder the directed metabolic engineering of algae for biofuels and the commercial viability of utilizing algae as a chas- sis for the synthesis of high-value products. However, a better understanding of the controls of the cell division cycle in response to nutrient shortage and the signal- ing pathways coupling the cellular growth to energy and lipid homeostasis has the potential to improve the future metabolic engineering strategies of algae. Indeed, emerg- ing evidence suggests that the manipulation of signaling pathways, such as TOR, represents a viable approach to increasing the lipid productivity in algae with little to no growth penalties [35, 268]. Thus, further studies of the signaling networks and the downstream components mediating and linking these biological processes are cru- cial in bridging a critical knowledge gap, which currently prevents us from achieving the optimal balance between the production of biofuels and biomass in algae employ- ing simple and robust culturing conditions. Acknowledgements 13. Woertz I, Feffer A, Lundquist T, Nelson Y. Algae grown on dairy and municipal wastewater for simultaneous nutrient removal and lipid production for biofuel feedstock. J Environ Eng. 2009;135(11):1115–22. Nothing to report. Only the authors contributed to this review. Abbreviations ACC1 l C A 4. Barrera DJ, Mayfield S. High-value recombinant protein production in microalgae. In: Richmond A, Hu Q, editors. Handbook of microalgal cul- ture: applied phycology and biotechnology. 2nd ed. Oxford: Blackwell Publishing Ltd; 2013. p. 532–44. 4. Barrera DJ, Mayfield S. High-value recombinant protein production in microalgae. In: Richmond A, Hu Q, editors. Handbook of microalgal cul- ture: applied phycology and biotechnology. 2nd ed. Oxford: Blackwell Publishing Ltd; 2013. p. 532–44. ACC1: acetyl-CoA carboxylase 1; AMPK: AMP-activated kinase; ATG: autophagy-related protein; BN-PAGE: blue native polyacrylamide gel electro- phoresis; C: carbon; CDK: cyclin-dependent kinase; CDKN1A/B: cyclin-depend- ent kinase inhibitor 1A/B; CHR: cell cycle genes homology region; CHT7: Compromised Hydrolysis of Triacylglycerols 7; CKIN: Chlamydomonas kinase; CP: commitment point; DAG: diacylglycerols; DGAT/DGTT: diacylglycerol acyltransferase type 1/type 2; DP: dimerization partner; DREAM: DP, RB, E2F, Myb-MuvB; DYRK: dual-specificity tyrosine phosphorylation-regulated kinase; E2F: adenovirus early gene 2 binding factor; ER: endoplasmic reticulum; FAS1/2: fatty acid synthase 1/2; FKBP12: FK506-binding protein; FOXM1: fork- head box M1; FRB: FKBP12–rapamycin binding; GPAT: glycerol-3-phosphate acyltransferase; InsP: inositol polyphosphate; LIP1: lipase 1; LST8: lethal with SEC13 protein 8; MGDG: monogalactosyldiacylglycerols; MSA: mitosis-specific activator element; MuvB: multi-vulva class B; N: nitrogen; P: phosphorus; PDAT: phospholipid:diacylglycerol acyltransferase; PE: phosphatidylethanolamine; PGD1: Plastid Galactoglycerolipid Degradation 1; RAPTOR: regulatory-asso- ciated protein of TOR; RB: retinoblastoma tumor suppressor protein; RICTOR: rapamycin-insensitive companion of TOR; ROS: reactive oxygen species; S: sulfur; S6K: S6 kinase; SMR: Siamese-related; Snf1: sucrose nonfermenting 1; SnRK: sucrose nonfermenting-related kinase; TAG: triacylglycerols; TAR1: TAG accumulation regulator 1; TOR: target of rapamycin; TORC1/2: target of rapamycin complex 1/2; YAK1: Yet Another Kinase 1. 5. 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https://openalex.org/W3110565628	https://www.frontiersin.org/articles/10.3389/fcimb.2020.608283/pdf	English	null	A Comparison of Stage Conversion in the Coccidian Apicomplexans Toxoplasma gondii, Hammondia hammondi, and Neospora caninum	Frontiers in cellular and infection microbiology	2,020	cc-by	12,432	A Comparison of Stage Conversion in the Coccidian Apicomplexans Toxoplasma gondii, Hammondia hammondi, and Neospora caninum Sarah L. Sokol-Borrelli, Rachel S. Coombs and Jon P. Boyle* University of Pittsburgh, Department of Biological Sciences, Kenneth P. Dietrich School of Arts and Sciences, Pittsburgh, PA, United States Stage conversion is a critical life cycle feature for several Apicomplexan parasites as the ability to switch between life forms is critical for replication, dissemination, pathogenesis and ultimately, transmission to a new host. In order for these developmental transitions to occur, the parasite must ﬁrst sense changes in their environment, such as the presence of stressors or other environmental signals, and then respond to these signals by initiating global alterations in gene expression. As our understanding of the genetic components required for stage conversion continues to broaden, we can better understand the conserved mechanisms for this process and unique components and their contribution to pathogenesis by comparing stage conversion in multiple closely related species. In this review, we will discuss what is currently known about the mechanisms driving stage conversion in Toxoplasma gondii and its closest relatives Hammondia hammondi and Neospora caninum. Work by us and others has shown that these species have some important differences in the way that they (1) progress through their life cycle and (2) respond to stage conversion initiating stressors. To provide a speciﬁc example of species- speciﬁc complexities associated with stage conversion, we will discuss our recent published and unpublished work comparing stress responses in T. gondii and H. hammondi. Edited by: Mathieu Gissot, Centre National de la Recherche Scientiﬁque (CNRS), France Reviewed by: Se´ bastien Besteiro, Universite´ de Montpellier, France Carsten Lüder, Universitätsmedizin Göttingen, Germany Correspondence: Jon P. Boyle boylej@pitt.edu Specialty section: This article was submitted to Parasite and Host, a section of the journal Frontiers in Cellular and Infection Microbiology Specialty section: This article was submitted to Parasite and Host, a section of the journal Frontiers in Cellular and Infection Microbiology Received: 19 September 2020 Accepted: 06 November 2020 Published: 03 December 2020 REVIEW published: 03 December 2020 doi: 10.3389/fcimb.2020.608283 REVIEW published: 03 December 2020 doi: 10.3389/fcimb.2020.608283 Keywords: Toxoplasma gondii, Hammondia hammondi, Neospora caninum, bradyzoite, tissue cysts INTRODUCTION Received: 19 September 2020 Accepted: 06 November 2020 Published: 03 December 2020 Eukaryotic parasites with multi-host life cycles have the unique challenge of persisting in a variety of different environments. Parasites of this nature must exist in a life form that is permissible and optimized for growth, survival, and transmission in a given host. The ability to adopt these speciﬁc life forms is especially critical for tissue cyst forming coccidia in the Sarcocystidae family which includes Sarcocystis, Frenkelia, Besnoitia, Cystoisopora, Toxoplasma, Hammondia, and Neospora species (Smith, 1981; Duszynski et al., 2018). Here, we will discuss the current knowledge about how three closely related parasites from this family, Toxoplasma gondii, Hammondia hammondi, and Neospora caninum, approach the challenges associated with multi-host life cycles. These three mammalian parasites follow heteroxenous, two host life cycles (Frenkel et al., 1970; Frenkel and Citation: Despite these genetic similarities, they exhibit substantial differences in pathogenesis, host range, and life cycles. Despite being the nearest living relative of T. gondii, H. hammondi is avirulent in comparison to T. gondii. H. hammondi is not known to infect humans and is not known to cause natural disease in any animal model (Dubey and Sreekumar, 2003). H. hammondi has a restricted host range when compared to T. gondii, as its only known natural intermediate hosts are rodents (Mason, 1978), roe deer (Entzeroth and Scholtyseck, 1978), and goats (Shimura and Ito, 1987). Despite having a limited natural host range, H. hammondi has been shown to experimentally infect monkeys (Dubey and Wong, 1978), dogs (Dubey, 1975), rabbits, and pigs (Dubey and Sreekumar, 2003), yet is unable to infect birds (Wallace, 1975; Dubey and Streitel, 1976). Despite this slight expansion in experimental host range, nonmurine animals are considered poor hosts for H. hammondi as infections do not result in robust chronic infection as H. hammondi-infected tissues from these animals produce fewer oocysts compared to H. hammondi- infected tissues from murine hosts upon sexual reproduction (Dubey and Streitel, 1976). Toxoplasma gondii has a broad host range and can infect all warm-blooded animals including birds (Tenter et al., 2000). T. gondii is responsible for the human disease toxoplasmosis and has infected approximately 2 billion people worldwide (Furtado et al., 2011). Although infections are often asymptomatic, T. gondii infections persist in the host in the form of tissue cysts, the life stage that contributes to chronic infection (Remington and Cavanaugh, 1965). Tissue cysts cannot be cleared by the host immune response nor can they be eliminated by known antiparasitic drugs (Tomavo and Boothroyd, 1995; Sullivan and Jeffers, 2012). T. gondii infections are thought to be long term, as bradyzoite containing tissue cysts reside in host tissue for extended periods of time and can reactivate causing clinical disease (Tenter et al., 2000; Weiss and Kim, 2000; Rougier et al., In stark contrast to the avirulent nature of H. hammondi, N. caninum is the major cause of bovine abortion, resulting in losses of over a billion dollars worldwide in cattle industries (Dubey et al., 2007; Goodswen et al., 2013). Like with T. gondii, the formation of tissue cysts is also critical for the transmission and survival of N. caninum. The reactivation of bradyzoite-containing A B FIGURE 1 \| Relationships between Toxoplasma gondii, Hammondia hammondi, and Neospora caninum. Citation: Sokol-Borrelli SL, Coombs RS and Boyle JP (2020) A Comparison of Stage Conversion in the Coccidian Apicomplexans Toxoplasma gondii, Hammondia hammondi, and Neospora caninum. Front. Cell. Infect. Microbiol. 10:608283. doi: 10.3389/fcimb.2020.608283 December 2020 \| Volume 10 \| Article 608283 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Stage Conversion in Coccidian Apicomplexans Sokol-Borrelli et al. Dubey, 1975; McAllister et al., 1998; Dubey, 2009; Dubey and Ferguson, 2015) where they must survive in a variety of cell types with varying immune pressures. In order to be successful in their hosts, these parasites are capable of converting to life forms that each serve an important function for survival and ﬁtness. Although these parasites exhibit the same infectious life forms, they have fundamental differences in their life cycles and stage conversion strategies. This review will focus on the critical differences in stage conversion between tachyzoites, the rapidly replicating life form, and bradyzoites, the slower growing parasites comprising tissue cysts, in T. gondii, H. hammondi, and N. caninum. 2017). T. gondii tissue cysts can cause severe complications for immunocompromised individuals, such as HIV/AIDS and organ transplant patients, when latent infections reactivate into highly replicative life forms and result in tissue damage (Derouin et al., 1987; Derouin et al., 2008). Furthermore, bradyzoite containing tissue cysts maintained in the animal population contributes to the spread of T. gondii to both animals and humans, as T. gondii is the leading cause of death caused by a food borne illness in the United States (CDC - Toxoplasmosis; Tenter et al., 2000; Tenter, 2009). Individuals, even those who are immunocompetent, infected with T. gondii can also develop ocular toxoplasmosis. T. gondii is capable of invading and replicating in the retina which can result in severe retinal damage that can lead to blindness (Ozgonul and Besirli, 2017). T. gondii, H. hammondi, and N. caninum are all closely related (Figure 1A), obligate intracellular parasites that follow two-host life cycles (See detailed description in section 2). These parasites species also share the same class of organelles and have several shared genomic features. T. gondii and H. hammondi share >95% of their genomes in near perfect synteny (Walzer et al., 2013), while T. gondii and Neospora caninum genomes are >81% syntenic (Adomako-Ankomah et al., 2014). Conservative predictions have identiﬁed 7,095 shared orthologs between H. hammondi and T. gondii and 6,308 orthologs between T. gondii and N. caninum (Lorenzi et al., 2016). Citation: (A) Neighbor-joining tree depicting relationship between the ITS1 (internal transcribed spacer 1) sequences of T. gondii, H. hammondi, and N. caninum. P. falciparum is used as an outgroup. ITS1 sequences were chosen to highlight the relationship between these parasite species due to high variability often attributed to these non-coding sequences. Sequences were obtained from GenBank. Bootstrap values for 1000 replicates are indicted. Scale bar represents the substitutions per site. (B) Diagrams showing the life cycle of T. gondii, H. hammondi, and N. caninum. A A B A B FIGURE 1 \| Relationships between Toxoplasma gondii, Hammondia hammondi, and Neospora caninum. (A) Neighbor-joining tree depicting relationship between the ITS1 (internal transcribed spacer 1) sequences of T. gondii, H. hammondi, and N. caninum. P. falciparum is used as an outgroup. ITS1 sequences were chosen to highlight the relationship between these parasite species due to high variability often attributed to these non-coding sequences. Sequences were obtained from GenBank. Bootstrap values for 1000 replicates are indicted. Scale bar represents the substitutions per site. (B) Diagrams showing the life cycle of T. gondii, H. hammondi, and N. caninum. Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org December 2020 \| Volume 10 \| Article 608283 Stage Conversion in Coccidian Apicomplexans Sokol-Borrelli et al. tissue cysts into fast replicating tachyzoites during bovine pregnancy results in efﬁcient transmission to the fetus. Efﬁcient transmission, persistence, and recrudescence in asymptomatic cows is a signiﬁcant biotic constraint in these agriculturally important animals, and currently there is no treatment or vaccine for bovine neosporosis (Buxton et al., 2002; Dubey et al., 2006; Williams and Trees, 2006; González-Warleta et al., 2018). While cattle are considered the major intermediate host, N. caninum can also infect a number of other domestic and wild ruminant species where its infection can result in disease (Dubey et al., 2013). parasites to be continually propagated through asexual reproduction in intermediate hosts and underlies disease progression in immunocompromised hosts (Dubey et al., 1998). To date, the molecular determinants of this unique ability have not been elucidated, but it has likely had a dramatic impact on the global distribution and broad host range of T. gondii. In comparison to T. gondii, Hammondia hammondi follows a strict obligate heteroxenous life cycle (Figure 1B). Sexual reproduction only occurs within the intestinal epithelium of the deﬁnitive host, which, like T. gondii, includes felids. H. hammondi oocysts, like T. LIFE CYCLES OF TOXOPLASMA GONDII, HAMMONDIA HAMMONDI, AND NEOSPORA CANINUM Toxoplasma gondii follows a facultative homoxenous/ heteroxenous two host life cycle (Figure 1B). Sexual reproduction of T. gondii occurs in its deﬁnitive hosts which include members of the Felidae genus and produces millions of orally infectious oocysts that are environmentally stable. Each oocyst contains 8 sporozoites contained within 2 sporocysts that are surrounded by an oocyst wall (Frenkel et al., 1970; Dubey, 1998; Dubey, 2009). When oocysts are ingested by an intermediate host, the sporozoites will excyst within the host digestive system and invade the intestinal epithelium. Once in the intestinal epithelium, sporozoites differentiate into tachyzoites, which are the fast growing life stage responsible for acute infection. During a primary infection of a naïve intermediate host, T. gondii tachyzoites are capable of crossing the placenta, which can result in vertical transmission of this parasite (McAuley, 2014). Eventually, T. gondii tachyzoites differentiate into bradyzoites, the slow growing life stage associated with chronic infection. Bradyzoites are found within tissue cysts which typically reside in the central nervous system or in skeletal or cardiac muscle (Dubey et al., 1998). Bradyzoite tissue cysts are orally infectious to the deﬁnitive host. When ingested by a deﬁnitive host, tissue cysts will differentiate into the sexual stages resulting in the production of millions of oocysts, thus completing the life cycle (Dubey and Frenkel, 1972). Additionally, cats have been described as complete hosts for T. gondii because they support proliferation of tachyzoites and bradyzoites in extra-intestinal tissues in addition to supporting sexual life stages and sexual reproduction in intestinal tissues (Frenkel, 1977). One of the most critical components of the T. gondii life cycle is reactivation. Reactivation can occur when 1) a chronically infected host becomes immunocompromised which results in rapid proliferation and dissemination of tachyzoites or 2) when a naïve intermediate host ingests T. gondii tissue cysts, the bradyzoites contained within these tissue cysts are released during digestion and differentiate back to tachyzoites, which then disseminate and cause acute infection in the host before encysting during chronic infection. Reactivation results in expanded transmission for T. gondii, as it allows for the Like T. gondii, N. caninum also follows a complex facultative heteroxenous life cycle (Figure 1B). As described for T. gondii and H. hammondi, sexual reproduction for N. caninum infections in the deﬁnitive host (in this case canines rather than felines) results in oocyst production and subsequent excretion in host feces. N. Citation: gondii oocysts, are orally infectious to intermediate hosts (Frenkel and Dubey, 1975; Dubey and Ferguson, 2015). Unlike T. gondii infections, feline hosts can only support H. hammondi enteroepithelial stages and do not support extraintestinal infections (Dubey and Sreekumar, 2003). Furthermore, during asexual reproduction H. hammondi remain infectious to intermediate hosts until they terminally differentiate into tissue cyst stages (Dubey and Sreekumar, 2003; Sokol et al., 2018) that are only orally infectious to deﬁnitive feline hosts. It is hypothesized that terminally differentiated H. hammondi parasites are incapable of reactivation, however, this has yet to be tested in head-to-head comparisons with T. gondii. Furthermore, H. hammondi is not known to be capable of vertical transmission (Dubey and Streitel, 1976). Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Toxoplasma gondii Bradyzoites are necessary for transmission as their cyst wall provides protection during passage through the mammalian gut (Jacobs et al., 1960), and they are also required for sexual reproduction which dramatically expands the number of infectious oocysts available to infect new hosts. Even though the deﬁnitive hosts for T. gondii and N. caninum (felines and canines, respectively), are complete hosts (host that can support both sexual and asexual replication), bradyzoite formation within these animals is still needed in order for sexual reproduction to occur. Bradyzoites also allow the parasites to remain in a host for extended period of time as they cannot be cleared by the host immune system. Finally, interconversion that occurs in T. gondii and N. caninum allows for these parasites to be transmitted completely independently of the deﬁnitive host, a remarkable trait given the vastly different species that can serve as intermediate hosts for these parasites. For these reasons, understanding the mechanisms driving the conversion of tachyzoites to bradyzoites and how it compares between species is necessary to manage the many manifestations of disease caused by these organisms. Moreover, by identifying the developmental sensors, triggers, and components in these species and comparing their activities in each may lead to a better understanding of how this process is regulated on the molecular level. Host factors, speciﬁcally the differentiation state of a given host cell, can also impact T. gondii cystogenesis. T. gondii forms tissue cysts when grown in mouse primary skeletal muscle cells that have been differentiated into polynucleated myotubes, which are withdrawn from cell cycle progression (Ferreira-da-Silva et al., 2009; Swierzy and Lüder, 2015). When these host cells are genetically manipulated to knock-down Testis-speciﬁc Y- encoded-like protein 2 (Tspyl2), a negative cell cycle regulator that contributes withdraw from host cell cycle progression in these myotubes, T. gondii fails to form tissue cysts in these cells (Swierzy and Lüder, 2015). Additionally, expression levels of the host gene human cell division autoantigen-1 (CDA1) are important for bradyzoite development. T. gondii has been shown to grow slower and express bradyzoite speciﬁc genes when grown in host cells treated (pre-treatment or continuous treatment) with the trisubstituted pyrrole small molecule Compound 1, which upregulates expression of host CDA1 (Radke et al., 2006). CDA1 is a negative regulator of cell growth and has regions with homology to testis protein TSPY (Chai et al., 2001). Additionally, T. Toxoplasma gondii exogenous (primary) transplacental infections occur (Williams et al., 2009). During vertical transmission, N. caninum converts into fast-replicating tachyzoites which cross the placenta to transmit to offspring often resulting in abortion. Following transmission, N. caninum converts to bradyzoites to evade the host immune responses and tissue cysts form. It is thought the interconversion from bradyzoite to tachyzoite, allows for one infected animal to transmit the parasite to offspring repeatedly. In support of this, recrudescence and transmission have been described in cattle (Williams et al., 2009), sheep (Gutiérrez-Expósito et al., 2020) and in dogs (Cole et al., 1995; Barber and Trees, 1998; Heckeroth and Tenter, 2007). As for T. gondii, the means by which N. caninum is capable of moving both forwards and backwards in its life cycle is unknown. Toxoplasma gondii Bradyzoite formation in T. gondii has been extensively studied. An important, but underappreciated, fact is that most strains of T. gondii are capable of spontaneously converting into bradyzoites when grown in vitro in a variety of different host cells (Lindsay et al., 1991; Lindsay et al., 1993). When T. gondii infections are initiated with parasites derived from sporozoites or from tachyzoites or bradyzoites that have not be extensively passaged following isolation, the parasites will ﬁrst grow as tachyzoites but will later form tissue cysts that express bradyzoite markers and/or are able to induce oocyst shedding when fed to cats (Lindsay et al., 1991; Jerome et al., 1998). This phenomenon of spontaneous stage conversion in T. gondii was most thoroughly studied in T. gondii strain VEG. When infections are initiated with T. gondii VEG sporozoites, the parasites differentiate into a rapidly growing tachyzoite stage resembling many lab-adapted strains like T. gondii RH, and after ~20 divisions they begin to differentiate into slower growing parasites that express bradyzoite markers, suggesting that there may be a developmental clock controlling this spontaneous conversion (Jerome et al., 1998). Taken together, these ﬁndings suggest that there is some type of parasite intrinsic factor that enables these parasites to transition to bradyzoites following initial tachyzoite expansion. Regardless of parasite species, the formation of bradyzoites is a critical step in the transmission of T. gondii, H. hammondi, and N. caninum, despite clear differences in their life cycles. Toxoplasma gondii gondii forms tissue cysts when grown in HeLa cells that overexpress a transgene encoding CDA1 (Radke et al., 2006). The ability of cells that have withdrawn from their cell cycle to promote tissue cyst formation in T. gondii is a likely contributor to the preference T. gondii shows for muscular tissue and the tissues of the central nervous system. LIFE CYCLES OF TOXOPLASMA GONDII, HAMMONDIA HAMMONDI, AND NEOSPORA CANINUM caninum oocysts are environmentally stable and contain sporozoites that are orally infectious to intermediate hosts. N. caninum undergoes sexual reproduction in members of the Canis genus (Donahoe et al., 2015) and it is presumed that sexual reproduction occurs in enteroepithelial cells similar to T. gondii and H. hammondi in felid infections, however, little is known about the sexual life cycle of N. caninum infections. The ﬁrst report of enteroepithelial developmental stages of N. caninum was published in 2015 and identiﬁed oocyst and schizont-like structures in epithelia in a naturally infected dog (Kul et al., 2015) N. caninum tachyzoites and bradyzoites can be found in both intermediate and deﬁnitive host tissue, primarily in the central nervous system (Dubey et al., 2002; Dubey et al., 2007). N. caninum infections in canids are similar to T. gondii in felids in that dogs can be considered complete hosts for N. caninum and support replication of all three infectious life stages. As with T. gondii, transmission via carnivory of infectious tissue cysts is important for horizontal transmission of N. caninum to the deﬁnitive host. Although experimental infections demonstrate N. caninum tissue cysts are orally infectious (Lindsay and Dubey, 1990; Lindsay et al., 2001), the ingestion of sporulated oocysts is the only known natural mode of horizontal transmission to cattle (De Marez et al., 1999; Kano et al., 2005; Eastick and Elsheikha, 2010). Vertical transmission is the most frequent mode of transmission in bovine hosts and both endogenous (reactivated) and December 2020 \| Volume 10 \| Article 608283 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 3 Stage Conversion in Coccidian Apicomplexans Sokol-Borrelli et al. INDUCERS OF BRADYZOITE DEVELOPMENT While environmental signals are likely at the heart of stage conversion in T. gondii and its near relatives, the precise mechanisms used to respond to these signals have remained elusive. However, the effect of various signals on inducing life stage development, speciﬁcally tissue cyst formation, has been well studied in T. gondii. Furthermore, recent work has begun to identify distinct responses to these triggers in H. hammondi, indicating the presence of a divergent stress response in this species. Several well characterized stressors are known to induce T. gondii tachyzoite-to-bradyzoite stage conversion in vitro (summarized in Table 1). Perhaps the most well-known stressor that leads to robust formation of T. gondii tissue cysts in vitro is alkaline pH (pH ~8 as compared to the December 2020 \| Volume 10 \| Article 608283 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Stage Conversion in Coccidian Apicomplexans Sokol-Borrelli et al. TABLE 1 \| Summary of exogenous stressors that induce bradyzoite development in Toxoplasma gondii. INDUCERS OF BRADYZOITE DEVELOPMENT Stressor Parasite life stage used for infection Parasite strain Host cell Method used to determine bradyzoite formation Citation Alkaline pH (treatment of infected host cells) Sporozoites VEG Human foreskin ﬁbroblasts (HFFs) Bradyzoite-speciﬁc antibodies (Jerome et al., 1998) In vivo tachyzoites RH HFFs; Vero cells Bradyzoite-speciﬁc antibodies (Soête et al., 1994) Alkaline pH (extracellular parasites) In vitro tachyzoites ME49 HFFs Bradyzoite-speciﬁc antibodies (Weiss et al., 1998) Heat Shock (43 degrees C) In vivo tachyzoites RH Vero cells Bradyzoite-speciﬁc antibodies (Soête et al., 1994) Sodium arsenite In vivo tachyzoites RH Vero cells Bradyzoite-speciﬁc antibodies (Soête et al., 1994) Sodium nitroprusside (SNP) (extracellular parasites) In vitro tachyzoites ME49 HFF Bradyzoite-speciﬁc antibodies (Weiss et al., 1998) SNP (infected host cells) In vitro tachyzoites NTE Murine bone marrow-derived macrophages (BMDM) Bradyzoite-speciﬁc antibodies (Bohne et al., 1994) Interferon gamma In vitro derived tachyzoites NTE Murine peritoneal macrophages Bradyzoite-speciﬁc antibodies (Bohne et al., 1993) In vitro tachyzoites NTE Murine BMDM Bradyzoite-speciﬁc antibodies (Bohne et al., 1994) Antimycin A In vitro tachyzoites NTE Murine BMDM Bradyzoite-speciﬁc antibodies (Bohne et al., 1994) Oligomycin In vitro tachyzoites NTE Human ﬁbroblasts Bradyzoite-speciﬁc antibodies (Bohne et al., 1994) Atovaquone In vitro tachyzoites PLK HFFs Bradyzoite-speciﬁc antibodies (Tomavo and Boothroyd, 1995) Arginine starvation In vitro tachyzoites RH; PLK HFFs Dolichos biﬂorus agglutinin (Fox et al., 2004) Pyrimidine starvation In vitro tachyzoites RHdUPRT HFFs Bradyzoite-speciﬁc antibodies (Bohne and Roos, 1997) Cholesterol depletion (Lipoprotein depleted serum) In vitro tachyzoites ME49 Chinese hamster ovary cells Bradyzoite-speciﬁc antibodies (Ihara and Nishikawa, 2014) Compound 1 In vitro tachyzoites ME49B7; Pru; VEG; CTG HFFs Bradyzoite-speciﬁc antibodies; Dolichos biﬂorus agglutinin (Radke et al., 2006) TABLE 1 \| Summary of exogenous stressors that induce bradyzoite development in Toxoplasma gondii. standard pH growth conditions of 7.2–7.4). Multiple groups have shown that alkaline pH induces T. gondii cyst development either when applied to host cells after infection (Soête et al., 1994) or when applied to extracellular parasites prior to infection (Weiss et al., 1998). Despite robust induction of T. gondii tissue cyst development, the exact mechanism remains unknown. It is possible that alkaline pH derived stress induces a myriad of both host and parasite derived signals that are needed in order to initiate bradyzoite development in T. gondii. Furthermore, treatment of infected host cells with pH 6.8 media has also been shown to induce bradyzoite development (Weiss et al., 1995). Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org INDUCERS OF BRADYZOITE DEVELOPMENT Tumor necrosis factor-alpha (TNF-a) and inducible nitric oxide synthase (iNOS) may play a role in restricting cystogenesis, as TNF receptor p55- and p75- deﬁcient mice and iNOS deﬁcient mice develop more tissue cysts December 2020 \| Volume 10 \| Article 608283 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 5 Stage Conversion in Coccidian Apicomplexans Sokol-Borrelli et al. in the brain compared to wild type mice despite relatively equivalent parasite burden in peritoneal cells early in infection. Interestingly, mice deﬁcient for TNF-a receptors or iNOS succumb to chronic infection while the WT parasites survive (Scharton-Kersten et al., 1997; Yap et al., 1998). It is challenging to determine if the increase in tissue cysts in the brains of these mice is a result of TNF-a and iNOS restricting tissue cyst development, or if more parasites make it to the brain prior to tissue cyst formation. Future experiments using live imaging that quantiﬁes parasite burden could be useful to test these hypotheses. Additionally, CD4+ and CD8+ T-cells are important for the maintenance of chronic infection characterized by the bradyzoite/tissue cyst life stage. Depletion of these cells with neutralizing antibodies leads to reactivation of T. gondii infection leading to parasite proliferation (Gazzinelli et al., 1992). It is also thought that IFN-g may play a critical role in cystogenesis. It is hypothesized that IFN-g contributes to the initiation of tissue cyst formation in vivo, as it can induce cystogenesis in vitro (described above), however IFN-g knockout mice fail to control acute proliferation of parasites and succumb to acute infection (Suzuki et al., 1988; Suzuki et al., 1989) even when infected with avirulent strains of T. gondii (Coombs et al., 2020) making it difﬁcult to determine if IFN-g induces cystogenesis in vivo. It is also possible that T. gondii spontaneously forms tissue cysts during in vivo infections, however this spontaneous development is again challenging to observe experimentally due to the lethality of T. gondii infections in mice with disrupted immune systems. Linking what is known about in vitro cyst development in T. gondii to what happens in vivo is a signiﬁcant, but important, knowledge gap in the ﬁeld that will require new technological innovation to ﬁll. important because it suggested that the ability to constitutively respond to alkaline pH in T. gondii was a derived trait. Since this initial work we have also investigated if H. Hammondia hammondi In comparison to T. gondii, little is known about bradyzoite and tissue cyst formation in H. hammondi. When grown in vitro, H. hammondi fails to grow in continuous culture and spontaneously undergoes a terminal differentiation process where it completely converts to tissue cysts that are only infectious to deﬁnitive feline hosts (Shefﬁeld et al., 1976; Riahi et al., 1995; Dubey and Sreekumar, 2003; Sokol et al., 2018). The timing of tissue cyst formation corresponds to when H. hammondi parasites lose their ability to infect a new host cell (Sokol et al., 2018), demonstrating that H. hammondi follows a strict obligate heteroxenous life cycle even in in vitro growth conditions (a sharp contrast to both T. gondii and N. caninum). Furthermore, comparative transcriptomic analysis between replicating T. gondii and H. hammondi showed that the H. hammondi transcriptional proﬁle is enriched for genes that are typically reserved for expression during bradyzoite and sexual stages occurring in feline intestinal cells in T. gondii (Sokol et al., 2018). It is likely that H. hammondi follows a strictly regulated life cycle where it is poised to convert to its next life stages after a pre-deﬁned time as a given life form. INDUCERS OF BRADYZOITE DEVELOPMENT In addition to changes in pH, heat shock (43°C as opposed to 37°C) and sodium arsenite treatment of infected host cells have also been shown to induce stage conversion in T. gondii, however heat shock is not an optimal method for inducing bradyzoite development as it often results in decreased parasite invasion, parasite killing, and loss of host cell viability (Soête et al., 1994). Nutrient starvation can also induce stage conversion in T. gondii. Pyrimidine starvation achieved via deletion of the uracil phosphoribosyl transferase (UPRT) gene in combination with growth in atmospheric CO2 (0.03% compared to 5%) also leads to a parasite growth reduction and expression of bradyzoite markers (Bohne and Roos, 1997). Arginine starvation also decreases the replication of T. gondii and induces tissue cyst formation (Fox et al., 2004). Cholesterol depletion via growth in media supplemented with lipoprotein depleted serum (as compared to growth in 5% fetal bovine serum) has also been shown to induce bradyzoite gene expression (Ihara and Nishikawa, 2014). Additionally, interferon gamma (IFN-g) treatment of T. gondii infected macrophages results in expression of bradyzoite speciﬁc antigens (Bohne et al., 1994), however IFN-g is not capable of inducing expression of bradyzoite speciﬁc antigens in human ﬁbroblasts (Suzuki et al., 1989; Bohne et al., 1993; Soête et al., 1994; Weiss et al., 1995). Other in vitro stressors that may mimic IFN-g-driven immune pressure also induces T. gondii cystogenesis, such as the production of nitric oxide. Exogeneous nitric oxide produced from sodium nitroprusside (SNP) treatment can induce bradyzoite development (Bohne et al., 1994). Exogenous nitric oxide likely inhibits proteins involved in the electron transport chain, thus treatment with mitochondrial inhibitors oligomycin, antimycin A, (Bohne et al., 1994), and atovaquone (Tomavo and Boothroyd, 1995) can also induce expression of bradyzoite antigens in T. gondii. Overall, the numerous and diverse exogenous stressors capable of inducing bradyzoite development in T. gondii suggest that multiple signals can be used as triggers to induce the fundamental process of bradyzoite formation. In comparison to in vitro systems, in vivo factors that induce cystogenesis are much less clear. Neospora caninum Neospora caninum Like T. gondii, N. caninum alternates between two life stages presumably to survive host immune responses. The mechanisms underlying N. caninum tachyzoite to bradyzoite conversion remain largely unknown (to an even greater extent than for H. hammondi) due to difﬁculties in developing in vitro models for bradyzoite development. Methods used to obtain T. gondii bradyzoites in vitro are ineffective or inefﬁcient for N. caninum cyst formation. Although nitric oxide treatment of murine keratinocytes infected with N. caninum tachyzoites yields cysts, they are surrounded by thick keratin ﬁlament bundles and thus impede parasite puriﬁcation processes (Vonlaufen et al., 2002). Very few studies have investigated the effects of stress on stage conversion for N. caninum. One study suggests that nitric oxide, increasing pH, or increasing temperature can increase tachyzoite to bradyzoite conversion in N. caninum (Weiss et al., 1999). SNP has also been shown to increase expression of N. caninum bradyzoite and cyst wall markers (Risco-Castillo et al., 2004). However, there are no studies investigating any difference in gene expression during the tachyzoite to bradyzoite conversion process in N. caninum. Substantial work with regards to how to identify N. caninum bradyzoites and reliably produce this life stage in vitro and in vivo is still needed in order to expand our understanding of how stage conversion occurs in N. caninum. PARASITE INTRINSIC MOLECULAR MECHANISMS OF STAGE CONVERSION Even though H. hammondi completely and spontaneously forms tissue cysts when grown in vitro, it cannot be induced to form tissue cysts with alkaline pH, a robust inducer of stage conversion in T. gondii, at early time points following sporozoite- initiated infection (Sokol et al., 2018). This discovery was INDUCERS OF BRADYZOITE DEVELOPMENT hammondi is eventually able to form tissue cysts in response to alkaline pH derived stress applied at later stages in its in vitro cycle. Interestingly, we have found that when alkaline pH derived stress is applied for 48 h at later developmental time points (Day 13 post sporozoite-derived infection), signiﬁcantly more tissue cysts form in response to alkaline pH treatment than occur spontaneously at this time (Figure 2). This ﬁnding suggests that as H. hammondi progresses through a predeﬁned developmental program it differentiates from a life form that is incapable of responding to alkaline pH (unable to sense and/or respond) to a life form that is capable of sensing and/or responding. This hints at a conserved linkage between alkaline pH responsiveness and the tachyzoite to bradyzoite stage conversion. Future work investigating the differences in gene expression between these time points could be promising in uncovering additional components of the mechanisms that these parasites use to sense and respond to their environment. It is currently unknown as to how H. hammondi responds to other stressors known to induce tissue cyst development in T. gondii. Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Chromatin Stage conversion in eukaryotic parasites is accompanied by signiﬁcant changes in patterns of gene expression (Gomez et al., 2010; Chen et al., 2018). The chromatin landscape of a Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org December 2020 \| Volume 10 \| Article 608283 6 Stage Conversion in Coccidian Apicomplexans Sokol-Borrelli et al. A B FIGURE 2 \| H. hammondi can respond to stress conditions when exposed to alkaline pH 13 days post excystation (DPE) from oocysts. (A) Percentage of DBA positive vacuoles observed following 48 h of alkaline pH stress initially applied at D4, 5, 7, 13, and 15PE for T. gondii VEG. (B) Percentage of DBA positive vacuoles observed following 48 h of alkaline pH stress initially applied at D4, 5, 7, 13, and 15PE for H. hammondi American. Statistical signiﬁcance was determined by 2way ANOVA with Sidak’s multiple comparisons test of arcsine transformed data. (N = 2–3 biological replicates, P = 0.03, P < 0.01, P < 0.001, and **P < 0.0001) This experiment, with the exception of time of alkaline stress application at the time points mentioned above, was performed as previously described (Sokol et al., 2018). A B B FIGURE 2 \| H. hammondi can respond to stress conditions when exposed to alkaline pH 13 days post excystation (DPE) from oocysts. (A) Percentage of DBA positive vacuoles observed following 48 h of alkaline pH stress initially applied at D4, 5, 7, 13, and 15PE for T. gondii VEG. (B) Percentage of DBA positive vacuoles observed following 48 h of alkaline pH stress initially applied at D4, 5, 7, 13, and 15PE for H. hammondi American. Statistical signiﬁcance was determined by 2way ANOVA with Sidak’s multiple comparisons test of arcsine transformed data. (N = 2–3 biological replicates, P = 0.03, P < 0.01, P < 0.001, and **P < 0.0001) This experiment, with the exception of time of alkaline stress application at the time points mentioned above, was performed as previously described (Sokol et al., 2018). given life stage plays a large role in what genes are expressed, therefore playing a critical role in stage conversion. Several chromatin remodeling factors have been identiﬁed in T. gondii that play a role in altering the chromatin landscape during different life stages. One such factor is Histone Deacetylase 3 (HDAC3), which is a component of T. gondii’s corepressor complex. Chromatin HDAC3 is associated with bradyzoite speciﬁc promoters (Saksouk et al., 2005) and when its activity is inhibited with the compound FR235222, expression of bradyzoite genes increases (Bougdour et al., 2009). These ﬁndings suggest that histone deacetylation via HDAC3 functions to repress the expression of bradyzoite genes and keep T. gondii in a tachyzoite life form. HDAC3 has been shown to work with the recently discovered T. gondii microrchidia (MORC) protein. MORC interacts with several AP2 transcription factors (Farhat et al., 2020) [including AP2IX-4 and AP2XII-2 discussed below (Srivastava et al., 2020)] and recruits HDAC3 to chromatin, enabling the generation of hypo-acetylated chromatin which represses gene expression. When MORC is depleted in T. gondii, the parasites begin to express transcripts typically expressed in other life stages, such as merozoite and oocysts speciﬁc genes, that are restricted to the T. gondii sexual stages (Farhat et al., 2020). These ﬁndings suggest that MORC functions as a repressor of sexual development associated gene expression through chromatin modiﬁcation in T. gondii. Our transcriptional data from replicating H. hammondi on Day 4 (D4) and Day 15 (D15) post sporozoite infection, does not show any changes in transcriptional abundance of either of H. hammondi’s orthologs of HDAC3 or MORC (Figure 3). However, in comparison to T. gondii, H. hammondi’s transcriptional proﬁle is enriched for genes that are associated with sexual development in T. gondii (Sokol et al., 2018), a process which has been shown to be repressed by MORC (Farhat et al., 2020). Because of this similarity, we hypothesized that transcript abundance of MORC repressed genes would be enriched in H. hammondi at D15 compared to D4. To test this hypothesis, we conducted pre- ranked gene set enrichment analysis (GSEA) as previously described (Subramanian et al., 2005) and found signiﬁcant enrichment for MORC repressed transcripts [gene sets derived December 2020 \| Volume 10 \| Article 608283 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 7 Stage Conversion in Coccidian Apicomplexans Sokol-Borrelli et al. alkaline pH stress conditions, GCN5-A occupancy is enriched inthepromotorsofbradyzoitespeciﬁc genesthatareupregulatedin response to stress. When GCN5-A is knocked out in T. gondii, the parasitesareunabletoupregulate74%ofknownbradyzoitegenesin response to alkaline pH induced stress (Naguleswaran et al., 2010). Together, these ﬁndings demonstrate that GCN5-A plays a critical role in T. gondii’s ability to alter its gene expression in response to alkaline pH stress. Chromatin p Additional chromatin remodeling enzymes that play a role in the alteration of stage conversion associated gene expression have also been identiﬁed in T. gondii. These include TgCARM1 (Saksouk et al., 2005), TgSCRAP (Sullivan et al., 2003), and TgRSC8 (Craver et al., 2010; Rooney et al., 2011). TgCARM1 is a histone arginine methyltransferase protein that is essential for parasite replication. When N-methyltransferase activity is inhibited with the small molecule AMI-1 via pretreatment of extracellular parasites, T. gondii forms signiﬁcantly more bradyzoites compared to a vehicle control (Saksouk et al., 2005). TgSCRAP, a Snf2-related CBP activator protein and SNF/SWI chromatin remodeler, upregulates the expression of the known bradyzoite gene bradyzoite antigen 1 (BAG1) during alkaline pH induced stress, and has been shown to enhance CREB (cAMP response element binding protein) mediated transcription, which may suggest a role in the protein kinase A signaling pathway that has also been implicated in bradyzoite development for T. gondii (Sullivan et al., 2003). Another chromatin remodeling complex that contributes to bradyzoite development is TgRSC8, a homolog of the nucleosome remodeling complex Rsc8p protein in Saccharomyces cerevisiae. When this protein is mutated in T. gondii, some bradyzoite genes showed a signiﬁcant reduction in transcript abundance when exposed to alkaline pH stress conditions. However, these mutants did not show reduced Dolichos biﬂorus agglutinin (DBA) staining (Rooney et al., 2011), which speciﬁcally recognizes the glycosylated cyst wall protein CST1 (Zhang et al., 2001). In addition to chromatin remodelers, histone variants have also been implicated in stage conversion in T. gondii. The histone variant H2A.Z is expressed in mature, in vivo bradyzoites and H2AX is also expressed in mature, in vivo bradyzoites but also displays increased expression in vitro during alkaline pH stress (Dalmasso et al., 2009). All together, these ﬁndings suggest that the alteration of chromatin is an important contributor to stage conversion associated gene expression in T. gondii. FIGURE 3 \| Heatmap representing H. hammondi transcriptional abundance during spontaneous development of known regulators of bradyzoite formation. Heatmaps show mean centered Log2FPM values. Asterisk () represent genes with signiﬁcant differences between D4 (N = 2 biological replicates) and D15 (N = 3 biological replicates) samples. Signiﬁcance is deﬁned as \|Log2 Fold Change\| >1 and Padj < 0.01. T. gondii gene IDs for these genes and the gene IDs for each orthologs in H. hammondi and N. caninum are found in Table 2. The H. Transcription Factors in vitro upon knockdown in T. gondii, suggesting that this factor may be important for maintaining tachyzoites (Srivastava et al., 2020). Additionally, AP2 transcription factors have been identiﬁed in T. gondii that are involved in promoting bradyzoite development. These include AP2IV-4 (Radke et al., 2018), AP2IV-3 (Hong et al., 2017), AP2IX-4 (Huang et al., 2017), and AP2XI-4 (Walker et al., 2013). When these AP2 transcription factors are deleted, parasites have a decreased ability to form tissue cysts in vitro and/or in vivo. Furthermore, alteration (either deletion or overexpression) of these factors results in signiﬁcant differences in transcript abundance of known bradyzoite genes (Walker et al., 2013; Hong et al., 2017; Huang et al., 2017; Radke et al., 2018). Our H. hammondi transcriptional data does not show any signiﬁcant differences in transcript abundance in these AP2 factors during spontaneous development in H. hammondi (Figure 3). Transcription factors are an important class of proteins that play a signiﬁcant role in the control of stage conversion speciﬁc gene expression and have long been investigated for their role in stage conversion associated gene expression in T. gondii. One of the ﬁrst major classes of transcription factors investigated for their role in stage conversion in T. gondii were the AP2 transcription factors. AP2 factors were ﬁrst identiﬁed in Apicomplexans in 2005. These transcription factors share the Apetala2 (AP2) integrase DNA binding domain typically found in transcription factors of numerous plant species (Balaji et al., 2005). AP2 transcription factors are known to play a major role in stage conversion in Plasmodium species (Painter et al., 2011). There are currently 67 AP2 factors (66 annotated as AP2 domain transcription factors and 1 annotated as an AP2 domain- containing protein) in the T. gondii ME49 genome (Gajria et al., 2008). However, only 6 of these AP2 transcription factors have been tied to bradyzoite development in T. gondii. The AP2 transcription factor AP2IX-9 functions mainly as a repressor of bradyzoite development, maintaining parasites in an intermediate, pre-bradyzoite state. AP2IX-9 binds to the CAGTGT motif and functions to repress transcription (Radke et al., 2013). Furthermore, deletion of AP2IX-9 results in increased tissue cyst formation in parasites cultivated in normal growth conditions (Hong et al., 2017). During spontaneous development (D4 versus D15) in H. hammondi, we see signiﬁcant increases in transcriptional abundance of the H. hammondi ortholog of AP2IX-9 (\|Log2 Fold Change\| = 2.21, Padj = 0.009). Chromatin hammondi transcriptional data used to generate this ﬁgure was obtained from (Sokol et al., 2018). from data published in (Farhat et al., 2020)] (NES= -10.88, FDRq = ~0.00 ) in D15 H. hammondi (Figure 4). This analysis suggests that transcriptional changes in spontaneously developing H. hammondi resemble some of the transcriptional changes observed in MORC depleted T. gondii. It would be interesting to further investigate if altering MORC levels could alter H. hammondi’s terminal differentiation phenotype. Despite having orthologs of all of these chromatin remodeling enzymes and histone variants, our understanding of their role in bradyzoite formation in H. hammondi and N. caninum is mostly unclear. Our transcriptional data from replicating H. hammondi show no signiﬁcant changes in the transcript abundance for any of the H. hammondi orthologs during spontaneous development (Figure 3). However, these ﬁndings could suggest that transcriptional abundance of these genes is needed similarly in all life stages and that the mechanisms controlling their translation, activation, or their recruitment to speciﬁc genes are important for initiating the tachyzoite to bradyzoite developmental transitions. Another histone remodeling enzyme found in T. gondii is GCN5-A, a lysine histone acetyltransferase. This enzyme plays an oppositional role to HDAC3. GCN5-A is found at tachyzoite promoters when T. gondii is grown under tachyzoite growth conditions (Saksouk et al., 2005). Additionally, ChIP qPCR experiments have shown that when T. gondii is exposed to December 2020 \| Volume 10 \| Article 608283 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Sokol-Borrelli et al. Stage Conversion in Coccidian Apicomplexans FIGURE 4 \| Spontaneously developing H. hammondi transcriptional abundance is enriched for genes regulated by MORC. Preranked GSEA, comparing D4 and D15 HhEth1, ranked listed was calculated from Log2 fold change between D4 and D15 spontaneous developing HhEth1 samples. The data used to generate this ﬁgure was obtained from (Sokol et al., 2018). Gene sets were created with data from (Farhat et al., 2020). FIGURE 4 \| Spontaneously developing H. hammondi transcriptional abundance is enriched for genes regulated by MORC. Preranked GSEA, comparing D4 and D15 HhEth1, ranked listed was calculated from Log2 fold change between D4 and D15 spontaneous developing HhEth1 samples. The data used to generate this ﬁgure was obtained from (Sokol et al., 2018). Gene sets were created with data from (Farhat et al., 2020). Transcription Factors Since AP2IX-9 is known to keep parasites in a pre- bradyzoite state, this data could indicate that D15 H. hammondi are being maintained in a pre-bradyzoite like state, prior to their complete terminal differentiation which is ﬁrst observed at D23 post sporozoite derived infection (Sokol et al., 2018). Another AP2 factor, AP2XII-2, has been shown to interact with the MORC protein and results in increased tissue cyst formation p p g In addition to AP2 transcription factors, T. gondii has additional transcription factors that are critical for stage conversion. One such transcription factor is Bradyzoite Formation Deﬁcient 1 (BFD1). BFD1 was recently identiﬁed as a master transcriptional regulator of bradyzoite development in T. gondii using a large-scale genetic screen. BFD1 is a nuclear localized Myb-like DNA binding protein that binds to the CACTGG motif near that transcriptional start site of differentially regulated genes. When BFD1 is deleted in T. gondii, these parasites fail to form tissue cysts in vitro in response to alkaline pH derived stress and treatment with Compound 1. These knockout parasites also fail to form tissue cysts in vivo. Furthermore, BFD1 knockout parasites fail to express several bradyzoite speciﬁc genes when treated with alkaline pH, suggesting that BFD1 plays a major role in initiating stage conversion associated gene expression (Waldman et al., 2020). Transcriptional abundance of the H. hammondi ortholog of BFD1 is modestly but signiﬁcantly upregulated (\|Log2 Fold Change\| = 1.47, Padj = <0.001) during December 2020 \| Volume 10 \| Article 608283 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 9 Stage Conversion in Coccidian Apicomplexans Sokol-Borrelli et al. TABLE 2 \| Summary of genes known to play a role in stage conversion in T. gondii. TABLE 2 \| Summary of genes known to play a role in stage conversion in T. gondii. Gene Name Role in stage conversion associated gene expression Gene ID T. gondii (from Toxodb.org) Gene ID H. hammondi ortholog (from Toxodb.org) Gene ID N. Transcription Factors Additional experiments investigating HhIF2a phosphorylation and HhIF2K-A activity during bradyzoite formation in H. hammondi would be helpful in identifying translation control as a conserved mechanism of stage conversion. spontaneous development (Figure 3), suggesting that increases in BFD1 expression in H. hammondi may play an important role in H. hammondi’s terminal differentiation phenotype. Additional T. gondii proteins that may play a role in stage conversion are the glycolytic enzymes, Enolase1 (ENO1) and Enolase 2 (ENO2). ENO1 is speciﬁcally expressed in bradyzoites and is a known bradyzoite speciﬁc gene. ENO2 is expressed in tachyzoites and can be found localized in the cytoplasm and nucleus (Ferguson et al., 2002). When ENO1 is deleted in T. gondii, the ability of the parasite to form in vivo cysts is impaired. Both ENO1 and ENO2 have been shown to bind promoters in ChIPseq and ChIP qPCR experiments and ENO1 has been shown to bind the ENO2 promoter. Furthermore, analysis of the ENO1 promoter revealed a stress response element that could be activated with nuclear extracts puriﬁed from bradyzoites (Kibe et al., 2005). Together, this data demonstrates a role as transcription factors implicated in stage conversion for ENO1 and ENO2. ENO1 is signiﬁcantly upregulated during spontaneous development in H. hammondi (Figure 3), however it is possible that this increase could also be due to ENO1’s metabolic role for bradyzoites. Transcription Factors caninum ortholog (from Toxodb.org) HDAC3 Represses transcription TgME49_227290 HHA_227290 NCLIV_045860 MORC Works with HDA3 and AP2 factors to repress transcription TgME49_305340 HHA_305340 NCLIV_043930 GCN5- A Activates transcriptions TgME49_254555 HHA_254555 NCLIV_008840 CARM1 Activity needed to maintain tachyzoites TgME49_294270 HHA_294270 NCLIV_001020 SCRAP Upregulates BAG1 expression TgME49_280800 HHA_280800 NCLIV_019390 RSC8 Regulates transcription of bradyzoite genes TgME49_286920 HHA_286920 NCLIV_013840 H2A.Z Expressed in bradyzoites; function in stage conversion is unknown TgME49_300200 HHA_300200 NCLIV_064530 H2AX Expressed in bradyzoites; function in stage conversion is unknown TgME49_261580 HHA_261580 NCLIV_025910 AP2XI-4 Promotes bradyzoite formation TGME49_315760 HHA_315760 NCLIV_058430 AP2IX-9 Maintains pre-bradyzoites TgME49_306620 HHA_306620 NCLIV_044800 AP2IX-4 Promotes bradyzoite formation TgME49_288950 HHA_288950 NCLIV_041340 AP2IV-4 Promotes bradyzoite formation TgME49_318470 HHA_318470 NCLIV_011080 AP2IV-3 Promotes bradyzoite formation TgME49_318610 HHA_318610 NCLIV_010930 AP2XII- 4 Interacts with MORC, maintains tachyzoites TgME49_217700 HHA_217700 NCLIV_062490 BFD1 Required for bradyzoite formation TgME49_200385 HHA_200385 NCLIV_038230 ENO1 Promotes bradyzoite formation TgME49_268860 HHA_268860 NCLIV_037490 ENO2 Expressed in tachyzoite nuclei, function in stage conversion is unknown TgME49_268850 HHA_268850 NCLIV_037500 IF2a Phosphorylated in bradyzoites, leads to increase in bradyzoite speciﬁc gene expression TgME49_258740 HHA_258740 NCLIV_027770 IF2K-A Phosphorylates IF2a, activity promotes bradyzoite formation TgME49_229630 HHA_229630 NCLIV_030460 Alba1 Promotes bradyzoite formation TgME49_221380 HHA_221380 NCLIV_004920 Alba2 Promotes bradyzoite formation TgME49_218820 HHA_218820 NCLIV_061560 Gene ID T. gondii (from Toxodb.org) Gene ID H. hammondi ortholog (from Toxodb.org) Gene ID N. caninum ortholog (from Toxodb.org) have been implicated in the conversion between tachyzoites and bradyzoites. Such factors include TgIF2a/eIF2 (eukaryotic initiation factor 2 alpha) and TgIF2K-A (initiation factor 2 kinase -A). TgIF2K-A is responsible for phosphorylating and activating TgIF2a, which functions as an inhibitor of translation initiation. TgIF2a is phosphorylated in response to stress derived from both alkaline pH and heat shock (Sullivan et al., 2004). TgIF2a phosphorylation is maintained in bradyzoites. When dephosphorylation of TgIF2a is inhibited, parasites both increase the transcriptional abundance of known bradyzoites genes and form DBA positive tissue cysts (Narasimhan et al., 2008). Furthermore, when the activity of TgIF2K-A is inhibited in T. gondii, thus preventing TgIF2a phosphorylation, these parasites form signiﬁcantly fewer tissue cysts when exposed to alkaline pH stress (Augusto et al., 2018). Our transcriptional data from replicating H. hammondi shows that transcript abundance of HhIF2K-A is signiﬁcantly upregulated during spontaneous development ((\|Log2 Fold Change\| = 1.22, Padj = <0.001) (Figure 3) (Sokol et al., 2018). This increase in transcriptional abundance could correlate to increased IF2a phosphorylation and translational inhibition in H. hammondi. REFERENCES Buxton, D., McAllister, M. M., and Dubey, J. P. (2002). The comparative pathogenesis of neosporosis. Trends Parasitol. 18, 546–552. doi: 10.1016/ s1471-4922(02)02414-5 Adomako-Ankomah, Y., Wier, G. M., Borges, A. L., Wand, H. E., and Boyle, J. P. (2014). Differential locus expansion distinguishes Toxoplasmatinae species and closely related strains of Toxoplasma gondii. MBio 5, e01003–e01013. doi: 10.1128/mBio.01003-13 CDC - Toxoplasmosis. Available at: https://www.cdc.gov/parasites/toxoplasmosis/ index.html (Accessed December 3, 2018). Chai, Z., Sarcevic, B., Mawson, A., and Toh, B. H. (2001). SET-related cell division Chai, Z., Sarcevic, B., Mawson, A., and Toh, B. H. (2001). SET-related cell division autoantigen-1 (CDA1) arrests cell growth. J. Biol. Chem. 276, 33665–33674. doi: 10.1074/jbc.M007681200 Augusto, L., Martynowicz, J., Staschke, K. A., Wek, R. C., and Sullivan, W. J. (2018). Effects of PERK eIF2a Kinase Inhibitor against Toxoplasma gondii. Antimicrob. Agents Chemother. 62 (11), e01442-18. doi: 10.1128/AAC.01442-18 autoantigen-1 (CDA1) arrests cell growth. J. Biol. Chem. 276, 33665–33674. doi: 10.1074/jbc.M007681200 Chen, L.-F., Han, X.-L., Li, F.-X., Yao, Y.-Y., Fang, J.-P., Liu, X.-J., et al. (2018). Comparative studies of Toxoplasma gondii transcriptomes: insights into stage conversion based on gene expression proﬁling and alternative splicing. Parasitol. Vectors 11, 402. doi: 10.1186/s13071-018-2983-5 Balaji, S., Babu, M. M., Iyer, L. M., and Aravind, L. (2005). Discovery of the principal speciﬁc transcription factors of Apicomplexa and their implication for the evolution of the AP2-integrase DNA binding domains. Nucleic Acids Res. 33, 3994–4006. doi: 10.1093/nar/gki709 Parasitol. Vectors 11, 402. doi: 10.1186/s13071-018-2983-5 Cole, R. A., Lindsay, D. S., Blagburn, B. L., Sorjonen, D. C., and Dubey, J. P. (1995). Vertical transmission of Neospora caninum in dogs. J. Parasitol. 81, 208–211. doi: 10.2307/3283921 Barber, J. S., and Trees, A. J. (1998). Naturally occurring vertical transmission of Neospora caninum in dogs. Int. J. Parasitol. 28, 57–64. doi: 10.1016/s0020- 7519(97)00171-9 Coombs, R. S., Blank, M. L., English, E. D., Adomako-Ankomah, Y., Urama, I.-C. S., Martin, A. T., et al. (2020). Immediate interferon gamma induction determines murine host compatibility differences between Toxoplasma gondii and Neospora caninum. Infect. Immun. 8 (4), e00027–20. doi: 10.1128/IAI.00027-20 Bohne, W., Heesemann, J., and Gross, U. (1993). Induction of bradyzoite-speciﬁc Toxoplasma gondii antigens in gamma interferon-treated mouse macrophages. Infect. Immun. 61, 1141–1145. doi: 10.1128/IAI.61.3.1141-1145.1993 aninum. Infect. Immun. 8 (4), e00027–20. doi: 10.1128/IAI.00027-2 Bohne, W., Heesemann, J., and Gross, U. (1994). Reduced replication of Toxoplasma gondii is necessary for induction of bradyzoite-speciﬁc antigens: a possible role for nitric oxide in triggering stage conversion. Infect. Immun. 62, 1761–1767. RNA-Binding Proteins While stage conversion is typically accompanied by global changes in transcript abundance in Apicomplexan parasites, translation control is an additional component needed for stage conversion associated gene expression. Several RNA binding proteins have been identiﬁed in T. gondii and some Additionally, T. gondii encodes two RNA binding proteins related to Alba proteins in archaea, TgAlba1 and TgAlba2. In response to alkaline pH derived stress, both TgAlba1 and December 2020 \| Volume 10 \| Article 608283 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 10 Stage Conversion in Coccidian Apicomplexans Sokol-Borrelli et al. gondii (summarized in Table 2), suggesting that they may also play a role in stage conversion in these species as well. However, only a few of these genes have altered transcriptional abundance in spontaneously differentiating H. hammondi. These observations from H. hammondi suggest that the mechanisms controlling stage conversion may function differently in H. hammondi and may rely on more than transcriptional regulation alone in order to induce bradyzoite development, which is not surprising given the importance of the tachyzoite to bradyzoite transition and the complexity of stage conversion associated gene expression. Together, these studies indicate that future work aimed at linking these factors in gene regulatory networks, in addition to identifying new factors, is needed to contribute to a better understanding of how these parasites initiate the global changes in stage conversion associated gene expression on a mechanistic level. TgAlba2 colocalize with RNA granules. These proteins bind greater than 30 RNAs, including their own. TgAlba1 binds to the promoter of TgAlba2. Upon deletion of TgAlba1, TgAlba2 is no longer translated. Furthermore, deletion of TgAlba1 in T. gondii also leads to decreased tissue cyst formation in vitro (in response to alkaline pH stress) and in vivo (Gissot et al., 2013). However, we do not see signiﬁcant changes in transcriptional abundance in the H. hammondi orthologs of TgAlba1 and TgAlba2 (Figure 3). Together, these examples of RNA binding proteins found in T. gondii demonstrate that translational control plays a critical role in stage conversion associated gene expression. FUNDING This work was supported by grants F31AI140529 to SS-B and R01AI116855 to JB. This work was supported by grants F31AI140529 to SS-B and R01AI116855 to JB. AUTHOR CONTRIBUTIONS SS-B, RC, and JB conceptualized, wrote, and edited this manuscript. All authors contributed to the article and approved the submitted version. CONCLUSIONS The tachyzoite to bradyzoite transition is a fundamental developmental process for the success of T. gondii and its closest relatives H. hammondi and N. caninum, as it is necessary for the survival and transmission of these parasite species. Some similarities in exogenous stressors that induce bradyzoite formation exist between species, indicating that there is some conservation in the way that these parasites sense stress. Moreover, the genetic components involved in the mechanisms governing stage conversion between tachyzoite and bradyzoite life stages are only starting to be uncovered for T. gondii, but so far include several factors that are needed for both transcriptional and translational control. Both H. hammondi and N. caninum have syntenic orthologs of all of these genetic components identiﬁed in T. REFERENCES History of the discovery of the life cycle of Toxoplasma gondii. Int. J. Parasitol. 39, 877–882. doi: 10.1016/j.ijpara.2009.01.005 Goodswen, S. J., Kennedy, P. J., and Ellis, J. T. (2013). A review of the infection, genetics, and evolution of Neospora caninum: from the past to the present. Infect. Genet. Evol. 13, 133–150. doi: 10.1016/j.meegid.2012.08.012 Dubey, J. P., and Ferguson, D. J. P. (2015). Life Cycle of Hammondia hammondi (Apicomplexa: Sarcocystidae) in Cats. J. Eukaryot. 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https://openalex.org/W2606971005	https://research.monash.edu/files/254719202/254656401_oa.pdf	English	null	Effect of a low intensity, self-management lifestyle intervention on knee pain in community-based young to middle-aged rural women: a cluster randomised controlled trial	Osteoarthritis and cartilage	2,017	cc-by	8,004	Effect of a low-intensity, self-management lifestyle intervention on knee pain in community-based young to middle-aged rural women: a cluster randomised controlled trial Yuanyuan Wang1* , Catherine Lombard2,3, Sultana Monira Hussain1, Cheryce Harrison2, Samantha Kozica2, Sharmayne R. E. Brady1, Helena Teede2,4† and Flavia M. Cicuttini1† Trial registration: ACTRN12612000115831, registered 24 January 2012. Trial registration: ACTRN12612000115831, registered 24 January 2012. Keywords: Randomised controlled trial, Lifestyle program, Knee pain, Rural women, Weight Abstract Background: Knee pain is common with obesity and weight gain being important risk factors. Previous clinical trials have focused on overweight or obese adults with knee pain and osteoarthritis and demonstrated modest effects of intense weight loss programs on reducing knee pain despite very significant weight loss. There has been no lifestyle intervention that targets community-based adults to test its effect on prevention of knee pain. We aimed to determine the effect of a simple low-intensity self-management lifestyle intervention (HeLP-her), proven in randomised controlled trials to improve lifestyle and prevent weight gain, on knee pain in community-based young to middle-aged rural women. Methods: A 1-year pragmatic, cluster randomised controlled trial was conducted in 649 community-based women (aged 18–50 years) to receive either the HeLP-her program (consisting of one group session, monthly SMS text messages, one phone coaching session, and a program manual) or one general women’s health education session. Secondary analyses were performed in 390 women who had knee pain measured using the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) at baseline and 12-month follow-up. “Any knee pain” was defined as a WOMAC pain score ≥1. Knee pain worsening was defined as an increase in WOMAC pain score over 12 months. Results: Thirty-five percent of women had “any knee pain” at baseline. The risk of knee pain worsening did not differ between the intervention and control groups over 12 months. For women with any knee pain at baseline, those in the intervention arm had a lower risk of knee pain worsening compared with those in the control arm (OR 0.37, 95% CI 0.14–1.01, p = 0.05), with a stronger effect observed in women with body mass index ≥25 kg/m2 (OR 0.28, 95% CI 0.09–0.87, p = 0.03). Conclusions: In community-based young to middle-aged women, a simple low-intensity lifestyle program reduced the risk of knee pain worsening in those with any knee pain at baseline, particularly in those overweight or obese. Pragmatic lifestyle programs such as HeLP-her may represent a feasible lifestyle intervention to reduce the burden of knee pain in the community. Wang et al. Arthritis Research & Therapy (2018) 20:74 https://doi.org/10.1186/s13075-018-1572-5 Wang et al. Arthritis Research & Therapy (2018) 20:74 https://doi.org/10.1186/s13075-018-1572-5 Open Access Background and long-term outcomes are often poor [12, 15]. There is an urgent and unmet need for a new approach aimed at preventing knee pain at a very early stage since there is evidence that any knee pain is a predictor of further episodes of knee pain [10]. Such a disease cycle is con- sistent with the causes of knee pain being multifactorial [3, 5] so that knee pain per se is also associated with weight gain and quadriceps muscle wasting [10, 16], with each also a risk factor for knee pain [6, 17]. This disease paradigm is also on the pathway to developing knee osteoarthritis [11]. Knee pain is a common problem which can be experi- enced by people of all ages, leading to physical disability and impaired quality of life [1, 2]. In the general popula- tion approximately one in five people report knee pain lasting at least 1 day during the past month [3]. In pri- mary care, the knee is the second most common individ- ual region of musculoskeletal pain, after the back, accounting for 10% of all musculoskeletal consultations [4]. Knee pain is more prevalent and severe in women than men [2]. The causes of knee pain are complex and multifactor- ial, with strong evidence suggesting that obesity is an important risk factor with a large population-attributable risk [3, 5]. We have shown in community-based adults that moderate weight gain (5%) was associated with de- velopment and worsening of knee pain [6]. Adults tend to gain weight progressively through middle age, with the modest accumulation of weight over time increasing obesity in the community [7]. For example, the average weight gain in the US population has been reported to be 0.45–0.9 kg [8] or 0.5–1.0 kg [7] per year based on data from different studies. In Australia, young and middle-aged women gain an average of 0.57 kg and 0. 5 kg weight per year, respectively [9]. Given the steady weight gain that is occurring in many countries and the role of weight gain in the development and worsening of knee pain, community interventions aimed at weight loss or preventing weight gain may have a role in redu- cing the prevalence and burden of knee pain. Moreover, early intervention for the prevention and treatment of knee pain is particularly important, as an episode of knee pain predicts future recurrence [10]. Background Given the role of weight gain in the development and worsening of knee pain [6], a low-intensity intervention aiming at preventing weight gain has the potential to be of benefit in reducing knee pain. There is some evidence that a healthy lifestyle (healthy diet and exercise) may help prevent excess weight gain and maintain body weight at healthy levels, potentially providing a feasible approach to preventing and treating early knee pain be- fore it becomes a major clinical problem [18]. However, there has been no lifestyle intervention that targets community-based adults to test its effect on prevention of knee pain. There is an urgent and unmet need for a simple low-intensity intervention for preventing weight gain and reducing knee pain along with other comorbid- ities, to be applied in community settings, with the aim of reducing long-time pain and disability. g g p y A low-intensity, self-management lifestyle intervention, the Healthy Lifestyle Program for women (HeLP-her), was designed to prevent weight gain based on the self- determination and social cognitive behavioural theory, with motivational interviewing the primary method of interaction with participants [19, 20]. The HeLP-her has been tested in two prior randomised controlled trials in reproductive aged women both showing efficacy in pre- vention of weight gain compared with controls who re- ceived general health information only [20, 21]. Our pragmatic, cluster randomised controlled trial showed that HeLP-her again prevented weight gain and improved diet quality and self-management behaviours over 1 year in community-based young to middle-aged women in rural settings (HeLP-her rural) [19]. The mean weight change was −0.48 kg in the intervention group and + 0.44 kg in the control group, with a between group difference −0. 92 kg [19]. The intervention group reported increased self-management strategy use related to diet and physical activity [19]. Therefore, the aim of the current knee pain substudy was to examine whether HeLP-her rural had an effect on reducing knee pain in community-based young to middle-aged women. The hypothesis was that the HeLP-her program would prevent knee pain in community-based women who were selected without ref- erence to knee symptoms, via its effect on preventing weight gain. We tested this hypothesis by examining the Obesity is a well-established major modifiable risk fac- tor for knee osteoarthritis [11]. * Correspondence: yuanyuan.wang@monash.edu † †Equal contributors 1Department of Epidemiology and Preventive Medicine, School of Public Health and Preventive Medicine, Monash University, 553 St Kilda Road, Melbourne, VIC 3004, Australia 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. Wang et al. Arthritis Research & Therapy (2018) 20:74 Page 2 of 9 Wang et al. Arthritis Research & Therapy (2018) 20:74 Page 2 of 9 Background The available evidence for weight loss is based on previous randomized con- trolled trials examining the effect of weight loss pro- grams on knee pain and structural progression in populations of obese adults with established symptom- atic knee osteoarthritis. Despite achieving substantial weight loss of 10% or approximately 10 kg, these trials have demonstrated modest effects on knee pain [12, 13] and no effect on structural progression of osteoarthritis [14]. For example, less than 40% of individuals reported no or little knee pain at the end of the trial, and only 14% of participants had a 50% reduction of their knee pain [12]. By the time an individual has symptomatic knee osteoarthritis, they are late in the trajectory from health through to end-stage joint disease where costly knee replacements are the only treatment option. Fur- thermore, these programs involve very intensive and complex interventions delivered by a multidisciplinary team of professionals to achieve significant weight loss, and therefore affordability and adherence are affected Wang et al. Arthritis Research & Therapy (2018) 20:74 Page 3 of 9 Page 3 of 9 facilitator-led 60-min interactive group session held with 8–15 women at community locations. The group session aimed to generate a shared understanding and knowledge of health, healthy eating behaviours and physical activity be- haviours. Simple messages regarding eating and physical ac- tivity behaviours provided an achievable context in which to begin to formulate their own priorities, rather than when or how to perform these behaviours. Facilitators using an interactive model and supported by the program manual, worked through examples of behavioural self-management skills including setting health priorities, problem solving, and self-monitoring, focusing on small changes to behav- iour. (ii) Week 2–4: women continue with program manual at home at own pace. The interactive program manual con- tained assessments, health information, personal stories, ac- tivities to challenge personal beliefs and behaviours and opportunities to develop self-management skills such as problem solving and action planning, and tools to self- monitor and assess progress that aimed to develop and im- prove skills in self-management. (iii) Weeks 2–52: monthly SMS text messages consistent with program messages to remind participants of key behaviours. (iv) Week 12: one 20-min phone coaching session based on motivation inter- viewing delivered by trained coaches and aimed to assist completion of manual activities and inforce intervention messages and generate action plans [19, 22]. Intervention The details of the 1-year self-management lifestyle inter- vention (HeLP-her) have been published previously [19, 22]. Its key features included community integration, non- prescriptive simple health messages, small changes to be- haviour, low participant burden, goal setting, self- monitoring including self-weighing, and delivery including a mix of a single face-to-face group session, one session of phone coaching, and mobile health with SMS text re- minders [19]. The program content is nonprescriptive; it provided general health messages and focused on small achievable changes around physical activity and eating to enhance self-efficacy and sustainability of behaviour change [22]. There was no reference to knee pain in the interven- tion. Briefly, the program consisted of (i) week 1: one At baseline and 1-year, weight was measured using cali- brated digital scales with participants in light clothing, with an empty bladder, and without shoes, and height was measured using a portable stadiometer. Body mass index (BMI, weight/height2, kg/m2) was calculated. Background All program activities focused on increasing awareness through personal stories, identifying personal barriers and enablers and de- veloping goals through activities and self-assessments de- signed to enhance intrinsic motivation and increase self- confidence [22]. Facilitators delivered the intervention in- cluding phone coaching after they had completed program- specific training. They were required to have a tertiary qualification in health sciences and undergo 1-day training [22]. The control group received one general women’s health education session based on national healthy diet and activity recommendations, held with 8–15 women at com- munity locations [19, 22]. effect of a sample, low-intensity intervention, which has shown an effect on weight gain prevention [19], on pre- vention of knee pain in community-based rural women. We selected rural women as they have a high prevalence of knee pain and reduced access to healthcare resources. Study design, participants, and randomisation Study design, participants, and randomisation A 1-year pragmatic, cluster randomised controlled trial was performed in 41 Australian towns (clusters) which were randomised using a computer-generated random- isation list for intervention (n = 21) or control towns (n = 20) [19]. Participants were recruited as clusters accord- ing to the town of residence between September 2012 and April 2013 [19]. Eligibility criteria included females, age 18–50 years, and residing in or near participating towns. Exclusion criteria were minimal and included pregnancy or serious medical conditions that would in- hibit full participation in the program [19]. There was no reference to musculoskeletal conditions, including knee pain, in the inclusion or exclusion criteria. Further details were described in the trial protocol [22] and the Consolidated Standards of Reporting Trials (CONSORT) flow diagram [19]. Due to the nature of the trial, re- searchers were aware of group allocation at baseline. Participants were not aware of group assignment, al- though they were aware that they were participating in a healthy lifestyle research program. At the 1-year data collection point, both participants and new field re- searchers were blinded to group allocation and previous anthropometric measures, with statistical analysis com- pleted by a blinded biostatistician [19]. The study was approved by the Monash Health Human Research Ethics Committee (Project No. 12034B). All participants gave written informed consent. The trial was registered with the Australian New Zealand Clinical Trials Registry (ACTRN12612000115831, registered 24 January 2012) prior to recruitment. Knee pain At baseline and 1-year follow-up, knee pain was assessed with the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) [23], using a Likert scale (0–4) for each of the five knee pain questions with the WOMAC knee pain score ranging from 0 to 20. Knee pain worsening was defined as any increase in WOMAC pain score from baseline to follow-up. In Wang et al. Arthritis Research & Therapy (2018) 20:74 Page 4 of 9 Page 4 of 9 women without knee pain at baseline (WOMAC pain score = 0), incidence of knee pain was defined if they de- veloped any knee pain at follow-up (WOMAC pain score ≥1). In those with any knee pain at baseline (WOMAC pain score ≥1), knee pain increasing was de- fined if their follow-up knee pain score was greater than the baseline measure, while knee pain improvement was defined if their follow-up knee pain score was less than the baseline measure. younger and had greater BMI and lower education level compared with those who provided the data, with no differ- ences observed for employment status and baseline knee pain between the two groups (Additional file 2: Table S2). p g p The effect of HeLP-her on knee pain change over 1 year is presented in Table 2. Worsening of knee pain was ob- served in 28 (13.6%) women in the intervention group and 33 (17.9%) women in the control group (p = 0.24). After adjusted for age, BMI, town cluster, and baseline knee pain, the risk of worsening knee pain did not differ be- tween the intervention and control groups (odds ratio [OR] 0.67, 95% CI 0.38–1.17, p = 0.16). There was evi- dence for a potential interaction between intervention and the presence of baseline knee pain for the association with worsening of knee pain (p = 0.12). For women with no knee pain at baseline, incident knee pain was observed in 20 (14.4%) women in the intervention group and 18 (15. 0%) women in the control group (p = 0.89); the risk of in- cident knee pain did not differ between the two groups after adjusted for age, BMI, and town cluster (OR 0.92, 95% CI 0.45–1.84, p = 0.81). Statistical analysis With 390 women who provided knee pain data at both baseline and 1-year follow-up, this study had 80% power to detect an absolute risk reduction of 10% in worsening of knee pain between intervention and control groups (10% vs. 20%) with 5% significance level. Baseline char- acteristics were compared using independent samples t test or Mann-Whitney U test for continuous variables where appropriate, and chi-squared test for categorical variables between the intervention and control groups. The effect of intervention on knee pain change out- comes (all dichotomous variables) was examined using chi-squared test and binary logistic regression adjusted for age, BMI, town cluster, and baseline knee pain. The interaction of intervention and the presence of baseline knee pain, and interaction of intervention and the base- line overweight status, for their association with knee pain worsening were examined. Subgroup analysis of women who were overweight or obese (BMI ≥25 kg/m2) at baseline was performed. The number needed to treat, i.e. the estimated number of people who need to be treated in order for one additional person to benefit, was calculated. P values of less than 0.05 (two-tailed) were considered statistically significant. All statistical analyses were performed using Stata (Intercooled Stata 12, StataCorp LP., College Station, TX, USA). Knee pain For women with knee pain at baseline, knee pain increasing was observed in 8 (11.9%) women in the intervention group and 15 (23.4%) women in the control group (p = 0.08), while knee pain improvement was observed in 43 (64.4%) women in the intervention group and 41 (64.1%) women in the control group (p = 0.99). After adjusted for age, BMI, town cluster, and baseline knee pain, women in the intervention group had a lower risk of knee pain increasing compared with those in the control group (OR 0.37, 95% CI 0.14–1.01, p = 0.05; number needed to treat nine), while the likelihood of knee pain im- provement did not differ between the two groups (OR 1.13, 95% CI 0.53–2.43, p = 0.75). When the data for the intervention and control arms were pooled and analysed, 38.7% of women had weight gain and 59.9% had weight loss. Weight gain was non- significantly associated with an increased risk of knee pain increasing (OR 1.15 for every 1 kg weight gain, 95% CI 0.99–1.34, p = 0.07), and weight loss was non- significantly associated with a reduced risk of knee pain increasing (OR 0.87 for every 1 kg weight loss, 95% CI 0.75–1.01, p = 0.07), adjusted for baseline age, height, weight, knee pain, and town cluster. Results The flowchart for the knee pain substudy is presented in Fig. 1. A total of 649 women were recruited for the original study, of whom 525 (81%) women had complete data for age, BMI, and WOMAC and were included in the current knee pain substudy. There were no significant differences in baseline characteristics between the intervention and control groups (Table 1). There were no significant differ- ences in baseline characteristics between those who were included in the current study and those who were not, ex- cept for a difference in BMI (Additional file 1: Table S1). There were 390 (74%) women who had knee pain data col- lected at 1 year. Of these 390, there were no significant dif- ferences in baseline characteristics between the intervention and control groups except for that women in the intervention group were older (p = 0.04) (Table 1). Those who did not provide 1-year knee pain data were There was evidence for a potential interaction between intervention and the overweight/obese status for the as- sociation with worsening of knee pain (p = 0.03). The ef- fect of HeLP-her on knee pain change over 1 year was further examined in the subgroup of women who were overweight or obese at baseline (n = 242) (Table 3). After adjustment for age, BMI, town cluster, and baseline knee pain, women in the intervention arm had a significantly lower risk of knee pain worsening (OR 0.45, 95% CI 0. 23–0.87, p = 0.02; number needed to treat eight) and Wang et al. Arthritis Research & Therapy (2018) 20:74 Page 5 of 9 Fig. p g y y cDifferences between intervention and control groups using independent samples t-test, chi squared test, or Mann-Whitney U test where appropriate dDefined as baseline WOMAC pain score (range 0–20) ≥1 aWomen with complete data at baseline for age, body mass index, town cluster, and WOMAC b te data at baseline for age, body mass index, town cluster, and WOMAC Results 1 Flowchart of the knee pain substudy Table 1 Characteristics of study participants Baseline characteristics Interventiona (n = 277) Controla (n = 248) P valuec Interventionb (n = 206) Controlb (n = 184) P valuec Age, years 40.0 (6.1) 39.0 (7.1) 0.11 40.7 (5.7) 39.4 (6.8) 0.04 Body mass index, kg/m2 28.5 (6.1) 28.1 (6.7) 0.50 28.4 (6.3) 27.2 (5.8) 0.06 Employment, n (%) 0.64 0.49 Full-time paid work 52 (19.0) 41 (16.7) 38 (18.5) 27 (14.8) Part time/casual work 144 (52.6) 139 (56.5) 110 (53.7) 108 (59.0) No paid work 78 (28.5) 66 (26.8) 57 (27.8) 48 (26.2) Education, n (%) 0.20 0.05 No post school qualification 42 (15.3) 50 (20.3) 29 (14.2) 38 (20.8) Certificate/diploma/apprenticeship 127 (46.2) 116 (47.2) 80 (39.2) 81 (44.3) Bachelor degree or higher 106 (38.6) 80 (32.5) 95 (46.6) 64 (35.0) WOMAC pain, median (range) 0 (0–20) 0 (0–15) 0.77 0 (0–20) 0 (0–15) 0.76 Any self-reported knee paind, n (%) 98 (35.4) 85 (34.3) 0.79 67 (32.5) 64 (34.8) 0.64 Data presented as mean (standard deviation), median (range), or n (%) aWomen with complete data at baseline for age, body mass index, town cluster, and WOMAC bWomen with complete data for age, body mass index, town cluster, and WOMAC at baseline, and WOMAC at 1 year cDifferences between intervention and control groups using independent samples t-test, chi squared test, or Mann-Whitney U test where appropriate dDefined as baseline WOMAC pain score (range 0–20) ≥1 Table 1 Characteristics of study participants Wang et al. Results Two randomised controlled trials of overweight or obese participants with osteoarthritis and knee pain demonstrated modest improvement in knee pain after very intensive weight loss programs resulting in 10% or approximately10 kg weight loss over 12–18 months [12, 13]. In overweight or obese adults with diabetes, an intensive lifestyle intervention which resulted in a knee pain increasing (OR 0.28, 95% CI 0.09–0.87, p = 0. 03; number needed to treat five) compared with those in the control arm, while the incidence (OR 0.68, 95% CI 0. 29–1.62, p = 0.39) or improvement (OR 2.16, 95% CI 0. 87–5.32, p = 0.10) of knee pain did not differ between the two groups. We found similar results for women who were overweight and those who were obese, but the results were less significant due to reduced sample size. For example of the effect of intervention on knee pain worsening with the same adjustment, the OR was 0.45 (95% CI 0.17–1.16, p = 0.10) for overweight women and 0.46 (95% CI 0.18–1.20, p = 0.11) for obese women. nine women with any knee pain needing to engage in the HeLP-her program to prevent one additional woman having an increase in knee pain, and only five over- weight/obese women with any knee pain need to engage in the HeLP-her program to prevent one additional woman having an increase in knee pain. The HeLP-her has been established with the aim to prevent weight gain, and tested in community-based young to middle- aged urban (community and antenatal populations and settings) and in rural women [19–21]. The HeLP-her program prevented weight gain with a mild effect on weight loss over 1 year where women in the control group gained weight, resulting in a between-group dif- ference in weight change of approximately 1 kg at 12 months [19, 20]. Obesity and weight gain are estab- lished risk factors for knee pain [3, 5, 6]. However the evidence for weight loss and reduced knee pain is lim- ited and largely in populations with established osteo- arthritis [12, 13, 18] or co-morbidities such as diabetes [24]. Two randomised controlled trials of overweight or obese participants with osteoarthritis and knee pain demonstrated modest improvement in knee pain after very intensive weight loss programs resulting in 10% or approximately10 kg weight loss over 12–18 months [12, 13]. In overweight or obese adults with diabetes, an intensive lifestyle intervention which resulted in a Results Arthritis Research & Therapy (2018) 20:74 Page 6 of 9 Table 2 Effect of intervention on change in knee pain over 1 year Intervention, n (%) Control, n (%) P valuea Odds ratio (95% CI)b P valueb Whole population, n = 390 n = 206 n = 184 Knee pain worsening 28 (13.6) 33 (17.9) 0.24 0.67 (0.38, 1.17) 0.16 Subgroup with no knee pain at baseline, n = 259 n = 139 n = 120 Incidence of knee pain 20 (14.4) 18 (15.0) 0.89 0.92 (0.45, 1.84) 0.81 Subgroup with knee pain at baseline, n = 131 n = 67 n = 64 Knee pain increasing 8 (11.9) 15 (23.4) 0.08 0.37 (0.14, 1.01) 0.05 Knee pain improvement 43 (64.2) 41 (64.1) 0.99 1.13 (0.53, 2.43) 0.75 aDifferences between intervention and control groups using chi squared test bLogistic regression, intervention vs control group, adjusted for age, body mass index, town cluster, and baseline WOMAC pain score Table 2 Effect of intervention on change in knee pain over 1 year aDifferences between intervention and control groups using chi squared test bLogistic regression, intervention vs control group, adjusted for age, body mass index, town cluster, and baseline WOMAC pain score nine women with any knee pain needing to engage in the HeLP-her program to prevent one additional woman having an increase in knee pain, and only five over- weight/obese women with any knee pain need to engage in the HeLP-her program to prevent one additional woman having an increase in knee pain. The HeLP-her has been established with the aim to prevent weight gain, and tested in community-based young to middle- aged urban (community and antenatal populations and settings) and in rural women [19–21]. The HeLP-her program prevented weight gain with a mild effect on weight loss over 1 year where women in the control group gained weight, resulting in a between-group dif- ference in weight change of approximately 1 kg at 12 months [19, 20]. Obesity and weight gain are estab- lished risk factors for knee pain [3, 5, 6]. However the evidence for weight loss and reduced knee pain is lim- ited and largely in populations with established osteo- arthritis [12, 13, 18] or co-morbidities such as diabetes [24]. bLogistic regression, intervention vs control group, adjusted for age, body mass index, town cluster, and baseline WOMAC pain score Discussion As a result, the adherence to intervention activities is often poor (approximately 50% for adherence to exercise and approximately 60% for adherence to diet intervention over 18 months) and long-term outcomes are disap- pointing [12, 15, 24]. In contrast, the simple low- intensity HeLP-her program targeted community-based women with the aim of preventing weight gain. While successfully preventing weight gain over 1 year, it was highly acceptable to participants and achievable with low participation burden and good retention at approxi- mately 80% [19, 25]. By performing a substudy with sec- ondary analysis on the original randomised controlled trial [19] with knee pain data prospectively collected using a validated tool, we found a favourable effect of the HeLP-her program on reducing the risk of knee pain worsening over 1 year in community-based young to middle-aged women selected without reference to knee pain, supporting its potential for knee pain prevention. behaviours. No component of the program targeted joint health or pain management. The effect of HeLP-her pro- gram on reducing knee pain is most likely via its benefi- cial effect on weight. There is no clear evidence suggesting an effect of leisure time physical activity on knee pain [27, 28]. Although the HeLP-her program showed a modest non-significant effect on leisure time activity and sitting time [19], any effect of the HeLP-her program on reducing knee pain by preventing inactivity is likely to be very small. Knee pain is a common musculoskeletal complaint, particularly in women [2] with no effective preventive treatments. Obesity is a major public health problem with its rate increasing globally and evidence of a link between excess weight and knee pain [18]. Given adults progressively gain weight, increasing the risk of knee pain and osteoarthritis and that knee pain episode pre- dicts future knee pain [6, 10], early interventions pre- venting weight gain and knee pain in the community are very important in order to reduce the burden of knee pain and disability. There is an unmet need for a simple intervention that targets prevention of both weight gain and knee pain in the community settings. Rural-dwelling women are disadvantaged with higher rates of weight gain and joint pain [29, 30]. The social inequality is reflected by their poorer health outcomes and limited access to health care. In our study of community-based young to middle-aged rural women, 35% reported any knee pain. Discussion The HeLP-her program is a simple low- intensity lifestyle intervention that is feasible to deliver, highly acceptable to participants, and requires few re- sources and health practitioner time, thus it is highly feasible to be implemented in the community settings. In a series of randomised controlled trials HeLP-her has been shown to prevent weight gain in young to middle- aged women [19–21], with extension out to 2 years and a health economic analysis of HeLP-her rural still under- way. Our study showed a favourable effect of this life- style program in the HeLP-her rural study on reducing the risk of knee pain worsening in community-based young to middle-aged women who were selected with- out reference to knee pain or disease. There is an urgent and unmet need for a new approach aimed at preventing knee pain since knee pain is a fluctuating condition and episodes of knee pain predict future knee pain both in terms of number of episodes and increasing severity [10]. Knee osteoarthritis is a major cause of pain, disabil- ity and healthcare costs, with no treatments that slow the disease progression. Knee pain is part of a disease cycle such that knee pain is associated with weight gain and quadriceps muscle weakness [10, 16], each in turn risk factors for knee pain [6, 17], structural knee damage, onset and progression of knee osteoarthritis [11], and so further pain and disability. Furthermore, once knee The study has limitations. Women who provided base- line knee pain data and were included in the current knee pain study had lower BMI than those who were not included; women who completed the 1-year follow- up of current knee study were older, higher educated, and had lower BMI than those who did not complete follow-up. The potential selection bias resulted in those of lower BMI tending to take part in this study so may have underestimated the effect of the interventions on knee pain. Data were not collected regarding work ab- sence or other metrics to illustrate the effect of the Help-her program on function and quality of life. The intervention was over 12 months. While the HeLP-her program showed a beneficial effect on reducing the risk of knee pain worsening at 1 year, its longer term effect will need to be determined. Discussion In community-based young to middle-aged rural women who were selected without reference to knee symptoms, a simple low-intensity lifestyle intervention (HeLP-her) reduced the risk of knee pain getting worse in those who had any knee pain, particularly in those who were over- weight or obese, although no effect of the HeLP-her life- style intervention was demonstrated on the overall knee pain change for the whole study sample. We found the HeLP-her lifestyle intervention reduced the risk of knee pain increasing in women who had any knee pain regardless of their BMI, with a stronger effect shown in overweight or obese women. This equated to ntion on change in knee pain over 1 year in women with baseline BMI ≥25 kg/m2 Table 3 Effect of intervention on change in knee pain over 1 year in women with baseline BMI ≥25 kg/m2 Intervention, n (%) Control, n (%) P valuea Odds ratio (95% CI)b P valueb Women who were overweight or obese, n = 242 n = 137 n = 105 Knee pain worsening 19 (13.9) 27 (25.7) 0.02 0.45 (0.23, 0.87) 0.02 Subgroup with no knee pain at baseline, n = 147 n = 85 n = 62 Incidence of knee pain 13 (15.3) 13 (21.0) 0.37 0.68 (0.29, 1.62) 0.39 Subgroup with knee pain at baseline, n = 95 n = 52 n = 43 Knee pain increasing 6 (11.5) 14 (32.6) 0.01 0.28 (0.09, 0.87) 0.03 Knee pain improvement 36 (69.2) 23 (53.5) 0.12 2.16 (0.87, 5.32) 0.10 aDifferences between intervention and control groups using chi squared test bLogistic regression, intervention vs control group, adjusted for age, body mass index, town cluster, and baseline WOMAC pain score Table 3 Effect of intervention on change in knee pain over 1 year in women with baseline BMI ≥25 kg/m2 Wang et al. Arthritis Research & Therapy (2018) 20:74 Page 7 of 9 Page 7 of 9 reduction of 3.1 kg/m2 in BMI on average over 12 months, reduced the risk of developing knee pain by 15% at 1 year compared with diabetes support and education, which led to an average of 0.2 kg/m2 reduction in BMI which attenuated at 4 years [24]. Such significant weight loss requires intensive, prolonged contact, supervised exercise and dietary modification delivered by a multidisciplinary team [12, 15]. Discussion The loss to follow-up of the current knee pain study was 26%, which was within the anticipated range and less than other lifestyle interven- tions [26]. The strengths of the trial include a community-based rural population which was selected without reference to the presence of knee symptoms, the pragmatic design with few exclusion criteria and high external validity, the large scale of the trial enabling valid subgroup analysis which identified a subgroup of women mostly likely to benefit from the HeLP-her program. The HeLP-her program targets multiple behaviours with the aim to prevent weight gain through its focus on achievable changes in healthy eating and physical activity Wang et al. Arthritis Research & Therapy (2018) 20:74 Page 8 of 9 Page 8 of 9 Wang et al. Arthritis Research & Therapy (2018) 20:74 Page 8 of 9 osteoarthritis is established, even major weight loss of over 10% did not slow the structural disease progression [14]. Thus intervening at an early stage of this cycle is important and likely to have a major effect on reducing long-term pain and disability as knee pain and each of the above consequences are associated with the develop- ment and progression of knee osteoarthritis [11]. It is well-established that increased weight is a risk factor for knee osteoarthritis [11] and that over midlife women tend to steadily gain weight [7]. In this study we showed that the HeLP-her had an effect on preventing knee pain in community-based women with low numbers needed to treat (from five to nine) in order to prevent one woman from having an increase in her knee pain, as de- scribed above. As there have been no previous lifestyle intervention programs that are effective in reducing knee pain in community-based population of individuals, the HeLP-her program integrated with a whole population approach may provide a novel, simple feasible strategy of early intervention for reducing the burden of knee pain in the community, especially where resources are limited in rural communities. design, collection, analysis and interpretation of data, preparation of the manuscript, or decision to submit the manuscript for publication. YW is the recipient of NHMRC Career Development Fellowship (Clinical level 1, APP1065464). SMH is the recipient of NHMRC Early Career Fellowship (APP1142198). CH is the recipient of a National Heart Foundation Fellowship. SREB is the recipient of an NHMRC Clinical Postgraduate Research Scholarship (APP1074979). Conclusions The authors declare that they have no competing interests. Our results showed that a simple low-intensity lifestyle program was able to reduce the risk of knee pain wors- ening in a general population of young to middle-aged rural women who had any knee pain, particularly in those overweight or obese. While the favourable effect of the program will need to be confirmed in other popula- tions and to be tested over a longer time period, prag- matic lifestyle programs such as HeLP-her may represent a novel, feasible lifestyle intervention to reduce the burden of knee pain in the community. Availability of data and materials All data generated or analysed during this study are included in this published article (and its Additional files). Received: 30 March 2017 Accepted: 19 March 2018 Received: 30 March 2017 Accepted: 19 March 2018 Discussion HT is the recipient of an NHMRC Practitioner Fellowship. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Consent for publication Not applicable. Consent for publication Not applicable. Abbreviations 3. Webb R, Brammah T, Lunt M, Urwin M, Allison T, Symmons D. Opportunities for prevention of ‘clinically significant’ knee pain: results from a population- based cross sectional survey. J Public Health (Oxf). 2004;26:277–84. 3. Webb R, Brammah T, Lunt M, Urwin M, Allison T, Symmons D. Opportunities for prevention of ‘clinically significant’ knee pain: results from a population- based cross sectional survey. J Public Health (Oxf). 2004;26:277–84. 4. Jordan KP, Kadam UT, Hayward R, Porcheret M, Young C, Croft P. Annual consultation prevalence of regional musculoskeletal problems in primary care: an observational study. BMC Musculoskelet Disord. 2010;11:144. 5. Jinks C, Jordan K, Croft P. Disabling knee pain–another consequence of obesity: results from a prospective cohort study. BMC Public Health. 2006;6:258. BMI: Body mass index; CI: Confidence interval; CONSORT: Consolidated Standards of Reporting Trials; HeLP-her: Healthy Lifestyle Program for women; OR: Odds ratio; WOMAC: Western Ontario and McMaster Universities Osteoarthritis Index 4. Jordan KP, Kadam UT, Hayward R, Porcheret M, Young C, Croft P. Annual consultation prevalence of regional musculoskeletal problems in primary care: an observational study. BMC Musculoskelet Disord. 2010;11:144. References Additional file 1: Characteristics of study participants, according to whether or not they had baseline knee pain data. (DOCX 18 kb) Additional file 1: Characteristics of study participants, according to whether or not they had baseline knee pain data. (DOCX 18 kb) 1. Ayis S, Dieppe P. The natural history of disability and its determinants in adults with lower limb musculoskeletal pain. J Rheumatol. 2009;36:583–91. 1. Ayis S, Dieppe P. The natural history of disability and its determinants in adults with lower limb musculoskeletal pain. J Rheumatol. 2009;36:583–91. Additional file 2: Characteristics of study participants with baseline knee data, according to whether or not they had knee pain data at 1 year follow-up. (DOCX 18 kb) Additional file 2: Characteristics of study participants with baseline knee data, according to whether or not they had knee pain data at 1 year follow-up. (DOCX 18 kb) 2. Urwin M, Symmons D, Allison T, Brammah T, Busby H, Roxby M, Simmons A, Williams G. Estimating the burden of musculoskeletal disorders in the community: the comparative prevalence of symptoms at different anatomical sites, and the relation to social deprivation. Ann Rheum Dis. 1998;57:649–55. Acknowledgements 5. Jinks C, Jordan K, Croft P. Disabling knee pain–another consequence of obesity: results from a prospective cohort study. BMC Public Health. 2006;6:258. We sincerely thank the HeLP-her participants and communities who took part in this trial. Nicole Ng for her support in trial delivery, Linda Downes for project management and field staff who conscientiously delivered and col- lected data in this complex trial. 6. Tanamas SK, Wluka AE, Davies-Tuck M, Wang Y, Strauss BJ, Proietto J, Dixon JB, Jones G, Forbes A, Cicuttini FM. Association of weight gain with incident knee pain, stiffness, and functional difficulties: A longitudinal study. Arthritis Care Res (Hoboken). 2013;65:34–43. Authors’ contributions CL and HT designed the study. CL, CH, and SK conducted the study. CL trained the facilitators to deliver the program. FMC and SREB contributed to knee pain data collection. YW and FMC analysed and interpreted the data and drafted the manuscript. CL, SMH, CH, SK, SREB, and HT contributed to the interpretation of results. All authors reviewed the manuscript for important intellectual content and approved the final manuscript. HT and FMC had equal contributions as senior authors. Ethics approval and consent to participate Ethics approval was obtained from the Monash Health Human Research Ethics Committee (Project No. 12034B). All participants gave written informed consent. Author details 1 1Department of Epidemiology and Preventive Medicine, School of Public Health and Preventive Medicine, Monash University, 553 St Kilda Road, Melbourne, VIC 3004, Australia. 2Monash Centre for Health Research and Implementation, Monash University, Melbourne, Australia. 3Department of Nutrition and Dietetics, Monash University, Melbourne, Australia. 4Diabetes and Vascular Medicine Unit, Monash Health, Melbourne, Australia. Funding Thi k g This work was funded by the National Health and Medical Research Council (NHMRC) Australia (Project Grant 1022951). The funder had no role in study 7. Williamson DF. Descriptive epidemiology of body weight and weight change in U.S. adults. Ann Intern Med. 1993;119:646–9. Page 9 of 9 Page 9 of 9 Page 9 of 9 Page 9 of 9 8. Hill JO, Wyatt HR, Reed GW, Peters JC. Obesity and the environment: where do we go from here? Science. 2003;299:853–5. 9. Women's Health Australia. Women's weight: Findings from the Australian Longitudinal Study on Women’s Health. Report prepared for the Australian Government Department of health and Ageing June 2007. http://www. alswh.org.au/images/content/pdf/major_reports/2007_major_report_b.pdf. Accessed 3 Apr 2018. 10. Soni A, Kiran A, Hart DJ, Leyland KM, Goulston L, Cooper C, Javaid MK, Spector TD, Arden NK. Prevalence of reported knee pain over twelve years in a community-based cohort. Arthritis Rheum. 2012;64:1145–52. 11. Felson DT, Lawrence RC, Dieppe PA, Hirsch R, Helmick CG, Jordan JM, Kington RS, Lane NE, Nevitt MC, Zhang Y, et al. Osteoarthritis: new insights. Part 1: the disease and its risk factors. Ann Intern Med. 2000;133:635–46. 12. 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Hall MC, Mockett SP, Doherty M. Relative impact of radiographic osteoarthritis and pain on quadriceps strength, proprioception, static postural sway and lower limb function. Ann Rheum Dis. 2006;65:865–70. 17. Glass NA, Torner JC, Frey Law LA, Wang K, Yang T, Nevitt MC, Felson DT, Lewis CE, Segal NA. The relationship between quadriceps muscle weakness and worsening of knee pain in the MOST cohort: a 5-year longitudinal study. Osteoarthr Cartil. 30. Aoyagi K, Ross PD, Huang C, Wasnich RD, Hayashi T, Takemoto T. Prevalence of joint pain is higher among women in rural Japan than urban Japanese- American women in Hawaii. Ann Rheum Dis. 1999;58:315–9. Wang et al. Arthritis Research & Therapy (2018) 20:74 28. Miranda H, Viikari-Juntura E, Martikainen R, Riihimaki H. A prospective study on knee pain and its risk factors. Osteoarthr Cartil. 2002;10:623–30. 28. Miranda H, Viikari-Juntura E, Martikainen R, Riihimaki H. A prospective study on knee pain and its risk factors. Osteoarthr Cartil. 2002;10:623–30. 29. Befort CA, Nazir N, Perri MG. Prevalence of obesity among adults from rural and urban areas of the United States: findings from NHANES (2005-2008). J Rural Health. 2012;28:392–7. 30. Aoyagi K, Ross PD, Huang C, Wasnich RD, Hayashi T, Takemoto T. Prevalence of joint pain is higher among women in rural Japan than urban Japanese- American women in Hawaii. Ann Rheum Dis. 1999;58:315–9. 28. Miranda H, Viikari Juntura E, Martikainen R, Riihimaki H. A prospective study on knee pain and its risk factors. Osteoarthr Cartil. 2002;10:623–30. 29. Befort CA, Nazir N, Perri MG. Prevalence of obesity among adults from rural and urban areas of the United States: findings from NHANES (2005-2008). J Rural Health. 2012;28:392–7. 29. Befort CA, Nazir N, Perri MG. Prevalence of obesity among adults from rural and urban areas of the United States: findings from NHANES (2005-2008). J Rural Health. 2012;28:392–7. Funding Thi k 2013;21:1154–9. 18. 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https://openalex.org/W3197665727	https://europepmc.org/articles/pmc8467707?pdf=render	English	null	New Insights into the Methylation of Mycobacterium tuberculosis Heparin Binding Hemagglutinin Adhesin Expressed in Rhodococcus erythropolis	Pathogens	2,021	cc-by	12,293	4 Department of Biochemistry and Structural Biology, Instituto de Fisiología Celular, Univers Autónoma de México, Ciudad de México 04510, Mexico; torres@ifc.unam.mx , , ; 5 Biotecnología Médica y Farmacéutica, Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, A.C., Guadalajara 44270, Mexico; mj.aceves.s@gmail.com (M.d.J.A.S.); ﬂoresv@ciatej.mx (M.A.F.V.) * Correspondence: espitia@iibiomedicas.unam.mx Citation: Parada, C.; Neri-Badillo, I.C.; Vallecillo, A.J.; Segura, E.; Silva-Miranda, M.; Guzmán-Gutiérrez, S.L.; Ortega, P.A.; Coronado-Aceves, E.W.; Cancino-Villeda, L.; Torres-Larios, A.; et al. New Insights into the Methylation of Mycobacterium tuberculosis Heparin Binding Hemagglutinin Adhesin Expressed in Rhodococcus erythropolis. Pathogens 2021, 10, 1139. https://doi.org/ 10.3390/pathogens10091139 A d i Edit R b t L H t Abstract: In recent years, knowledge of the role that protein methylation is playing on the phys- iopathogenesis of bacteria has grown. In Mycobacterium tuberculosis, methylation of the heparin binding hemagglutinin adhesin modulates the immune response, making this protein a subunit vaccine candidate. Through its C-terminal lysine-rich domain, this surface antigen interacts with hep- aran sulfate proteoglycans present in non-phagocytic cells, leading to extrapulmonary dissemination of the pathogen. In this study, the adhesin was expressed as a recombinant methylated protein in Rhodococcus erythropolis L88 and it was found associated to lipid droplets when bacteria were grown under nitrogen limitation. In order to delve into the role methylation could have in host–pathogen interactions, a comparative analysis was carried out between methylated and unmethylated protein produced in Escherichia coli. We found that methylation had an impact on lowering protein isoelectric point, but no differences between the proteins were found in their capacity to interact with heparin and A549 epithelial cells. An important ﬁnding was that HbhA is a Fatty Acid Binding Protein and differences in the conformational stability of the protein in complex with the fatty acid were observed between methylated and unmethylated protein. Together, these results suggest that the described role for this mycobacteria protein in lipid bodies formation could be related to its capacity to transport fatty acids. Obtained results also provide new clues about the role HbhA methylation could have in tuberculosis and point out the importance of having heterologous expression systems to obtain modiﬁed proteins. Received: 18 July 2021 Accepted: 30 August 2021 Published: 4 September 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Keywords: Mycobacterium tuberculosis; recombinant HbhA; Rhodococcus erythropolis methylation Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). New Insights into the Methylation of Mycobacterium tuberculosis Heparin Binding Hemagglutinin Adhesin Expressed in Rhodococcus erythropolis Cristina Parada 1, Isabel Cecilia Neri-Badillo 1, Antonio J. Vallecillo 1,2 , Erika Segura 1, Mayra Silva-Miranda 1,3, Silvia Laura Guzmán-Gutiérrez 1,3, Paola A. Ortega 1 , Enrique Wenceslao Coronado-Aceves 1, Laura Cancino-Villeda 1, Alfredo Torres-Larios 4 , Michel de Jesús Aceves Sánchez 5, Mario Alberto Flores Valdez 5 and Clara Espitia 1,* 1 Departamento de Inmunología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad de México 04510, Mexico; mariac@iibiomedicas.unam.mx (C.P.); cecineribadillo@hotmail.com (I.C.N.-B.); antonio.vallecillo@ucuenca.edu.ec (A.J.V.); eseguras@iibiomedicas.unam.mx (E.S.); msilvami@conacyt.mx (M.S.-M.); laura.guzman@iibiomedicas.unam.mx (S.L.G.-G.); paoandortega@hotmail.com (P.A.O.); wenceslao.coronadoa@iibiomedicas.unam.mx (E.W.C.-A.); anacv1009@gmail.com (L.C.-V.) 2 Facultad de Ciencias Agropecuarias, Universidad de Cuenca, Cuenca 010220, Ecuador 3 Consejo Nacional de Ciencia y Tecnología, CONACyT, Ciudad de México 03940, Mexico 4 Department of Biochemistry and Structural Biology, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad de México 04510, Mexico; torres@ifc.unam.mx 5 Biotecnología Médica y Farmacéutica, Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, A.C., Guadalajara 44270, Mexico; mj.aceves.s@gmail.com (M.d.J.A.S.); ﬂoresv@ciatej.mx (M.A.F.V.) * Correspondence: espitia@iibiomedicas.unam.mx 1 Departamento de Inmunología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad de México 04510, Mexico; mariac@iibiomedicas.unam.mx (C.P.); cecineribadillo@hotmail.com (I.C.N.-B.); antonio.vallecillo@ucuenca.edu.ec (A.J.V.); eseguras@iibiomedicas.unam.mx (E.S.); msilvami@conacyt.mx (M.S.-M.); laura.guzman@iibiomedicas.unam.mx (S.L.G.-G.); paoandortega@hotmail.com (P.A.O.); wenceslao.coronadoa@iibiomedicas.unam.mx (E.W.C.-A.); anacv1009@gmail.com (L.C.-V.) 1 Departamento de Inmunología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad de México 04510, Mexico; mariac@iibiomedicas.unam.mx (C.P.); cecineribadillo@hotmail.com (I.C.N.-B.); antonio.vallecillo@ucuenca.edu.ec (A.J.V.); eseguras@iibiomedicas.unam.mx (E.S.); msilvami@conacyt.mx (M.S.-M.); laura.guzman@iibiomedicas.unam.mx (S.L.G.-G.); paoandortega@hotmail.com (P.A.O.); wenceslao.coronadoa@iibiomedicas.unam.mx (E.W.C.-A.); anacv1009@gmail.com (L.C.-V.) g 2 Facultad de Ciencias Agropecuarias, Universidad de Cuenca, Cuenca 010220, Ecuador Citation: Parada, C.; Neri-Badillo, I.C.; Vallecillo, A.J.; Segura, E.; Silva-Miranda, M.; Guzmán-Gutiérrez, S.L.; Ortega, P.A.; Coronado-Aceves, E.W.; Cancino-Villeda, L.; Torres-Larios, A.; et al. New Insights into the Methylation of Mycobacterium tuberculosis Heparin Binding Hemagglutinin Adhesin Expressed in Rhodococcus erythropolis. Pathogens 2021, 10, 1139. https://doi.org/ 10.3390/pathogens10091139 Academic Editor: Robert L. Hunter Received: 18 July 2021 Accepted: 30 August 2021 Published: 4 September 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2021 by the authors pathogens pathogens pathogens pathogens 1. Introduction Methylation is a common post-translation modiﬁcation, occurring in a wide range of prokaryotic and eukaryotic proteins, e.g., cytochrome c, histones, actin, myosin, myelin, ﬂagella proteins, ribosomal proteins, heterogeneous nuclear ribonucleoproteins and trans- lation factors [1]. Methylation of non-histone prokaryotic proteins modulates diverse https://www.mdpi.com/journal/pathogens Pathogens 2021, 10, 1139. https://doi.org/10.3390/pathogens10091139 Pathogens 2021, 10, 1139 2 of 17 functions. For instance, pilin methylation of Synechocystis sp. regulates cell motility and methylation of EF-Tu factor in Escherichia coli decreases its GTPase activity, stimulating its dissociation from membrane [2]. Furthermore, trimethylated lysine residues of outer membrane protein B (OmpB) of Rickettsia typhi are associated with virulence, while protein from non-virulent strains contains mainly monomethyl lysine. In addition, methylation of OmpB is involved in immunogenicity in all rickettsia species, serving as a protective envelope that mediates host cell adhesion and invasion [3,4]. p Two adhesins of M. tuberculosis, the heparin binding hemagglutinin (HbhA, Rv0475) and the histone-like protein (Hlp/HupB, Rv2986), are molecules modiﬁed by methylation. At least 13 out of 16 lysine residues in the C-terminus of M. tuberculosis HbhA were shown to be mono or dimethylated [5,6], while in HupB, ﬁve lysine and two arginine residues are modiﬁed by acetylation or methylation [7]. On the other hand, methylation of HbhA is associated with the induction of a protective T cell immune response in mice [8]. In humans, a higher IFN-γ response to methylated protein was observed in individuals with latent tuberculosis, compared to the response of patients with active tuberculosis [9]. Even more, there is evidence that a methylated HbhA T cell peptide epitope is recognized by T cells from humans latently infected with M. tuberculosis as was evidenced by IFN- γ release upon peptide stimulation. The unmethylated peptide did not induce IFN-γ, arguing that the methyl lysine is part of the T cell epitope [10]. HbhA is also involved in the extrapulmonary dissemination of mycobacteria [11]. The protein interacts with epithelial cells through Syndecans, a protein family of four members (Sdc1–Sdc4) that possesses domains covalently attached to heparan sulfate chains [12]. Binding of HbhA to proteoglycans is mediated by the lysine-rich C-terminal, which contains the domain where methylation takes place, but the role of lysine methylation in this interaction is not completely understood. p y In this study, R. erythropolis, a bacterium that belongs to a genus genetically closely related to M. tuberculosis [13], was used as a surrogate host to express M. 1. Introduction tuberculosis HbhA. We were prompted to use this system due to the identiﬁcation of an HbhA or- thologous in Rhodococcus opacus PD630, the triacylglycerol accumulation deﬁcient protein (TadA) involved in the assembly and maturation of lipid droplets (LD) [14]. Later, it was demonstrated that through this protein, LD Rhodococcus jostii RHA1 bind to genomic DNA, increasing the survival rate of bacteria under nutritional and genotoxic stress [15]. More re- cently, the participation of mycobacteria HbhA in LD formation was demonstrated, as well as the capacity of HbhA to speciﬁcally binds to a eukaryotic lipid, 4,5 di-phosphorylated phosphatidylinositol [16]. Here, we report that recombinant HbhA expressed in R. erythro- polis was methylated, and importantly, as native mycobacterial HbhA, the recombinant in R. erythropolis was found associated to LD induced when recombinant bacteria were grown under nitrogen deprivation. Differences between methylated HbhA expressed in R. erythropolis and unmethylated expressed in E. coli were found, with regard to their isoelectric point (pI) and in their capacity to bind fatty acids (FA). Small differences in interaction of both methylated and unmethylated proteins with proteoglycans and with A549 epithelial cells were found. Together, the results contribute to our understanding of the putative role that methylation of HbhA could have in M. tuberculosis. This work points out R. erythropolis as a surrogate bacterium to express methylated M. tuberculosis proteins, providing a system to study their putative physiopathological roles. 2. Results 2.1. Expression and Puriﬁcation of M. tuberculosis HbhA from Recombinant R. erythropolis R. erythropolis L88 grown in Luria Bertani medium supplemented with Chlorampheni- col (LB/Chl) was transformed with pTip-QC1 with and without hbha insert. Recombinant extracts and puriﬁed recombinant HbhA were resolved by SDS-PAGE and transferred to PDVF membranes. Coomassie blue staining of whole sonicated extract (WSE) and insoluble fraction (IF) from transformed bacteria are shown in Figure 1a. As expected, no expression of rRhoHbhA was observed in the control without insert, in contrast with Pathogens 2021, 10, 1139 3 of 17 3 of 17 bands of ~28 kDa present in WSE and IF from bacteria transformed with the plasmid with insert (lanes 2 and 3, respectively). Unspeciﬁc reactivity was observed by Western blot (WB), with both rabbit anti-HbhA polyclonal antibody (R-anti-HbhA) and with anti-6xHis- tag-horseradish peroxidase (HRP) antibody (anti-His6-HRP) in WSE from negative control without insert. bands of ~28 kDa present in WSE and IF from bacteria transformed with the plasmid with insert (lanes 2 and 3, respectively). Unspeciﬁc reactivity was observed by Western blot (WB), with both rabbit anti-HbhA polyclonal antibody (R-anti-HbhA) and with anti-6xHis- tag-horseradish peroxidase (HRP) antibody (anti-His6-HRP) in WSE from negative control without insert. Figure 1. Expression of rRhoHbhA from R. erythropolis transformed with pTip-QC1-HbhA vector. (a) Coomassie blue staining of whole sonicated extract proteins from R. erythropolis L88 transformed with pTip QC1vector without insert (lane 1). Whole sonicated extract and insoluble fraction of R. erythropolis transformed with pTip-QC1-hbha (lanes 2 and 3). Negative controls: Western blot of whole sonicated extract from R erythropolis transformed with pTip QC1 without insert incubated Figure 1. Expression of rRhoHbhA from R. erythropolis transformed with pTip-QC1-HbhA vector. (a) Coomassie blue staining of whole sonicated extract proteins from R. erythropolis L88 transformed with pTip QC1vector without insert (lane 1). Whole sonicated extract and insoluble fraction of R. erythropolis transformed with pTip-QC1-hbha (lanes 2 and 3). Negative controls: Western blot of whole sonicated extract from R. erythropolis transformed with pTip-QC1 without insert, incubated with rabbit-anti-HbhA and anti-His6-HRP antibodies (lanes 4 and 5, respectively). (b) Puriﬁed rRhoHbhA (lane 1) and rE.coliHbhA (lane 2). Proteins were resolved by PAGE-SDS and stained with Coomassie blue. By Western blot, both proteins were detected with anti-His6-HRP and R-anti- HbhA antibodies, but only rRhoHbhA was recognized by R-anti-methylLys-HRP. 2. Results (c) Coomassie blue staining of soluble extract and cell wall proteins from rBCGHbhA transformed with pMV361 vector (lanes 1 and 2). Soluble extract and cell wall from rBCGHbhA proteins were detected by Western blot with R-anti-HbhA (lanes 3 and 4) and with R-anti-methylLys-HRP (lanes 5 and 6). Recombinant HbhA is indicated with an arrow. Figure 1. Expression of rRhoHbhA from R. erythropolis transformed with pTip-QC1-HbhA vector. (a) Coomassie blue staining of whole sonicated extract proteins from R. erythropolis L88 transformed with pTip QC1vector without insert (lane 1). Whole sonicated extract and insoluble fraction of R. erythropolis transformed with pTip-QC1-hbha (lanes 2 and 3). Negative controls: Western blot of whole sonicated extract from R. erythropolis transformed with pTip-QC1 without insert, incubated with rabbit-anti-HbhA and anti-His6-HRP antibodies (lanes 4 and 5, respectively). (b) Puriﬁed rRhoHbhA (lane 1) and rE.coliHbhA (lane 2). Proteins were resolved by PAGE-SDS and stained with Coomassie blue. By Western blot, both proteins were detected with anti-His6-HRP and R-anti- HbhA antibodies, but only rRhoHbhA was recognized by R-anti-methylLys-HRP. (c) Coomassie blue staining of soluble extract and cell wall proteins from rBCGHbhA transformed with pMV361 vector (lanes 1 and 2). Soluble extract and cell wall from rBCGHbhA proteins were detected by Western blot with R-anti-HbhA (lanes 3 and 4) and with R-anti-methylLys-HRP (lanes 5 and 6). Recombinant HbhA is indicated with an arrow. M. tuberculosis HbhA was also expressed in M. bovis BCG. Soluble extract (SE) and cell wall (CW) proteins were resolved by SDS-PAGE and transferred to PDVF membranes. Coomassie blue staining of WSE and SE are shown in Figure 1c. HbhA proteins were recognized by WB, with R-anti-HbhA. The presence of methyl groups was evidenced in both WSE and SE with R-anti-methylLys-HRP. 2.2. Molecular Mass Determination By mass spectrometry of rRhoHbhA, a major peak with a MM of 22,828 Da was observed. Removing the six-histidine tag and amino acids prom linker peptide adding by plasmid sequences, the MM should be 21,817.091 Da, which is very close to the 21,796 Da derived of mass spectrometry analysis of puriﬁed native HbhA. Comparing with the theoretical mass of the native protein of 21,475.39 Da, the estimated difference of 341 Da would correspond to 24.4 methyl groups, very similar to the value previously reported for methylated native M. tuberculosis HbhA [6]. 2.3. Isolation and Characterization of Lipid Droplets Induced in Recombinant R. erytropolis Strain Grown in M9 Medium with Low Nitrogen and Carbon Excess WB are shown in Figure 2a. 2.3. Isolation and Characterization of Lipid Droplets Induced in Recombinant R. erytropolis Strain Grown in M9 Medium with Low Nitrogen and Carbon Excess R. erythropolis recombinant strains rRhoHbhA, rRhoPstS1 and rRhoApa were grown under nitrogen starvation conditions. The IF from those strains were obtained to conﬁrm the expression of recombinant proteins from bacteria grown under this condition. Coomassie blue staining of IF extracts from recombinant strains resolved by SDS- PAGE, and recombinant proteins from the same extracts recognized by anti-His6-HRP by WB are shown in Figure 2a. Coomassie blue staining of IF extracts from recombinant strains resolved by SDS- PAGE, and recombinant proteins from the same extracts recognized by anti-His6-HRP by 4 of 17 Pathogens 2021, 10, 1139 Figure 2. Expression of M. tuberculosis HbhA, Apa and PstS-1 in R. erythropolis grown in M9GhiNlow (a) Coomassie blue staining of IF from rRhoHbhA (lane 1), rRhoApa (lane 2) and rRhoPstS-1 (lane 3). WB of the same proteins detected with anti-His6-HRP (lanes 4, 5 and 6). (b) Coomassie blue staining of proteins extracted from LD puriﬁed from rRhoHbhA (lane 1), rRhoApa (lane 2) and rRhoPstS-1 (lane 3). WB of the same proteins detected with anti-His6-HRP (lanes 4, 5 and 6). (c) TLC of LD extracted from rRhoHbhA strain, an arrow (line 2) indicates TAG. Glyceryl trioleate and glyceryl tripalmitate used as standards are shown in lane 1 and 3, respectively. Figure 2. Expression of M. tuberculosis HbhA, Apa and PstS-1 in R. erythropolis grown in M9GhiNlow (a) Coomassie blue staining of IF from rRhoHbhA (lane 1), rRhoApa (lane 2) and rRhoPstS-1 (lane 3). WB of the same proteins detected with anti-His6-HRP (lanes 4, 5 and 6). (b) Coomassie blue staining of proteins extracted from LD puriﬁed from rRhoHbhA (lane 1), rRhoApa (lane 2) and rRhoPstS-1 (lane 3). WB of the same proteins detected with anti-His6-HRP (lanes 4, 5 and 6). (c) TLC of LD extracted from rRhoHbhA strain, an arrow (line 2) indicates TAG. Glyceryl trioleate and glyceryl tripalmitate used as standards are shown in lane 1 and 3, respectively. Growing recombinant strains in M9 low nitrogen and carbon excess medium, supple- mented with 4% glucose and 0.15% of NH4SO2. (M9GHiNLow), induced the production of LD, which were puriﬁed by a sucrose gradient and treated to eliminate unspeciﬁc bound proteins. Lipids present in LD were solubilized and proteins were precipitated and separated by SDS-PAGE. Several protein bands from LD of recombinant rRhoHbhA, rRhoPsts, and rRhoApa between 50 and 25 kDa were observed by Coomassie blue-staining (Figure 2b). 2.3. Isolation and Characterization of Lipid Droplets Induced in Recombinant R. erytropolis Strain Grown in M9 Medium with Low Nitrogen and Carbon Excess After the extensive treatment to eliminate unspeciﬁc bound proteins, only rRhoHbhA was recognized by anti-His6-HRP by WB, indicating that the binding of HbhA to LD was highly speciﬁc (Figure 2b). Solubilized lipids present in LD were separated by thin-layer chromatography (TLC) (Figure 2c). This fraction contains triacylglycerol (indicated by an arrow), diacylglycerol, and free FA that occurred with other components (lane 2). Glyceryl trioleate and glyceryl tripalmitate were used as standards. It is worth pointing out that LD were only found in both wild-type and recombinant strains but only when bacteria were grown under nitrogen starvation conditions. 2.4. Characterization of Recombinant Proteins by 2-DE To study the pI pattern, puriﬁed rE.coliHbhA, rRhoHbhA and SE from rBCGHbhA were resolved by 2-DE in pH range between 3 to 10. Coomassie blue staining of rEcoliHbhA showed several spots concentrated in the basic pH region, many of them with lower molec- ular weights that migrated along the pH gradient, probably due to protein degradation. All the spots were recognized by R-anti-HbhA, but not by R-anti-methylLys-HRP, conﬁrming the absence of methylation of rHbHA expressed in E. coli (Figure 3a). y p g In contrast, when puriﬁed rRhoHbhA was resolved by 2-DE, spots were preferentially concentrated in the pH acid region, as shown by Coomassie blue staining. R-anti-HbhA recognized all the spots by WB, but R-anti-methylLys-HRP only recognized the spots with low pI, located in the acid pH region (Figure 3b). Since it is known that M. tuberculosis HbhA expressed in M. bovis BCG is methylated, we also studied the 2-DE pattern of the protein from SE of recombinant strain as a positive control of protein methylation. By WB, R-anti-HbhA recognized spots in both the acid and basic pH zone. However, R-anti-methylLys-HRP only recognized the spots located in the acid pH zone, conﬁrming that methylated rBCGHbhA also showed a low pI (Figure 3c). 5 of 17 Pathogens 2021, 10, 1139 Figure 3. Two-dimensional electrophoresis and SDS-PAGE. Recombinant puriﬁed rE.coliHbhA (a), rRhoHbhA (b) and SE from rBCGHbhA (c) are shown in each column. The ﬁrst row corresponds to Coomassie blue staining of the proteins, the second and third row show the recognition of the spots with R-anti-HbhA R-anti-methylLys-HRP antibodies by WB. Figure 3. Two-dimensional electrophoresis and SDS-PAGE. Recombinant puriﬁed rE.coliHbhA (a), rRhoHbhA (b) and SE from rBCGHbhA (c) are shown in each column. The ﬁrst row corresponds to Coomassie blue staining of the proteins, the second and third row show the recognition of the spots with R-anti-HbhA R-anti-methylLys-HRP antibodies by WB. 2.5. Binding of rE.coliHbhA and rRhoHbhA to Heparin Gray bars are controls without R-anti- HbhA and bar c indicates the control without rHbhA protein. The results represent 2 independent experiments. The ANOVA and Tukey’s statistical analysis did not show signiﬁcant differences. Figure 5. ELISA ligand assay. Interaction of recombinant rE.coliHbhA and rRhoHbhA with bi- otinylated heparin bound to streptavidin-coated plates. Binding was detected with R-anti-HbhA. Black bars represent the interaction signal towards heparin. Gray bars are controls without R-anti- HbhA and bar c indicates the control without rHbhA protein. The results represent 2 independent experiments. The ANOVA and Tukey’s statistical analysis did not show signiﬁcant differences. It is important to mention that although a higher reactivity with heparin of methylated rRhoHbhA was consistently observed in all the experiments we carried out, no signiﬁcant differences were found. 2.6. Binding of rE.coli HbhA and rRhoHbhA to A549 Cells and Post-Infection of Cells with BCG In order to study the interaction of rE.coliHbhA and rRhoHbhA with epithelial cells, both proteins were incubated with the cells and a pre-absorbed R-anti-HbhA was used to detect the proteins. The binding of both proteins to A549 as detected by ﬂuorescence microscopy was very similar, as shown in Figure 6a. Figure 6. Immunoﬂuorescence microscopy analysis. (a) Binding of puriﬁed rRhoHbhA and rE.coli HbhA to human A549 cells. Bound proteins were detected using a R-anti-rHbhA followed by incubation with anti-IgG coupled to Alexa Fluor 488 as secondary antibody. As negative control, cells without R-anti-HbhA were used. More than 200 ﬁelds were examined for each condition using the 100× magniﬁcation by ﬂuorescence microscopy. (b) Infection of A549 cells previously incubated with rE.coliHbhA and rRhoHbhA and then with M. bovis BCG; as negative control, the R-anti-rHbhA was omitted. Binding Data represent three independent experiments; asterisks describe statical signiﬁcance differences between M. bovis BCG control vs M. bovis BCG/rE.coliHbhA * (p = 0.0457) and M. bovis BCG vs M. bovis BCG/rRhoHbhA ** (p = 0.0127) by one-way ANOVA. Figure 6. Immunoﬂuorescence microscopy analysis. (a) Binding of puriﬁed rRhoHbhA and rE.coli HbhA to human A549 cells. Bound proteins were detected using a R-anti-rHbhA followed by incubation with anti-IgG coupled to Alexa Fluor 488 as secondary antibody. As negative control, cells without R-anti-HbhA were used. More than 200 ﬁelds were examined for each condition using the 100× magniﬁcation by ﬂuorescence microscopy. (b) Infection of A549 cells previously incubated with rE.coliHbhA and rRhoHbhA and then with M. 2.5. Binding of rE.coliHbhA and rRhoHbhA to Heparin The measure of afﬁnity of biotinylated heparin for recombinant proteins rE.coliHbhA and rRhoHbhA by biolayer interferometry was performance. The measure of afﬁnity of biotinylated heparin for recombinant proteins rE.coliHbhA and rRhoHbhA by biolayer interferometry was performance. y y y p Recombinants HbhA interacted with heparin in a concentration-dependent manner and allowed us to calculate a dissociation constant value of 0.28 µM and 0.62 µM for the unmethylated rE.coliHbhA and methylated rRhoHbhA, respectively (Figure 4). Figure 4. Real-time biolayer interferometry sensorgrams for determination of binding afﬁnity (a,c) Sensorgrams showing binding on streptavidin biosensors of biotinylated heparin with increased concentrations of recombinants HbhA. (b,c) Steady- state analysis of the binding response (nm) as a function of the concentration of HhbA. Calculated KD = 0.28 µM (R2 = 0.92) and 0.62 µM (R2 = 0.95) for the non-methylated rE.coliHbhA (a,b) and methylated rRhoHbhA (c,d). Figure 4. Real-time biolayer interferometry sensorgrams for determination of binding afﬁnity (a,c) Sensorgrams showing binding on streptavidin biosensors of biotinylated heparin with increased concentrations of recombinants HbhA. (b,c) Steady- state analysis of the binding response (nm) as a function of the concentration of HhbA. Calculated KD = 0.28 µM (R2 = 0.92) and 0.62 µM (R2 = 0.95) for the non-methylated rE.coliHbhA (a,b) and methylated rRhoHbhA (c,d). Two reference controls were used, one that did not include the protein, only biotiny- lated heparin, and another that used biotinylated BSA instead of biotinylated heparin. No binding of biotinylated heparin to the biosensor or binding to biotinylated BSA to HbhA was detected. The speciﬁcity of rE.coliHbhA and rRhoHbhA to heparin was also studied by an enzyme-linked immunosorbent assay (ELISA). The results are shown in Figure 5, with both unmethylated rE.coliHbhA and methylated rRhoHbhA (black bars) bound to heparin. 6 of 17 Pathogens 2021, 10, 1139 Figure 5. ELISA ligand assay. Interaction of recombinant rE.coliHbhA and rRhoHbhA with bi- otinylated heparin bound to streptavidin-coated plates. Binding was detected with R-anti-HbhA. Black bars represent the interaction signal towards heparin. Gray bars are controls without R-anti- HbhA and bar c indicates the control without rHbhA protein. The results represent 2 independent experiments. The ANOVA and Tukey’s statistical analysis did not show signiﬁcant differences. Figure 5. ELISA ligand assay. Interaction of recombinant rE.coliHbhA and rRhoHbhA with bi- otinylated heparin bound to streptavidin-coated plates. Binding was detected with R-anti-HbhA. Black bars represent the interaction signal towards heparin. 2.7. rE.coli HbhA and rRhoHbhA Lipid-Binding Assays Degradation pattern and stability of apo- and holo-forms of recombinant rE.coliHbhA and rRhoHbhA, after incubation with and without Glu C for 15 and 120 min. (a,c) SDS-PAGE, gels were stained with Coomassie blue. Lane 1 (c), control protein without any treatment, only one sample was running for both treatment times. Line 2, proteins incubated at 37 ◦C for 15 and 120 min. Line 3, with oleic acid (W/OA), proteins were incubated with oleic acid at RT for 30 min and then at 37 ◦C for 15 and 120 min. Line 4, (W/protease), proteins incubated only with Glu C at 37 ◦C for 15 and 120 min. Line 5, (W/ OA+ protease), proteins incubated with oleic acid, and Glu C at 37 ◦C for 15 and 120 min. (b,d) Each protein band was analyzed by using ImageJ software. The results represent 2 independent experiments. Signiﬁcant differences: * (p < 0.05), (p < 0.005) and ** ( p < 0.0001). Figure 7. Lipid-binding assay of puriﬁed soluble rHbhA. Degradation pattern and stability of apo- and holo-forms of recombinant rE.coliHbhA and rRhoHbhA, after incubation with and without Glu C for 15 and 120 min. (a,c) SDS-PAGE, gels were stained with Coomassie blue. Lane 1 (c), control protein without any treatment, only one sample was running for both treatment times. Line 2, proteins incubated at 37 ◦C for 15 and 120 min. Line 3, with oleic acid (W/OA), proteins were incubated with oleic acid at RT for 30 min and then at 37 ◦C for 15 and 120 min. Line 4, (W/protease), proteins incubated only with Glu C at 37 ◦C for 15 and 120 min. Line 5, (W/ OA+ protease), proteins incubated with oleic acid, and Glu C at 37 ◦C for 15 and 120 min. (b,d) Each protein band was analyzed by using ImageJ software. The results represent 2 independent experiments. Signiﬁcant differences: * (p < 0.05), (p < 0.005) and ** ( p < 0.0001). Samples collected at different times of protease exposition were analyzed by SDS- PAGE. Proteins incubated or not with SA were rapidly degraded by exposition to Glu C independently of treatment, suggesting that in the solution, rE.coliHbhA and rRhoHbhA did not bind to SA (data not shown). In contrast, speciﬁc binding of both recombinant proteins was observed with OA. 2.5. Binding of rE.coliHbhA and rRhoHbhA to Heparin bovis BCG; as negative control, the R-anti-rHbhA was omitted. Binding Data represent three independent experiments; asterisks describe statical signiﬁcance differences between M. bovis BCG control vs M. bovis BCG/rE.coliHbhA * (p = 0.0457) and M. bovis BCG vs M. bovis BCG/rRhoHbhA ** (p = 0.0127) by one-way ANOVA. Pathogens 2021, 10, 1139 7 of 17 7 of 17 In order to ﬁnd out if the presence of either rE.coliHbhA or rRhoHbhA could have an effect on the internalization of M. bovis BCG, epithelial cells pre-incubated with puriﬁed recombinant proteins were infected. The results in Figure 6b show that there were no differences in the number of M. bovis BCG internalized when A549 cells were pre-incubated with the proteins, but there was a signiﬁcant inhibition of bacterial entrance with regard to the control without pre-incubation. 2.7. rE.coli HbhA and rRhoHbhA Lipid-Binding Assays 2.7. rE.coli HbhA and rRhoHbhA Lipid-Binding Assays As FA are important constituents of triacylglycerol (TAG), they were used as targets for rHbhA. The interaction of rE.coliHbhA and rRhoHbhA with FA was evaluated by a dot lipid-binding assay. TAG and FA were spotted on PDVF membranes, and stained with phosphomolybdic acid. Glyceryl tristearate and palmitic acid (PA) were not stained and were excluded. From lipids tested for binding to rHbhA, only stearic acid (SA) interacted with both proteins and rE.coliHbhA only bound to oleic acid (OA), as shown in Figure S1. p y ( ) g We also studied the binding to FA of soluble puriﬁed rE.coliHbhA and rRhoHbhA by using a lipid-binding assay that involved the exposition of soluble recombinant pro- teins and their complexes formed with SA and OA to endoproteinase Glu C, for 15 and 120 min (Figure 7). The band in lane 1 of Figure 7a,c is the same for both treatment times. This band was superimposed in the experiment performed at 120 min, in order to have the same reference of HbhA protein concentration to be analyzed with ImageJ software (https://imagej.nih.gov/ij/, accessed on 12 June 2021) (Figure 7b,d). Figure 7. Lipid-binding assay of puriﬁed soluble rHbhA. Degradation pattern and stability of apo- and holo-forms of recombinant rE.coliHbhA and rRhoHbhA, after incubation with and without Glu C for 15 and 120 min. (a,c) SDS-PAGE, gels were stained with Coomassie blue. Lane 1 (c), control protein without any treatment, only one sample was running for both treatment times. Line 2, proteins incubated at 37 ◦C for 15 and 120 min. Line 3, with oleic acid (W/OA), proteins were incubated with oleic acid at RT for 30 min and then at 37 ◦C for 15 and 120 min. Line 4, (W/protease), proteins incubated only with Glu C at 37 ◦C for 15 and 120 min. Line 5, (W/ OA+ protease), proteins incubated with oleic acid, and Glu C at 37 ◦C for 15 and 120 min. (b,d) Each protein band was analyzed by using ImageJ software. The results represent 2 independent experiments. Signiﬁcant differences: * (p < 0.05), (p < 0.005) and ** ( p < 0.0001). binding assay of puriﬁed soluble rHbhA. Degradation pattern and stability of apo- and holo-forms of Figure 7. Lipid-binding assay of puriﬁed soluble rHbhA. Degradation pattern and stability of apo- an Figure 7. Lipid-binding assay of puriﬁed soluble rHbhA. 3. Discussion Lysine methylation of histones in eukaryotes has been considered as one of the most important post-translational modiﬁcations, being methylation an epigenetic mark essential for gene regulation and development. Although lysine methylation of non-histone proteins in bacteria is restricted to very few molecules, this modiﬁcation can play different physio- logical roles [17]. Even more, methylation of some bacterial proteins is directly related to their immunogenicity and virulence [3,4,8]. g y In mycobacteria, Hlp/HupB is post-translationally modiﬁed by lysine acetylation and lysine methylation. Mutation of lysine 86 results in the speciﬁc loss of the ability of Mycobacterium smegmatis to survive when exposed to isoniazid [7]. On the other hand, M. tuberculosis HbhA possesses a C-terminal moiety that com- promises a heparin-binding domain consisting of lysine-rich repeated motifs that are involved in the ability of bacteria to invade host cells [11]. This domain undergoes mono or di-methylation, containing 20–26 methyl groups on residues 159–199 [6]. Although methylation of HbhA had been associated with the induction of both T and B cell re- sponses [8,18–20], the physiopathological role of this modiﬁcation on the host–pathogen relationship in tuberculosis is unknown. p In this work, M. tuberculosis hbha gene was expressed in E. coli and R. erythropolis, and the latter bacteria were used as alternative host for expression of modiﬁed HbhA, based on a previous work in our laboratory where M. tuberculosis proteins modiﬁed by glycosylation were produced in R. erythropolis [21]. The rRhoHbhA was expressed as methylated protein in R. erythropolis and the presence of methyl groups was evidenced with a R-methylLys-HRP antibody and by mass spectrometry of puriﬁed recombinant protein, which showed an increase in MM that could corresponded with the presence of methyl groups. The expression of methylated M. tuberculosis HbhA protein in R. erythropolis offers some advantages, such as optimization of production time and high level of expression, almost ﬁve times higher than the recombinant expressed in M. smegmatis [22]. Although it is well known that HbhA is a mycobacterial adhesin that binds to heparan sulfate-containing proteoglycans [5,23], the role of methylation in this interaction has not been established. Previous studies identiﬁed the proteoglycans Sdc4 and Sdc1 present in epithelial cells as ligands for HbhA. 2.7. rE.coli HbhA and rRhoHbhA Lipid-Binding Assays The protector effect against Glu C activity was clearly seen in the complexes formed between rE.coliHbhA or rRhoHbhA with OA as shown in Pathogens 2021, 10, 1139 8 of 17 8 of 17 Figure 7 (lane 5). Signiﬁcant differences in protein degradation were observed between the proteins without treatment and those previously incubated with OA. Figure 7 (lane 5). Signiﬁcant differences in protein degradation were observed between the proteins without treatment and those previously incubated with OA. Interestingly, stability of the apo-forms of both proteins seems to be affected by the incubation at 37 ◦C. While the holo-forms of methylated rRhoHbhA with OA remain relatively stable during the times of treatment independently of the exposition to Glu C, both unmethylated and methylated apo-forms were degraded by activity of the protease. 3. Discussion Surface Plasmon resonance analyses showed that native HbhA expressed in BCG immobilized to Syndecan 1 and 4 showed a KD in the micromolar range (KD = 1.4 × 10−5 Sdc1; KD = 1 × 10−5 Sdc4), suggesting low-afﬁnity interactions between HbhA and both Syndecans [12]. In order to deepen this knowledge, we carried out a comparative ELISA ligand assay using rE.coliHbhA and rRhoHbhA and heparin bound to streptavidin-coated plates. No differences in heparin binding were found between the unmethylated and methylated proteins. In addition, the afﬁnity for heparin of both unmethylated and methylated rHbhA was also determined by BBI; the afﬁnities were in the micromolar range (KD = 0.28 µM rE.coliHbhA and KD = 0.68 µM rRhoHbhA) with no signiﬁcant differences between the proteins. Although in this work, small differences were found respect to protein interaction with proteoglycans between methylated and unmethylated proteins, the effect of those apparent small changes could be signiﬁcant in molecular interactions. In this line, we studied the entrance of M. bovis BCG to A549 epithelial cells previously incubated with either unmethylated or methylated proteins, and no differences were found. However, a signiﬁcant inhibition of bacterial entrance was observed with regard to the control cells without preincubation with recombinant proteins. Together, these Pathogens 2021, 10, 1139 9 of 17 results suggest that the interaction of HbhA with epithelial cells seems to be independent of methylation. results suggest that the interaction of HbhA with epithelial cells seems to be independent of methylation. y In this work, rRhoHbhA was also found in LD induced when recombinant R. ery- thropolis was grown in nitrogen-limited conditions, conﬁrming previous observations demonstrating that HbhA from M. tuberculosis was able to bind to LD of the recombinant M. bovis BCG strain and R. opacous PD63 [16,24]. The localization of this protein in LD had been associated with the assembling and maturation of those structures [14,16]. Biosyn- thesis and accumulation of TAG and other neutral lipids in LD seems to be a common feature of bacteria belonging to the actinomycetes group, such as Streptomyces, Rhodococcus and Mycobacterium [25]. M. tuberculosis stores FA in the form of TAG under carbon excess and nitrogen starvation [26] and during non-replicative states, such as hypoxia-induced dormancy [27]. LD have also been found in M. tuberculosis isolated from the sputum of tu- berculosis patients, highlighting the importance of TAG during infection [28]. Importantly, M. 3. Discussion tuberculosis acquires FA from the human host, which bacteria use to synthesize TAG, and subsequently stored in the form of LD [29]. Since the pI is a powerful tool to predict and understand interactions between proteins or proteins and membranes [30], we analyzed this biochemical marker by 2D-E in un- methylated and methylated recombinant proteins. The results were interesting: rRhoHbhA showed several spots distributed along the pH gradient, but the majority of them were concentrated in the acid pH region while the spots of unmethylated rE.coliHbhA that mi- grated to the basic pH region, showing lower molecular weight, which could be the result of proteolytic degradation, as has been previously observed for unmethylated HbhA [6]. In fact, the pI of 9.17 predicted for HbhA (ExPASy) corresponds to the pI experimentally determined for native HbhA identiﬁed by 2-DE from M. tuberculosis extract, obtained from bacteria grown under high iron condition, where this basic spot was identiﬁed as HbhA by N-terminal sequence [31]. The presence of methylated spots with low pI in rRhoHbhA and rBCGHbhA was also conﬁrmed by the recognition of the methyl groups with R-anti- methylLys-HRP. The biochemical consequence of the decreased of pI in methylated HbhA is unknown. It has been reported that one of the most notable characteristics of methylation of some enzymes, such as cytochrome C, is accompanied by lowering the pI. The authors put forward the hypothesis that an altered conformation of apocytochrome c induced by enzymatic methylation would favor its interaction with the receptor on mitochondrial membrane. Those changes only occur in proteins that are transported to intracellular sites, such as cytochrome C [32]. The ﬁnding of a putative lipid domain, predicted on the basis of primary amino acid sequence and structure similarity with apolipoprotein E2 (PBD:1NFO) and the ﬁnding that methylated HbhA bound to 4,5 di-phosphorylated phosphatidylinosi- tol [16], prompted us to use FA as the main constituents of TAG as targets for HbhA. The results showed that by dot lipid-binding assay, both rE.coliHbhA and rRhoHbhA bound to SA, but only rE.coliHbhA was able to bind to OA. Moreover, by using a lipid-binding assay with the soluble forms of both recombinant proteins, it was shown by SDS-PAGE that both proteins bound speciﬁcally to OA and the resultant holo-forms were more stable and protected from the enzyme activity. However, higher stability of the complex was observed with methylated protein. 3. Discussion Our results suggest that reduction of the pI of HbhA due to methylation could increase the afﬁnity of the protein by FA through an unknown mechanism that would favor a conformational change and stabilization of the protein after its interaction with the FA, allowing them to be safely delivered to LD in beneﬁt of the bacteria. Further quantitative studies will be necessary to evaluate the role of methylation in stabilization of FA–protein complex and its relationship with the low pI. Together, these results are showing for the ﬁrst time that HbhA is a fatty acid binding protein (FABP) that could have an important role in the transport of these molecules to my- cobacteria lipids storage compartments. FABP are intracellular molecules mainly identiﬁed in eukaryotes. In prokaryotes, they are present only within the order of Actinomycetales. In M. tuberculosis, Rv0813c protein was identiﬁed as a FABP, on the grounds of its crystal Pathogens 2021, 10, 1139 10 of 17 10 of 17 structure. These proteins in bacteria may have roles in the recognition, transport and/or storage of small molecules [33]. structure. These proteins in bacteria may have roles in the recognition, transport and/or storage of small molecules [33]. The ﬁndings obtained in this paper conﬁrm the complexity of HbhA as a moonlighting protein that serves several functions depending on its location [16], and probably on the biochemical changes, such as methylation, that could contribute to the functional complexity of the protein. It is worth mentioning that this protein has also been associated with the induction of endoplasmic reticulum stress-mediated apoptosis through the generation of reactive oxy- gen species and cytosolic Ca2ﬂin M. tuberculosis infected macrophages. The experiments reported were carried out with a recombinant M. tuberculosis HbhA methylated expressed in M. smegmatis [34]. Since the role of HbHA methylation in ROS generation is unknown, comparative assays with unmethylated protein would be important to deﬁne a putative role of this modiﬁcation in apoptosis and ROS generation. Furthermore, it is important to mention that although M. tuberculosis HbhA has a relevant role in infection and virulence, this protein is also present in non-pathogenic mycobacteria and in actinobacteria. 3. Discussion Based on phylogenetic analysis that showed that some regulatory sequences are present only in slow-growing mycobacteria, it has been proposed that the hbha gene underwent functional divergence during its evolutionary differentiation, acquiring a new function related to virulence and invasion of host but preserving its ancestral LD binding function, as well as its FA binding capacity, as we found in this work. In this line, and taking together these observations and our results, it is tempting to propose that methylation of HbhA could be another evolutionary acquisition of my- cobacteria that would allow this protein to have more efﬁcient binding to FA, ensuring its transport to cope with mycobacteria nutritional stress and survival. Although the differences found in this work regarding protein interaction with proteo- glycans and FA between methylated and unmethylated proteins are low, the effect of these apparent small changes in molecular interactions needs to be addressed in further studies. pp g Furthermore, our results are showing for the ﬁrst time that, as occurs in mycobacteria, Rhodococcus can also methylate proteins, pointing out that the heterologous production of the recombinant methylated HbhA of M. tuberculosis in R. erytropolis opens a door to study the role that methylation of this protein has in host–pathogen interaction in tuberculosis. 4.1. Bacterial Strains E. coli TOP10F’, DH5-α, Rosetta (DE3) (Novagen Inc., Madison, WI, USA), R. erythropo- lis strain L88 and Mycobacterium bovis BCG Pasteur were used for cloning and expression of hbha gene. E. coli strains and R. erythropolis were grown in LB medium (Difco, Sparks, MD, USA). For E. coli, medium was supplemented with 100 µg mL−1 of carbenicillin (Invitrogen, Carlsbad, CA USA) (LB/Car) and for R. erythropolis with 34 µg mL−1 of Chl (Sigma Aldrich, St. Louis, MO, USA). For the induction of LD, R. erythropolis recombinants strains were grown in M9GHiNLow [35]. 4.2. Cloning of M. tuberculosis HbhA Protein in E. coli and R. erythropolis 4.2. Cloning of M. tuberculosis HbhA Protein in E. coli and R. erythropolis The coding region of the hbhA gene was ampliﬁed by PCR with high ﬁdelity DNA polymerase Pfx (Invitrogen) from M. tuberculosis H37Rv genomic DNA with the oligonu- cleotide primers hbhA 15bF (5′-GCATATGGCTGAAAACTCGAACATTG-3′, NdeI site underlined) and hbhA 15bR (5′-CGGATCCTACTTCTGGGTGACCTTCTTGG-3′, BamHI site underlined). The PCR product (600 bp) was cloned into the pCR4 Blunt-TOPO vector (Invitrogen) with the use of TOP10F’ strain. Vector was digested with NdeI and BamHI and the released fragment was gel-puriﬁed and ligated into the NdeI and BamHI sites of pET15b (Novagen) and into the thiostrepton (Sigma) inducible pTip-QC1vector (kindly donated by Tomohiro Tamura National Institute of Advanced Industrial Science and Tech- Pathogens 2021, 10, 1139 11 of 17 11 of 17 nology, Japan) to generate pET15b-HbhA and pTip-QC1-HbhA, respectively. The identities and orientation of the inserts were conﬁrmed by restriction analysis and DNA sequencing. nology, Japan) to generate pET15b-HbhA and pTip-QC1-HbhA, respectively. The identities and orientation of the inserts were conﬁrmed by restriction analysis and DNA sequencing. 4.3. Expression of M. tuberculosis HbhA in E. coli, Puriﬁcation as N-Terminal His-Tagged Protein 4.3. Expression of M. tuberculosis HbhA in E. coli, Puriﬁcation as N-Terminal His-Tagged Protein E. coli Rosetta (DE3) was heat shock-transformed with pET15b-HbhA. A single recom- binant colony was grown in LB/Car overnight (ON) at 37 ◦C with shaking (200 rpm). The next day, ON culture was diluted 1/100 in LB/Car and incubated at 37 ◦C with shaking until OD600nm reached ~0.4. Then, culture was induced with 250 µM (ﬁnal concentration) of Isopropyl -β-D-1-galactopyranoside (IPTG) (Roche., Applied Science, Mannheim, Ger- many) and kept for 4 h at 37 ◦C. Recombinant Histidine-tagged HbhA (rE.coliHbhA) was puriﬁed from inclusion bodies (IB) by Nickel chromatography, as previously indicated [36]. 4.4. Expression of M. tuberculosis HbhA in R. erythropolis, Puriﬁcation as N-Terminal His-Tagged Protein pTip-QC1-HbhA was used to transform R. erythropolis L88 strain. Transformation, induction, expression and lysis were carried out as described before [21]. Brieﬂy, after growth in LB/Chl for 48 h at 26 ◦C, cells were harvested by centrifugation, re-suspended in lysis buffer (50 mM Tris–HCl, 50 mM NaCl, pH 8.0) and disrupted by sonication during 8 cycles of 1 min of activity and 2 min of rest for 2 h in ice, at 70% of sonicator (Sonics Vibro-cell, Newtown, CN, USA) potency, to obtain the WSE. 4.2. Cloning of M. tuberculosis HbhA Protein in E. coli and R. erythropolis After centrifugation of this extract, the pellet containing IF was washed twice with 1% Triton X-100 in phosphate buffered saline (PBS) and once with PBS. After that, the sample was solubilized in sample buffer (100 mM Tris-HCl, 50 mM NaCl, 8 M urea, pH 8) and the mixture was stirred ON at room temperature (RT). Protein was puriﬁed in an AKTA FPLC system (GE Healthcare Biosciences, Pittsburgh, PA, USA) using a 1 ml HistrapMT column (GE Healthcare), as has been previously described [19]. After washing and equilibration of Ni-NTA column with sample buffer, the sample was loaded and the column was washed with buffer (100 mM Tris-HCl, 50 mM NaCl, 25 mM Imidazole, urea 8 M, pH 8). Then, the protein was eluted with elution buffer (100 mM Tris-HCl, 50 mM NaCl, 500 mM Imidazole, urea 8 M, pH 8.0) at 1 mL/min, by using a gradient (0% to 100% of elution buffer). Fractions displaying the recombinant proteins were pooled and dialyzed against decreasing urea concentrations (from 4, 2, 1 and 0 M). The amount of protein was determined with BCA protein assay kit (Thermo Fisher, Waltham, MA, USA) Proteins were stored at −70 ◦C until needed. In all the steps, the presence of puriﬁed recombinant proteins was monitored by SDS-PAGE. 4.7. Expression of HbhA in R. erythropolis Grown in Low Nitrogen and Carbon Excess Identiﬁcation of Protein in Puriﬁed Lipid Bodies 4.7. Expression of HbhA in R. erythropolis Grown in Low Nitrogen and Carbon Excess Identiﬁcation of Protein in Puriﬁed Lipid Bodies rRhoHbhA and M. tuberculosis recombinant glycoproteins PstS-1 (rRhoPstS-1) and Apa (rRhoApa) previously expressed in R. erythropolis [21] were grown in LB/Chl for 48 h at 26 ◦C, then cells were harvested by centrifugation and for the induction of LD, they were re-suspended in M9GHiNLow. Cultures were induced with 1 µg mL−1 thiostrepton and bacteria were grown at 26 ◦C for an additional 96 h. After culture centrifugation, cell pellets were either treated as described above to obtain the IF or re-suspended in 25 mM Tricine, 250 mM sucrose, pH 7.8 for isolation of LD by ultracentrifugation, as has been previously described elsewhere, with some modiﬁcations [37]. Puriﬁed LD were then submitted to different treatments in order to eliminate proteins non-speciﬁcally bound [38]. After that, LD were treated with 1 ml of chloroform–acetone (Sigma) (1:1 v/v) to dissolve lipids and precipitate proteins. The sample was centrifuged at 20,000× g for 15 min at 4 ◦C. Supernatant fractions were dried, and lipids dissolved in chloroform were analyzed by TLC on silica gel 60 plates (Alugram® Xtra SIL G/UV254, Macherey-Nagel, Germany) applying the solvent system Hexane/Diethyl ether/Acetic acid 80:20:1. Lipid fractions were visualized by iodine vapor [39]. In this assay, glyceryl trioleate and glyceryl tripalmitate were used as standards. LD proteins and the IF initially obtained were loaded in 12% SDS-PAGE and transferred to PDVF membranes. Membranes were stained with Coomassie blue and proteins were identiﬁed by WB with anti-His6-HRP (Roche) as described below. 4.8. SDS-PAGE and Two-Dimensional Polyacrylamide Gel Electrophoresis 4.5. Molecular Mass Analysis of rRhoHbhA by MALDI-TOF The molecular mass (MM) of rRhoHbhA was determined by MALDI-TOF. A total of 0.8 g of protein was mixed with sinapinic acid saturated with dH2O 60%, Acetonitrile 40% and 0.1% triﬂuoroacetic acid. Mass detection was carried at 1000 to 100,000 Da intervals in microﬂex® LRF, nitrogen laser spectrometer (Bruker Daltonics, Bremen, Germany) at 337 nm. The parameters used were positive linear mode, 20 KV ionization force and 600 shots. 4.6. Cloning and Expression of M. tuberculosis HbhA Protein in Mycobacterium bovis BCG Pasteur The hbhA gene was ampliﬁed from M. tuberculosis H37Rv genomic DNA, using Pfu DNA polymerase (Promega., Madison, WC, USA) with primers hbhA-5F (5′- GGAGAATT CATGGCAGAAAACTC-3′, EcoRI site in underlined) and hbhA-3R (5′-AGCAATACGAGC ATGACGGT-3′). The 734 bp PCR product was digested with EcoRI and SalI, ligated into the pMV361 vector digested at the same sites. Ligation reactions were transformed into E. coli DH5-α and selected in LB with kanamycin (Kan) (MP Biomedicals, Irvine, CA, USA) at 50 µg mL−1. pMV361-based plasmids containing hbhA were isolated and veriﬁed by sequencing, then vector was used to transformed by electroporation into M. bovis BCG Pasteur and selected on 7H10/OADC (Difco) agar with 0.5% glycerol and 25 g mL−1 of Kan, for 3 weeks at 37 ◦C. After that, the recombinant mycobacteria were grown in 7H9/ADC (Difco) Kan Pathogens 2021, 10, 1139 12 of 17 12 of 17 20 g mL−1 for 28 days. Bacteria were harvested by centrifugation, washed with cold PBS, re-suspended in lysis buffer (50 mM Tris-HCL, 1 mM EDTA, pH 8.0) and disrupted by sonication at 30% of potency, with intervals of 1 min of activity and 1 min of rest for 2 h in ice. Then, the sample was centrifuged at 20,000× g for 20 min at 4 ◦C. The SE was ﬁltered by 0.45 µm and 0.22 µm ﬁlter (Corning, Camarillo, CA, USA). To obtain the CW, pellet was washed with PBS, re-suspended and incubated for 2 h at 50 ◦C in PBS with 2% of SDS. The sample was centrifuged at 20,000× g for 15 min at 4 ◦C. Supernatant was ﬁltered by as described above and proteins were quantiﬁed and stored until their use. 4.9. Binding of rE.coli HbhA and rRhoHbhA to Heparin The binding kinetics and the determination of the dissociation constant (KD) for the rE.coliHbhA and rRhoHbhA against biotinylated heparin (Sigma) was carried out by using Biolayer Interferometry (BLI) at 25 ◦C. Streptavidin biosensors in an Octet RED96 system (FortéBio Inc., San Jose, CA, USA) were used. The assays were performed on black bottom 96-well microplates (Greiner Bio-One 655209) (Thermo Fisher, Waltham, MA, USA) in a total volume of 200 µL with orbital shaking. Experiments were controlled with the software Data Acquisition 8.2 (ForteBio, Inc., San Jose, CA, USA). For the BLI experiment, a baseline was established using 10× Kinetics buffer (FortéBio Inc., San Jose, CA, USA) diluted to 1× in PBS buffer. Then, the biotinylated heparin at 10 µg mL−1 was allowed to bind to streptavidin sensor for 5 min, followed by washing with the same buffer to eliminate nonspeciﬁc binding. Next, the rE.coliHbhA and/or rRhoHbhA were bound to the bound heparin (association). In the last step, a wash (dissociation) was made. The BLI experiment was conducted with ﬁve different concentrations of both recombinant proteins. New streptavidin biosensors were used for each experiment. The data were processed using the Octet Data Analysis Software version 8.2 (FortéBio Inc., San Jose, CA, USA). ELISA was also carried out to evaluate the binding of recombinant proteins to heparin, 0.5 µg/well of biotinylated heparin in PBS was immobilized on streptavidin-coated plates (Thermo Scientiﬁc). Samples were incubated ON at 4 ◦C. The next day, plates were washed twice with PBS-T and then incubated 1 h at RT with 5 µg/well of rE.coliHbhA or rRhoHbhA diluted in PBS-T/BSA. After 3 washes with PBS-T, wells were incubated for 1 h with R-anti-HbhA diluted 1:2000 and then with Protein A-HRP diluted 1:2000 in PBS-T/BSA. The reaction was revealed with TMB (3,3′,5,5′-tetramethylbenzidine) (Thermo Fisher, Waltham, MA, USA) and stopped with 100 µL of 0.16 M H2SO4. Absorbance values were measured at OD450nm using an ELISA plate reader (Multiskan Go., Thermo Scientiﬁc, Ratastie I, Finland). 4.8. SDS-PAGE and Two-Dimensional Polyacrylamide Gel Electrophoresis Approximately 100 µg of SE from rBCGHbhA and ~50 µg of recombinant puriﬁed proteins were applied on Immobiline DryStrip pH 3–10, 7 cm linear-gradient strips (GE healthcare) for 16 h to dehydrate at room temperature, following the manufacturer’s instructions. Focusing was performed in an Ettan IPGphor 3 Isoelectric Focusing System (GE healthcare) starting at 500 V (for 5 h), increasing potential to 4500 V (90 min) and ﬁnally to 14,000 V. After focusing, the strips were equilibrated for 20 min in sample buffer (2% SDS, 50 mM Tris-HCl pH 8.8, 6 M urea, 30% glycerol, 0.002% bromophenol blue, 0.5% DTT). The strips were then loaded onto 12% SDS-PAGE. After electrophoresis, gels were transferred to PVDF membrane stained with Coomassie Brilliant Blue and for antibody detection, membranes were blocked for 1 h with PBS-BSA, and then incubated with R-anti-HbhA or with R-methylLys-HRP and developed as described above. 4.8. SDS-PAGE and Two-Dimensional Polyacrylamide Gel Electrophoresis Ten µg of recombinant extracts, WSE and IF from rRhoHbhA, SE and CW from rBCGHbhA and 5 µg of puriﬁed rE.coliHbhA and rRhoHbhA from IB and IF, respectively were separated by a 12% SDS-PAGE and transferred to PVDF membranes (Millipore, Burlington, MS USA) that were stained with Coomassie R-250 (Bio-Rad Laboratories, Hercules, CA, USA). For WB, membranes were blocked for 1 h with 3% bovine serum albumin (BSA) (Sigma) in PBS (PBS-BSA) at RT. After that, membranes were incubated 1 h with rabbit anti-HbhA polyclonal antibody (R-anti-HbhA) diluted 1:2000 or with anti-His6- HRP diluted 1:2000 in PBS containing 0.05% Tween 20 (PBS-T). After 3 washes with PBS-T, membranes were incubated 1 h with 1:2000 dilution of Protein A-HRP (Invitrogen), washed with PBS-T, and developed with 3 g mL−1 of 3,3-diaminobenzidine (DAB) (Sigma) in PBS and 30% hydrogen peroxide (Merck., Darmstadt, Alemania) diluted 1:1000. For detection of methylated lysine, membranes were blocked with 1% skim milk in PBS for 3 h at RT with a wash step with PBS-T, then incubated with rabbit anti-mono/dimethyl lysine horseradish peroxidase antibody (Abcam., Cambridge, UK) (R-methylLys-HRP) diluted 1:1000 in Tris buffer saline (TBS) (50 mM Tris, 150 mM NaCl) with 1% skim milk, ON at 23 ◦C with shaking. The next day, membranes were washed twice with TBS containing 0.05% Tween 20 and once with only TBS. Buffer was drained and membranes were incubated for a few seconds with SuperSignal West Pico chemiluminescent substrate (Thermo Scientiﬁc, Waltham, MA, USA) and developed by using a C-DiGit Blot Scanner (Li-COR, Lincoln, NE, USA). For 2D-PAGE analysis, isoelectric focusing was performed as described elsewhere, with some modiﬁcations [40]. SE from rBCGHbhA and puriﬁed rRhoHbhA and rE.coliHbhA from IF and IB, respectively, were desalted in an Illustra NAP 5 column (GE healthcare). Pathogens 2021, 10, 1139 13 of 17 13 of 17 Proteins were then concentrated by ﬁltration and precipitated by adding 2% sodium deoxycholate to a ﬁnal 0.02% concentration, 100% trichloroacetic acid to a ﬁnal 10% and 200 µL of ice-cold acetone. Protein pellets were re-suspended, and the ﬁnal volume was adjusted to 125 µL with rehydration buffer (8 M urea, 2% CHAPS, 0.5% IPG buffer pH 4–7, and 20 mM DTT). 4.10. Binding of rE.coli HbhA and rRhoHbhA to A549 Epithelial Cells To assess the binding of HbhA protein on cells surface, an ex vivo assay was performed using the human lung pneumocyte A549 cell line. The cells were cultured in RPMI 1640 medium, (Gibco, Grand Island, NB, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco) in 75 cm2 Falcon culture ﬂasks (Corning) under standard culture conditions (5% CO2 and 37 ◦C). Then, cells were collected by centrifugation and the viability was determined by Trypan blue exclusion. The incubation with HbhA proteins was performed as described elsewhere [21] with some changes; the cells were seeded at 1.5 × 103 per well in 8-well plates. (Nunc Lab-Tek, Greensburg, PA, USA) in 150 L of RPMI 10% FBS and incubated at 37 ◦C, 5% CO2, for 18 h. After 2 washes with RPMI free of FBS, cells were incubated with rE.coliHbhA and rRhoHbhA proteins at 26.8 g/L × 106 cells for Pathogens 2021, 10, 1139 14 of 17 14 of 17 2 h at 37 ◦C with 200 µL of RPMI supplemented with FBS. After 3 washes with PBS-T, cells were ﬁxed for 10 min with 1% Paraformaldehyde, washed 3 times with PBS-T, blocked with PBS-BSA at RT with shaking, and after washing with PBS-T, cells were incubated for 1 h with pre-adsorbed R-anti-HbhA. (R-anti-HbhA serum diluted 1/500 was adsorbed by incubation with 1.5 × 103 A549 cells previously adhered to a slide for 1 h at 37 ◦C at 5% CO2). After 4 washes with PBS-T, cells were incubated with anti-rabbit-AF488 (Invitrogen ) 1/2000 in PBS-BSA for 45 min in agitation and after 3 washes with PBS- T, nuclei were stained with Hoechst (Life Technologies., Bleiswijk, Netherland) 1/9000 for 10 min, then after 3 washes with con PBS-T, cells were covered with a coverslide using vectashield (Vector Laboratories, Burlingame, CA, USA). Fluorescence images were acquired with Olympus BX41 (Fluorescence-microscopy) (Olympus Corporation of the Americas Headquarters, Center Valley, PA, USA) with the 100× magnifying lens, using the program Zen blue 2.6 (Carl Zeiss GmbH, Promenade, Jena, Germany) for capturing digital images. 4.11. M. bovis BCG Infection of A549 Cells Previously Incubated with rHbhA Proteins For infection, A549 cells were pre-incubated with rE.coliHbhA and rRhoHbhA 10 g/L × 106 cells for 2 h at 37 ◦C, 5% CO2. After that, cells were washed twice with fresh medium and infected with M. 4.10. Binding of rE.coli HbhA and rRhoHbhA to A549 Epithelial Cells bovis BCG Danish strain (MOI 3:1) for 1 h in RPMI without FBS and antibiotics. Then, cells were washed 3 times with RPMI and incubated for 1 h in RPMI supplemented with amikacin at 200 µg mL−1. Finally, cells were washed and lysed with 100 µL of 0.05% SDS for 1 min and neutralized with 4% BSA. Lysed cells were plated in 7H11/OADC medium. After 21 days of incubation, colony-forming units (CFU) were determined. The assays were performed 3 times. 4.12. Fatty Acid Binding Assays 4.12. Fatty Acid Binding Assays Dot lipid-binding assay was carried out as described elsewhere [16], with some modiﬁcations. Brieﬂy, glyceryl tristearate, glyceryl trioleate, PA (C16:0), SA (C18:0), OA (C18:0) and mineral oil were purchased from Sigma. PVDF membranes previously wetted in methanol and dried at RT were spotted with 62 µg of each lipid diluted in ethanol while membranes were drying for 1 h at RT in the dark. Lipids were developed with 10% phosphomolybdic acid solution in ethanol, followed by heating to ~60 ◦C for ~2 min. Only lipids stained with this reagent were used for incubation with rHbhA proteins as follows: membranes were blocked for 1 h in PBS-T/BSA. Then, strips were incubated ON at 4 ◦C with 5.0 µg mL−1 of rE.coliHbhA and/or rRhoHbhA in PBS-T/BSA with shaking. The next day, after several washes with PBS-T, lipids bound to rHbhA were detected by incubation with R-anti-HbhA 1:3000 for 1 h at RT, followed by incubation with Protein A-HRP (Invitrogen) 1:2000. Strips were revealed with a chemiluminescence substrate of HRP as described above. We also studied the binding of puriﬁed recombinants methylated and un-methylated HbhA proteins to SA and OA by a lipid-binding assay that involved the exposition of soluble recombinant proteins and proteins in complex with FA to endoproteinase Glu C (Promega), which speciﬁcally hydrolyzes peptide and ester bonds at the carboxylic side of glutamic acid residues [41], with some modiﬁcations. Brieﬂy, samples of 3 µg of rE.coliHbhA and rRhoHbhA were incubated for 30 min at RT with OA or SA dissolved in ethanol at a ratio of 1:4 (protein:FA) in buffer 20 mM Tris–HCl, 50 mM NaCl, 1 mM DTT. After that, Glu C was added at a radio of 1:20 (enzyme:protein) and further incubated at 37 ◦C for 15 and 120 min. As controls, recombinant proteins without any treatment or treated with Glu C as well as proteins in complex with FA but without Glu C treatment were incubated at 37 ◦C for 15 and 120 min. After different times of treatment, samples were boiled in sample buffer and loaded in 12% SDS-PAGE. Gels were stained with Coomassie blue. Pathogens 2021, 10, 1139 15 of 17 15 of 17 References 1. Polevoda, B.; Sherman, F. Methylation of proteins involved in translation. Mol. Microbiol. 2007, 65, 590–606. [CrossRef] 2. Lanouette, S.; Mongeon, V.; Figeys, D.; Couture, J. The functional diversity of protein lysine methylation. Mol. Syst. Biol. 2014, 10, 724. [CrossRef] 1. Polevoda, B.; Sherman, F. Methylation of proteins involved in translation. Mol. Microbiol. 2007, 65, 590–606. [CrossRef] 2 Lanouette S ; Mongeon V; Figeys D ; Couture J The functional diversity of protein lysine methylation Mol Syst Biol 2014 10 1. Polevoda, B.; Sherman, F. Methylation of proteins involved in translation. Mol. Microbiol. 2007, 65, 590–606. [CrossRef] 1. Polevoda, B.; Sherman, F. Methylation of proteins involved in translation. Mol. Microbiol. 2007, 65, 590–606. [CrossRef] 2. Lanouette, S.; Mongeon, V.; Figeys, D.; Couture, J. The functional diversity of protein lysine methylation. Mol. Syst. 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Statistical Analysis 4.13. Statistical Analysis GraphPad Prims version 6.0 software was used to analyze the results shown in Figures 5–7. For the statistical analyses, one-way ANOVA multiple comparations with Tukey’s correction was used. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/pathogens10091139/s1. Figure S1: Dot Lipid-binding assays with TAG, glyceril trioleate (line 1) stearic acid (line 2), oleic acid, (line 3) and mineral oil (line 4) were spotted on PVDF membranes and incubated with rE.coliHbhA and rRhoHbhA. In control row, rHbhA proteins were omitted. Control of rHbhA protein used for the assay spotted to membrane and developed with R-antiHbhA is also shown. Phosphomolybdic acid stain was carried out to conﬁrm the presence of lipids on membrane. Author Contributions: Methodology, C.E., C.P., I.C.N.-B., P.A.O., A.J.V., E.S., M.S.-M., S.L.G.-G., E.W.C.-A., A.T.-L. and M.d.J.A.S.; software, L.C.-V.; validation, L.C.-V. and E.W.C.-A.; formal anal- ysis, M.S.-M.; investigation, C.E., I.C.N.-B., C.P., A.J.V., P.A.O., E.S., M.S.-M., S.L.G.-G. and A.T.-L.; resources, C.E.; writing—original draft preparation, C.E.; writing—review and editing, C.E., A.J.V. and M.A.F.V.; supervision, C.E.; project administration, C.P.; funding acquisition, C.E. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Universidad Nacional Autónoma de México [PAPIIT-UNAM IN207717] and by and Consejo Nacional de Ciencia y Tecnología [Conacyt PN-2017/4843]. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: We thank Miguel Cuevas Cruz for technical assistance and Institucional Pro gram “Nuevas Alternativas de Tratamiento para Enfermedades Infecciosas” (NUATEI). Instituto de Investigaciones Biomédicas UNAM. I.C.N.B was Master’s student from the program Maestria en Ciencias Bioquímicas at Universidad Nacional Autónoma de México (UNAM), and had an scholarship from the Consejo Nacional de Ciencia y Tecnología (CONACYT), (CVU/894564). Acknowledgments: We thank Miguel Cuevas Cruz for technical assistance and Institucional Pro gram “Nuevas Alternativas de Tratamiento para Enfermedades Infecciosas” (NUATEI). Instituto de Investigaciones Biomédicas UNAM. I.C.N.B was Master’s student from the program Maestria en Ciencias Bioquímicas at Universidad Nacional Autónoma de México (UNAM), and had an scholarship from the Consejo Nacional de Ciencia y Tecnología (CONACYT), (CVU/894564). Conﬂicts of Interest: The authors declare no conﬂict of interest. References Vallecillo, A.J.; Parada, C.; Morales, P.; Espitia, C. Rhodococcus erythropolis as a host for expression, s Mycobacterium tuberculosis proteins. Microb. Cell Factories 2017, 16, 1–7. [CrossRef] 22. Delogu, G.; Bua, A.; Pusceddu, C.; Parra, M.; Fadda, G.; Brennan, M.J.; Zanetti, S. Expression and puriﬁcation of recombinant methylated HBHA in Mycobacterium smegmatis. FEMS Microbiol. Lett. 2004, 239, 33–39. [CrossRef] 23. Pethe, K.; Aumercier, M.; Fort, E.; Gatot, C.; Locht, C.; Menozzi, F.D. Characterization of the Heparin-binding Site of the Mycobacterial Heparin-binding Hemagglutinin Adhesin. J. Biol. Chem. 2000, 275, 14273–14280. 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https://openalex.org/W3008525359	https://europepmc.org/articles/pmc7076352?pdf=render	English	null	Large Volume Direct Injection Ultra-High Performance Liquid Chromatography–Tandem Mass Spectrometry-Based Comparative Pharmacokinetic Study between Single and Combinatory Uses of Carthamus tinctorius Extract and Notoginseng Total Saponins	Pharmaceutics	2,020	cc-by	7,085	Received: 25 December 2019; Accepted: 17 February 2020; Published: 20 February 2020 Abstract: The combination of Carthamus tinctorius extract (CTE) and notoginseng total saponins (NGTS), namely, CNP, presents a synergistic eﬀect on myocardial ischemia protection. Herein, comparative pharmacokinetic studies between CNP and CTE/NGTS were conducted to clarify their synergistic mechanisms. A large volume direct injection ultra-high performance liquid chromatography–tandem mass spectrometry (LVDI-UHPLC-MS/MS) platform was developed for sensitively assaying the multi-component pharmacokinetic and in vitro cocktail assay of cytochrome p450 (CYP450) before and after compatibility of CTE and NGTS. The pharmacokinetic proﬁles of six predominantly eﬃcacious components of CNP, including hydroxysaﬄor yellow A (HSYA); ginsenosides Rg1 (GRg1), Re (GRe), Rb1 (GRb1), and Rd (GRd); and notoginsenoside R1 (NGR1), were obtained, and the results disclosed that CNP could increase the exposure levels of HSYA, GRg1, GRe, GRb1, and NGR1 at varying degrees. The in vitro cocktail assay demonstrated that CNP exhibited more potent inhibition on CYP1A2 than CTE and NGTS, and GRg1, GRb1, GRd, quercetin, kaempferol, and 6-hydroxykaempferol were found to be the major inhibitory compounds. The developed pharmacokinetic interaction-based strategy provides a viable orientation for the compatibility investigation of herb medicines. Keywords: Carthamus tinctorius extract; notoginseng total saponins; comparative pharmacokinetic study; large volume direct injection; compatibility mechanism pharmaceutics pharmaceutics pharmaceutics Large Volume Direct Injection Ultra-High Performance Liquid Chromatography–Tandem Mass Spectrometry-Based Comparative Pharmacokinetic Study between Single and Combinatory Uses of Carthamus tinctorius Extract and Notoginseng Total Saponins Jinfeng Chen 1, Xiaoyu Guo 1, Yingyuan Lu 1, Mengling Shi 1, Haidong Mu 1, Yi Qian 1, Jinlong Wang 1, Mengqiu Lu 1, Mingbo Zhao 1, Pengfei Tu 1,2, Yuelin Song 2,* and Yong Jiang 1,* 1 State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China; chenjinfeng0513@163.com (J.C.); guoxiaoyu@bjmu.edu.cn (X.G.); luyingyuan2005@126.com (Y.L.); shimenglingbj@126.com (M.S.); a1538933944@163.com (H.M.); yiqian@163.com (Y.Q.); wang-jinlong@pku.edu.cn (J.W.); mengqiulu@163.com (M.L.); zmb_77@163.com (M.Z.); pengfeitu@bjmu.edu.cn (P.T.) 1 State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China; chenjinfeng0513@163.com (J.C.); guoxiaoyu@bjmu.edu.cn (X.G.); luyingyuan2005@126.com (Y.L.); shimenglingbj@126.com (M.S.); a1538933944@163.com (H.M.); yiqian@163.com (Y.Q.); wang-jinlong@pku.edu.cn (J.W.); mengqiulu@163.com (M.L.); zmb 77@163 com (M Z ); pengfeitu@bjmu edu cn (PT ) 2 Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, China 2 Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, China * Correspondence: syltwc2005@163.com (Y.S.); yongjiang@bjmu.edu.cn (Y.J.); Tel.: + 86-10-82802719 (Y.J. Received: 25 December 2019; Accepted: 17 February 2020; Published: 20 February 2020 www.mdpi.com/journal/pharmaceutics 1. Introduction Myocardial ischemia-induced infarction is one of the leading causes of human death worldwide. The beneﬁts of either Carthamus tinctorius extract (CTE) or notoginseng total saponins (NGTS) towards myocardial ischemia injury on rats have been well deﬁned, and more interestingly, previous studies have demonstrated that better cardio-protective eﬀects were observed when using their combination Pharmaceutics 2020, 12, 180; doi:10.3390/pharmaceutics12020180 www.mdpi.com/journal/pharmaceutics Pharmaceutics 2020, 12, 180 2 of 12 preparation (CNP) [1–3]. However, the underlying synergetic mechanisms of CTE and NGTS combination, their pharmacokinetic (PK) interactions in particular, still remain unclear. It is widely accepted that the drug–drug interactions (DDIs) and herb–herb interactions (HHIs) can cause changes of pharmacokinetic proﬁles, which result in the possible improvement of drug eﬃcacy and in the decrease of side eﬀects, or vice versa [4,5]. However, most of the literature has merely focused on the pharmacokinetic proﬁle variations of these primary components between individual dosing and combined use, but has overlooked the reasons responsible for the changed pharmacokinetic behaviors, which may be caused, at least in part, by cytochrome p450 (CYP450)- and/or transporter-mediated HHIs [6]. Therefore, the objective of this study was to gain insight into the synergistic actions between CTE and NGTS by determination of the pharmacokinetic proﬁles of six major active components from CTE and NGTS, as well as their CYP450-based synergetic mechanisms. An in vitro cocktail assay, which is an eﬃcient and widely favored approach for CYP450-mediated HHIs, was employed to pursue the factors accounting for the diﬀerent pharmacokinetic patterns before and after compatibility. Our previous pharmacological evaluations optimized a relatively low dosage CNP for the anti-myocardial ischemia eﬀect [2]. Furthermore, the cocktail method usually suﬀers from extensive CYP450 crossover within the probe substrates [7]. Therefore, the emerging demand is to develop a sensitive and eﬃcient method for reliable detection and determination of the trace ingredients for the PK and cocktail studies. Attempts were made herein to propose and apply a large volume direct injection ultra-high performance liquid chromatography–tandem mass spectrometry (LVDI-UHPLC-MS/MS) method for direct and sensitive multiple-component PK and cocktail studies. 2.2. Animals and Rat Liver Microsomes Male Sprague-Dawley (SD) rats (12–14 weeks, 200–240 g) were provided by the Experimental Animal Center, Peking University Health Science Center. All animal experimental protocols were approved by the Biomedical Ethical Committee of Peking University Health Science Center (SYXK (Jing) 2016-0041, 23 December 2016). The animal experiments were carried out in accordance with the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications no. 8023, revised 1978). Pooled SD rat liver microsome (RLM, 20 mg/mL, LM-DS-02M, BDVH) were purchased from the Research Institute for Liver Diseases (Shanghai, China) Co. Ltd. 2.3. Plasma Pharmacokinetic Studies CTE, NGTS, and CNP powders were dissolved using saline, and were orally dosed at 50 mg·kg−1, 60 mg·kg−1, and 50 mg·kg−1 CTE + 60 mg·kg−1 NGTS, respectively, whereas saline was administered to the vehicle group. Six rats were used in each group, and all rats fasted overnight but had free access to water prior to treatment. A terminal sampling design was used to collect blood samples at 0 (pre-dose), 0.08, 0.16, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12, 24, 48, 72, and 96 h. At each time, 0.5 mL of blood was collected in the heparin sodium tubes. Plasma was separated by centrifugation at 4000 rpm for 10 min and stored below −80 ◦C until bioanalysis. Oasis PRiME HLB SPE cartridges (1 cc/30 mg, Waters, Milford, MA, USA) were used to process the plasma samples (Supplementary Materials). 2.1. Plant Materials Notoginseng total saponins (NGTS), containing ginsenoside Rg1 (GRg1, A1, 26.6%), ginsenoside Rb1 (GRb1, A2, 32.5%), ginsenoside Rd (GRd, A3, 6.6%), ginsenoside Re (GRe, A4, 4.1%), and notoginsenoside R1 (NGR1, A5, 6.2%), prepared according to the Monograph of NGTS recorded in Chinese Pharmacopoeia [8], was purchased from Yunnan Plant Pharm. Co., Ltd. (Yunnan, China). Besides NGR1, GRg1, GRe, GRb1, and GRd (Han et al., 2010), the remaining amount of around 25% ginsenosides in NGTS were further clariﬁed as previously described [9]. Carthamus tinctorius extract, containing hydroxysaﬄor yellow A (HSYA; A9, 8.0%) and kaempferol-3-O-rutinoside (A11, 0.2%) was prepared following the protocol described in a previous report [1]. The chemical structures of the main components contained in CNP are shown in Figure 1. The chemical proﬁling based on LC–MS (Figure S1) and the detail chemical composition information were reported in our previous research papers [10]. The detailed information of other chemicals and reagents is shown in the Supplementary Materials section. 3 of 12 Pharmaceutics 2020, 12, 180 harmaceutics 2020, 12, 180 3 of 12 Figure 1. The chemical structures of the main components contained in the combination of Carthamus tinctorius extract and notoginseng total saponins (CNP). Figure 1. The chemical structures of the main components contained in the combination of C h i i d l (C ) Figure 1. The chemical structures of the main components contained in the combination of Carthamus tinctorius extract and notoginseng total saponins (CNP). 2.2. Animals and Rat Liver Microsomes 2.4. Incubation Procedure and CYP450 Activity Assay The eﬀects of CTE, NGTS, CNP, and 18 representative compounds (A1–A18, Supplementary Materials) on CYP450 activities were investigated using a pool of SD RLM following the procedure in [11]. Incubations were conducted at 37 ± 1 ◦C in 200 µL of incubation mixtures containing RLM (0.2 mg/mL), phosphate buﬀer saline (PBS) (pH 7.4, 0.1 mM), MgCl2 (5 mM), nicotinamide adenine dinucleotide phosphate (NADPH) (1 mM), and CYP450 probe substrates (90/1.07/18/0.13/0.02/3.6/90 µM of phenacetin/omeprazole/ tolbutamide/dextromethorphan/midazolam/chlorzoxazone/ bupropion, respectively). CYP450 inhibitors (triethylenethiophosphoramide/sulfaphenazole/ticlopidine/furafylline/ P450 inhibitors (triethylenethiophosphoramide/sulfaphenazole/ticlopidine/furafylline 4 of 12 Pharmaceutics 2020, 12, 180 ketoconazole/quinidine/4-methylpyrazole for CYP2B6/2C9/2C19/1A2/3A4/2D6/2E1, respectively) were added as the positive control, and blank solvents (PBS containing methanol and/or dimethyl sulfoxide (DMSO)) were used as the negative control. The incubation mixtures also contained eight concentrations for CTE (2.5–200 µg/mL), NGTS (2.5–200 µg/mL), CNP (2.5–200 µg/mL), A1–A17 (5–200 µM), and A18 (0.25–10 µM). Reactions were initiated by adding the NADPH-generating system and terminated after 15 min by 200 µL of cold methanol containing 105 nM hydroxybupropion-[D6] (OHBUP-[D6]) and 5 nM 4′-hydroxydiclofenac-[13C6] (OHDIC-[13C6]) as internal standards (ISs). The mixture was placed in an ice bath for 30 min, and the precipitated protein was removed by centrifugation (12,000 rpm for 10 min at 4 ◦C) three times. Then an aliquot of 100 µL supernatant was diluted with 100 µL ultrapure water, and centrifuged at 12,000 rpm for 10 min, before being subjected to LVDI-UHPLC-MS/MS analysis. ketoconazole/quinidine/4-methylpyrazole for CYP2B6/2C9/2C19/1A2/3A4/2D6/2E1, respectively) were added as the positive control, and blank solvents (PBS containing methanol and/or dimethyl sulfoxide (DMSO)) were used as the negative control. The incubation mixtures also contained eight concentrations for CTE (2.5–200 µg/mL), NGTS (2.5–200 µg/mL), CNP (2.5–200 µg/mL), A1–A17 (5–200 µM), and A18 (0.25–10 µM). Reactions were initiated by adding the NADPH-generating system and terminated after 15 min by 200 µL of cold methanol containing 105 nM hydroxybupropion-[D6] (OHBUP-[D6]) and 5 nM 4′-hydroxydiclofenac-[13C6] (OHDIC-[13C6]) as internal standards (ISs). The mixture was placed in an ice bath for 30 min, and the precipitated protein was removed by centrifugation (12,000 rpm for 10 min at 4 ◦C) three times. Then an aliquot of 100 µL supernatant was diluted with 100 µL ultrapure water, and centrifuged at 12,000 rpm for 10 min, before being subjected to LVDI-UHPLC-MS/MS analysis. 3.1. Injection Solvent Optimization It is well known that the sample solvent and volume have large eﬀects on the peak asymmetry and column eﬃciency [13]. Herein, we established a LVDI-UHPLC-MS/MS method, assisted by injection solvent optimization, for sensitive bioassays of the pharmacokinetic interactions and cocktail analysis of CTE and NGTS. Considering the solubility and the sample pretreatment methods (Supplementary Materials) of the target analytes, the mixed standard solution of the six reference compounds was diluted with methanol (MeOH)/water (H2O) in a ratio of 20% increment ranging from 100% H2O to 100% MeOH for the PK study, and all the six standards of the PK study showed the highest responses when using 40% or 60% aqueous MeOH as the injection solvent. The CYP450 probe substrates and their corresponding metabolites for the in vitro cocktail assay were chosen according to the FDA guide [14]. Likewise, the six metabolites for the cocktail assay showed the best chromatograms when the 25% aqueous MeOH was served as the sample solvent, with exception of 1′-hydroxymidazolam, for which 50% aqueous MeOH was selected as the injection solvent (Figure S2). 2.5. LVDI-UHPLC-MS/MS Analysis The generic layout of instrumentation setup of the LVDI-UHPLC-MS/MS was conducted on a Shimadzu UHPLC system consisting of two LC-20ADXR pumps, a DGU-20A3R degasser, and a CBM-20A controller (Kyoto, Japan) with a SCIEX 4500 QTRAP mass spectrometer mounted with an electron spray ionization (ESI) interface as well as an electronic 6-port/2-channel valve (Foster City, CA, USA). Analyst software package (version 1.6.2, SCIEX) was implemented to control and synchronize the entire system, and also for data acquisition and processing. A single analytical run was fragmented into two phases, namely, loading and elution phases, by switching the electronic valve (Figure 2). At the loading phase, the valve was maintained at A-channel. The specimen aliquot of large volume delivered from the auto-sampler was captured onto a guard column. Meanwhile, the pumps were responsible for delivering the mobile phase at a high ﬂow rate, aiming to dilute the sample solvent, thus facilitating the candidate constituents to concentrate on the pre-guard column. Then, the valve was automatically switched to the B-channel to trigger the elution phase. The trapped components were ﬂushed from the pre-guard column into an analytical column, and underwent multiple reaction monitoring (MRM) analysis on the optimized LC gradients. The detailed information of LVDI-UHPLC-MS/MS analysis for pharmacokinetic and cocktail studies is shown in the Supplementary Materials section. Figure 2. Connectivity sketch of the six-port/two-channel switching valve controlling the large volume direct injection ultra-high performance liquid chromatography–tandem mass spectrometry (LVDI-UHPLC-MS/MS) system. At the loading phase, the valve was maintained at A-channel. The sample delivered from the auto-sampler was captured onto a pre-guard column. Meanwhile, the pumps delivered the mobile phase at a high ﬂow rate. Then, the valve was automatically switched to the B-channel to trigger the elution phase. The trapped components were ﬂushed from the pre-guard column into an analytical column. Figure 2. Connectivity sketch of the six-port/two-channel switching valve controlling the large volume direct injection ultra-high performance liquid chromatography–tandem mass spectrometry (LVDI-UHPLC-MS/MS) system. At the loading phase, the valve was maintained at A-channel. The sample delivered from the auto-sampler was captured onto a pre-guard column. Meanwhile, the pumps delivered the mobile phase at a high ﬂow rate. Then, the valve was automatically switched to the B-channel to trigger the elution phase. The trapped components were ﬂushed from the pre-guard column into an analytical column. 5 of 12 Pharmaceutics 2020, 12, 180 2.6. Method Validations Method validations for the pharmacokinetic and cocktail assays, in terms of speciﬁcity, linearity, and sensitivity; precision and accuracy; recovery and matrix eﬀect; and stability, were individually carried out by following the U.S. Food and Drug Administration (FDA) Guidance on Bioanalytical Method Validation and Drug Interaction Studies [12]. 2.7. Data Processing Calibration data were ﬁtted to linear calibration curves using 1/x2 weighting. For the PK study, the half-time (T1/2), maximum plasma concentration (Cmax), time to reach the maximum concentration (Tmax), and area under concentration-time curve (AUC) were determined by non-compartmental method using Drug and Statistics 3.0 (DAS 3.0, Mathematical Pharmacology Professional Committee of China, Shanghai, China). For the cocktail assay, the activity is expressed as the percentage of activity remaining comparing with that of a control sample containing no inhibitor. Substrate inhibition data were analyzed using GraphPad Prism 6 (version 6.01; San Diego, CA, USA) in logistic regression. All the data were described as mean ± standard deviation (SD). Normality assumptions were tested by the Kolmogorov–Smirnov statistic setting p = 0.10 as the limit for rejection of the null hypothesis of normality. If the distribution of the data was normal with equal variances, a two-sided test was performed at the 5% level of signiﬁcance. The Welch’s correction was then applied when the underlying variances were not equal. When the assumption of normality must be rejected, the Mann-Whitney test, a non-parametric equivalent of the independent-measures t-test, was used. 3.2. Optimization of the Loading Phase for the LVDI-UHPLC-MS/MS-Based Method The instrument stability of the LVDI-UHPLC-MS/MS setup was ﬁrst investigated, and the results indicated the LVDI-UHPLC-MS/MS could meet the demands of quantitation (Table S3). An analytical run of the LVDI-UHPLC-MS/MS-based method was fragmented into a loading phase and an elution phase. The gradient condition for the elution phase turned out to be the same as that of the regular UHPLC-MS/MS analysis. Thus, the optimization works were concentrated on the loading phase, including the ﬂow rate, mobile phase, and dilution time. According to the optimized gradient programs of the elution phases, both PK and cocktail studies employed water as the mobile phase for their loading phase after evaluating the solvents ramped from 0% to 20% aqueous acetonitrile. The ﬂow rate of the loading phase for the PK study was ﬁnally optimized as 3.0 mL·min−1 after assays of 0.4, 1.0, 2.0, and 3.0 mL·min−1 ﬂow rates. Because the higher the ﬂow rate, the lower the signal responses of paracetamol and 6-hydroxychlorzoxazone, the cocktail assay ﬁnally chose 0.4 mL·min−1 as the ﬂow rate of the loading phase. It turned out that the dilution time did not have a profound eﬀect on the 6 of 12 Pharmaceutics 2020, 12, 180 total chromatogram by comparing 0.5, 1, and 2 min. Thus, the shortest time, 0.5 min, was chosen to promote the analytical eﬃciency. The corresponding bioanalytical method validations were carried out by following the FDA guidance [12], and the results demonstrated that the newly developed LVDI-UHPLC-MS/MS-based methods enabled reliable detection and precise determination of the multiple-component in PK and cocktail studies. The lower limits of quantitation (LLOQ) of most analytes (except Rb1 and Rd) were lower than 60 pg/mL. 3.3. Comparative Multiple-Component PK Studies Pharmacokinetic parameters of hydroxysaﬄor yellow A (HSYA), ginsenoside Re (GRe), ginsenoside Rb1 (GRb1), ginsenoside Rd (GRd), ginsenoside Rg1 (GRg1), and notoginsenoside R1 (NGR1) after oral administration of CTE, NGTS, and CNP to rats. Each point represents the mean ± SD (n = 6). Table 1. Pharmacokinetic parameters of hydroxysaﬄor yellow A (HSYA), ginsenoside Re (GRe), ginsenoside Rb1 (GRb1), ginsenoside Rd (GRd), ginsenoside Rg1 (GRg1), and notoginsenoside R1 (NGR1) after oral administration of CTE, NGTS, and CNP to rats. Each point represents the mean ± SD (n = 6). Table 1. Pharmacokinetic parameters of hydroxysaﬄor yellow A (HSYA), ginsenoside Re (GRe), ginsenoside Rb1 (GRb1), ginsenoside Rd (GRd), ginsenoside Rg1 (GRg1), and notoginsenoside R1 (NGR1) after oral administration of CTE, NGTS, and CNP to rats. Each point represents the mean ± SD (n = 6) Table 1. Pharmacokinetic parameters of hydroxysaﬄor yellow A (HSYA), ginsenoside Re (GRe), ginsenoside Rb1 (GRb1), ginsenoside Rd (GRd), ginsenoside Rg1 (GRg1), and notoginsenoside R1 (NGR1) after oral administration of CTE, NGTS, and CNP to rats. Each point represents the mean ± SD (n = 6). Analyte Group t1/2 (h) Tmax (h) Cmax (ng·mL−1) AUC0–t (ng·h·mL−1) AUC0–∞ (ng·h·mL−1) HSYA CTE 2.01 ± 0.34 0.88 ± 0.54 12.16 ± 3.09 33.05 ± 10.70 33.85 ± 10.78 CNP 1.68 ± 0.79 1.17 ± 0.41 15.17 ± 4.39 38.33 ± 8.42 38.94 ± 8.60 NGR1 NGTS 10.53 ± 3.06 1.15 ± 1.06 0.64 ± 0.18 6.30 ± 2.41 8.14 ± 3.60 CNP 12.36 ± 4.48 1.04 ± 0.97 0.79 ± 0.18 8.12 ± 1.53 11.49 ±3.54 GRb1 NGTS 36.89 ± 9.65 5.50 ± 1.80 113.08 ± 41.78 2317.66 ± 682.70 2808.87 ± 617.99 CNP 34.47 ± 8.45 6.33 ± 3.45 129.00 ± 65.97 2472.33 ± 394.72 2816.01 ± 563.44 GRd NGTS 42.75 ± 8.84 * 4.50 ± 1.76 29.43 ± 10.69 460.90 ± 117.22 618.90 ± 157.23 CNP 27.79 ± 6.94 6.33 ± 3.44 31.07 ± 16.78 459.04 ± 51.04 549.18 ± 58.09 GRg1 NGTS 11.68 ± 2.09 * 4.71 ± 3.71 0.78 ± 0.13 13.99 ± 5.03 16.72 ± 3.93 CNP 15.11 ± 8.89 5.38 ± 4.66 1.04 ± 0.34 16.69 ± 3.42 20.23 ± 3.35 GRe NGTS 5.25 ± 2.27 2.60 ± 0.89* 0.29 ± 0.04* 1.82 ± 0.19 2.90 ± 0.60 CNP 5.99 ± 3.86 0.80 ± 0.41 0.39 ± 0.08 1.93 ± 0.19 3.33 ± 1.13 : p < 0.05, versus the combination group. 3.3. Comparative Multiple-Component PK Studies Tmax: the time of peak concentration; t1/2: half-life; Cmax: the peak or maximum concentration; AUC: area under concentration-time curve. For abbreviations of analytes A6–A18, please refer to the Supplementary Materials section. : p < 0.05, versus the combination group. Tmax: the time of peak concentration; t1/2: half-life; Cmax: the peak or maximum concentration; AUC: area under concentration-time curve. For abbreviations of analytes A6–A18, please refer to the Supplementary Materials section. 3.3. Comparative Multiple-Component PK Studies Considering the bioavailability improvement of active ingredients is a key point of traditional Chinese medicine compatibility, we executed a multiple-component PK study. HSYA, GRb1, GRd, GRe, GRg1, and NGR1 were the primary circulating compounds and the main cardio-protective components in CNP [15,16], and thus were chosen as the PK markers. Meanwhile, the optimized injection solvent and LVDI-UHPLC-MS/MS method were integrated to achieve a much more sensitive method for reliable quantiﬁcation of these components in vivo. After validation (Figure 3, Tables S4–S7), the developed method was ﬁrst applied to characterize the pharmacokinetic characters of HSYA, GRb1, GRd, GRe, GRg1, and NGR1 in rat plasma. Their plasma concentrations versus time proﬁles are displayed in Figure 4 and Table S8. The combination group showed greater Cmax and AUC0-t values (Table 1) of HSYA, GRg1, GRb1, NGR1, and GRe over the individual extract groups. Exceptionally, GRd exhibited considerable AUC0-t and Cmax between NGTS and CNP dosing groups, whereas signiﬁcantly diﬀerent T1/2 values, which indicated the combination use of CTE and NGTS, may have accelerated the elimination processes of GRd. The reason may have been due to the hydrolysis of GRb1 to GRd in vivo [17], resulting in a more complicated PK behavior of GRd than other compounds in CNP. Figure 3. Representative multiple reaction monitoring (MRM) chromatograms of target analytes in rat plasma in a positive mode: (A) blank plasma; (B) blank plasma spiked with six chemical standards and internal standards (IS); (C) plasma sample collected at 2 h following oral administration of extract CNP (Carthamus tinctorius extract (CTE) 50 mg/kg + notoginseng total saponins (NGTS) 60 mg/kg) to rats. Figure 3. Representative multiple reaction monitoring (MRM) chromatograms of target analytes in rat plasma in a positive mode: (A) blank plasma; (B) blank plasma spiked with six chemical standards and internal standards (IS); (C) plasma sample collected at 2 h following oral administration of extract CNP (Carthamus tinctorius extract (CTE) 50 mg/kg + notoginseng total saponins (NGTS) 60 mg/kg) to rats. 7 of 12 Pharmaceutics 2020, 12, 180 7 of 12 Figure 4. Mean plasma concentration-time proﬁles of the six analytes in rats after oral administration of CTE, NGTS, and CNP. Each point represents the mean ± SD (n = 6). Figure 4. Mean plasma concentration-time proﬁles of the six analytes in rats after oral administration of CTE, NGTS, and CNP. Each point represents the mean ± SD (n = 6). Table 1. 3.4. CYP450-Mediated Herb–Herb Interactions To investigate the possible HHIs between NGTS and CTE, an in vitro cocktail assay involving seven probe substrates was conducted, with the assistance of LVDI-UHPLC-MS/MS method to avoid CYP450 crossovers (Figure 5, Tables S9–S11). The incubation system was optimized in the aspects of substrate choice, enzyme concentration, incubation time, and substrate concentration (Figures S3–S4, Table S12), following the guidance of the FDA [14]. According to the half inhibiting concentration (IC50) values of CTE, NGTS, and CNP (Table 2), CTE showed weak inhibition on CYP1A2, CYP2D6, and CYP2C9 and moderate inhibition on CYP2B6 and CYP2E1, whereas NGTS presented much more potent inhibitions on all these detected CYP450s than CTE (Table 2). After combination, CNP showed 8 of 12 Pharmaceutics 2020, 12, 180 more potent inhibition on CYP1A2 than CTE and NGTS, and more potent inhibition on CYP2C9, CYP2C19, CYP3A4, and CYP2D6 than CTE (Table 2). Figure 5. Representative MRM chromatograms of all probe metabolites and two ISs in the incubated rat microsomal sample with no substrate cocktails: (A) blank microsomal sample, (B) blank microsomal sample spiked with seven chemical standards and two ISs monitored in a polarity switching mode. Figure 5. Representative MRM chromatograms of all probe metabolites and two ISs in the incubated rat microsomal sample with no substrate cocktails: (A) blank microsomal sample, (B) blank microsomal sample spiked with seven chemical standards and two ISs monitored in a polarity switching mode. 9 of 12 Pharmaceutics 2020, 12, 180 Table 2. IC50 values of CTE, NGTS, CNP, and the 18 representative compounds for inhibiting cytochrome p450 (CYP450) isozymes. NGTS, CNP, and the 18 representative compounds for inhibiting cytochrome p450 (CYP450) isozymes. Table 2. IC50 values of CTE, NGTS, CNP, and the 18 representative compounds for inhibiting cytochrome p450 (CYP450) isozymes. No. 3.4. CYP450-Mediated Herb–Herb Interactions IC50 (µg·mL−1/µM) # CYP2C9 CYP2E1 CYP2C19 CYP2D6 CYP2B6 CYP1A2 CYP3A4 CNP 21.66 ± 1.15 31.85 ± 4.15 62.95 ± 1.17 25.27 ± 0.25 52.19 ± 2.24 26.36 ± 6.98 91.77 ± 5.16 CTE 183.30 ± 17.98 35.47 ± 2.98 >200 126.17 ± 17.63 58.09 ± 13.41 138.60 ± 14.36 >200 NGTS 9.32 ± 0.56 16.12 ± 2.59 40.85 ± 6.98 12.23 ± 4.25 43.90 ± 7.91 60.68 ± 2.26 106.27 ± 14.00 GRg1 (A1) 36.49 ± 1.32 34.08 ± 4.76 78.12 ± 5.25 55.73 ± 6.08 11.02 ± 0.87 13.09 ± 8.24 39.25 ± 4.57 GRb1 (A2) 17.57 ± 2.03 27.96 ± 2.39 16.80 ± 2.52 17.06 ± 1.29 120.83 ± 10.89 5.84 ± 0.14 30.04 ± 2.47 GRd (A3) 73.01 ± 3.71 43.46 ± 0.06 71.36 ± 14.28 64.81 ± 11.68 >200 4.36 ± 2.31 61.43 ± 14.32 GRe (A4) >200 25.59 ± 9.03 50.53 ± 15.39 >200 39.40 ± 4.81 94.41 ± 4.52 75.11 ± 7.24 NGR1 (A5) 154.47 ± 16.65 121.70 ± 8.96 107.59 ± 29.58 20.42 ± 3.45 >200 112.07 ± 7.72 84.59 ± 1.79 A6 55.84 ± 3.40 40.76 ± 1.65 53.67 ± 0.22 19.46 ± 2.83 114.70 ± 3.04 52.77 ± 1.93 26.45 ± 1.39 A7 80.47 ± 1.05 13.59 ± 0.60 18.50 ± 1.60 31.02 ± 4.17 177.17 ± 4.21 153.27 ± 8.73 126.70 ± 4.24 A8 1.21 ± 0.51 9.06 ± 1.85 111.26 ± 21.57 10.54 ± 1.11 >200 >200 34.38 ± 9.16 A9 (HSYA) 0.17 ± 0.02 0.73 ± 0.15 49.33 ± 3.28 0.14 ± 0.06 >200 >200 9.42 ± 2.26 A10 39.06 ± 0.09 >200 >200 >200 >200 >200 >200 A11 24.92 ± 4.96 5.45 ± 1.37 >200 64.55 ± 7.71 17.33 ± 0.96 53.17 ± 6.12 16.18 ± 4.30 A12 >200 >200 >200 98.56 ± 12.30 >200 >200 >200 A13 93.00 ± 2.56 10.22 ± 0.65 48.91 ± 4.36 37.12 ± 8.85 21.02 ± 2.31 13.35 ± 2.27 58.52 ± 0.66 A14 >200 >200 >200 >200 >200 >200 >200 A15 >200 >200 >200 >200 >200 127.47 ± 6.55 >200 A16 13.48 ± 2.41 20.29 ± 4.13 46.35 ± 7.05 31.11 ± 3.91 68.48 ± 9.88 41.22 ± 3.18 73.74 ± 13.64 A17 14.92 ± 4.24 32.46 ± 5.79 70.38 ± 1.18 59.17 ± 1.01 >200 21.46 ± 4.50 117.00 ± 6.77 A18 2.13 ± 0.64 4.27 ± 1.30 1.79 ± 0.52 0.98 ± 0.38 4.42 ± 1.31 0.12 ± 0.01 2.48 ± 0.48 Values were obtained from triplicate tests, and presented as mean ± SD. #: Values were obtained from triplicate tests, and presented as mean ± SD. The units of IC50 values for CTE, NGTS Potent inhibitors (IC50 < 15 µM for 1A2 2C9 2C19 2D6 2B6 and 3A4) are presented in italic For abbreviations of #: Values were obtained from triplicate tests, and presented as mean ± SD. The units of IC50 values for CTE, NGTS, and CNP are µg·mL−1, whereas for the 18 single components are µM. Potent inhibitors (IC50 < 15 µM for 1A2, 2C9, 2C19, 2D6, 2B6, and 3A4) are presented in italic. For abbreviations of analytes A6–A18, please refer to the Supplementary Materials section. Values were obtained from triplicate tests, and presented as mean ± SD. The units of IC50 values for CTE, NGTS, and CN otent inhibitors (IC50 < 15 µM for 1A2, 2C9, 2C19, 2D6, 2B6, and 3A4) are presented in italic. For abbreviations of analytes 3.5. Discussion Herbal pair, the most fundamental and simplest form of Chinese herbal medicine formula, has been favored for centuries because of its better therapeutic outcomes and fewer side eﬀects [18]. Herein, we primarily aimed to clarify the compatibility mechanisms between NGTS and CTE from PK interactions. Given the low dosage, more eﬀorts were paid onto the detection and quantiﬁcation of trace CNP-derived components in vivo. We found the injection solvent extensively aﬀected the chromatographic performances. Increasing the injection volume could advance the sensitivity owing to the subjection of larger amounts of analytes [19]. However, the solvent eﬀect might be initiated by directly injecting large volume of solution onto the chromatographic column without any additional treatment. Increasing the ﬂow rate of the mobile phase could guarantee the dilution of the injection solvent and retention of the target analytes, which should be a practical choice to minimize, or even avoid the solvent eﬀect. However, a rapid ﬂow rate gave rise to a higher back pressure. Therefore, the evaporation–reconstitution step often involved loading the suﬃcient quantity of sample onto the column for LC–MS analysis. Fortunately, the electronic six-port/two-channel valve mounted on the QTRAP system can be applied as a viable solution to split the back pressure. Therefore, LVDI-UHPLC-MS/MS method was proposed. With the LVDI-UHPLC-MS/MS method, the sample can be directly injected into LC-MS analysis without any evaporation–reconstitution step, which is time-consuming and risks crucial chemical degradation during the evaporation procedure. Under the assistance of the injection solvent optimization, the validated LVDI-UHPLC-MS/MS-based method turned out to be extremely sensitive, accurate, and qualiﬁed for the bioassay measurement. The pharmacokinetic results of HSYA, GRg1, GRe, GRb1, and NGR1 indicated that after combination, the absorption of these active components was increased inferred from their higher Cmax and AUC0-t values over that of the individual extract groups (Table 1). The increment of Cmax and AUC0-t values suggested that CYP450-mediated HHIs between CTE and NGTS may primarily account for the compatibility mechanisms of CTE and NGTS. An in vitro cocktail assay was then carried out to ﬁnd the clues being responsible for HHIs between CTE and NGTS. The results showed CNP exhibited more potent inhibition on CYP1A2 (Table 2), the key enzyme involved in the oxidation reactions of most xenobiotics [20], compared with CTE and NGTS. 3.4. CYP450-Mediated Herb–Herb Interactions The units of IC50 values for CTE, NGTS, and CNP are µg·mL−1, whereas for the 18 single components ar otent inhibitors (IC50 < 15 µM for 1A2, 2C9, 2C19, 2D6, 2B6, and 3A4) are presented in italic. For abbreviations of analytes A6–A18, please refer to the Supplementary Materials se 10 of 12 Pharmaceutics 2020, 12, 180 10 of 12 To identify the major CNP components responsible for the inhibition, 18 representative components from CNP were evaluated by the cocktail assays. The results (Table 2) showed that HSYA and ginsenosides showed high or moderate inhibition activities on the seven CYP450 isozymes. Although ﬂavonoid glycosides showed moderate or weak inhibition activities, or even no inhibition activities (IC50 values of >200 µM, Table 2), the ﬂavonoid aglycones quercetin (A16), kaempferol (A17), and 6-hydoxykaempferol (A18), in contrast, exhibited potent inhibitory activities against the seven CYP450 isozymes. In particular, 6-hydroxykaempferol (A18) showed remarkably stronger inhibitory activities on the seven CYP450 isozymes (IC50 < 5 µM, Table 2). In conclusion, the ginsenosides GRg1 (A1), GRb1 (A2), and GRd (A3), and the ﬂavonoids 6-hydoxykaempferol-3-O-glucoside (A6), kaempferol-3-O-glucoside (A7), anhydroxysaﬄor yellow B (AHSYB, A8), hydroxysaﬄor yellow A (HSYA, A9), 6-hydroxykaempferol-3,6-di-O-glucoside (A13), quercetin (A16), kaempferol (A17), and 6-hydroxykaempferol (A18) were presented as being the intensive inhibitors to diﬀerent CYP450s (IC50 ≤15 µM). Among them, GRg1 (A1), GRb1 (A2), GRd (A3), quercetin (A16), kaempferol (A17), and 6-hydroxykaempferol (A18) were the main active components of CNP for the inhibition of CYP1A2. 4. Conclusions In conclusion, the ﬁndings gained from the comparative pharmacokinetic investigations revealed there were pharmacokinetic interactions between CTE and NGTS, which may explain the integrative mechanisms of CNP and provide the experimental data and theoretical basis for further development and clinical applications of CNP. The developed LVDI-UHPLC-MS/MS method and pharmacokinetic interaction-based strategy provide a viable orientation for the compatibility investigation of herb medicines. Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4923/12/2/180/s1: Figure S1. The total ion current chromatogram (TIC) of CTE; the corresponding chemical composition information was reported in previous research (Chen, et al., 2014; Analyst 139, 6474–6485). Figure S2. The optimization of sample solvents for the pharmacokinetic analysis (A) and the cocktail assay (B) (n = 3). Figure S3. Kinetic proﬁles for the enzymatic turnover of CYP450-mediated probe reactions. Figure S4. Inhibition curves of the seven positive inhibitors obtained from the substrate cocktail incubation. Table S1. Multiple reaction monitoring transitions and fragmentation parameters of six standards and IS1 for PK analysis. Table S2. Multiple reaction monitoring transitions and fragmentation parameters of seven metabolites and two internal standards (IS2 and IS3) for cocktail assay. Table S3. The instrument stability of the LVDI-UHPLC-MS/MS setup. Table S4. Regression equations, linear ranges, and low limits of quantiﬁcation (LLOQ) of the six standards in rat plasma for the PK study. Table S5. Intra- and inter-day precisions and determination accuracies of six standards for the pharmacokinetic study. Table S6. Extract recoveries and matrix eﬀects of six target constituents in rat plasma samples for the PK study. Table S7. Stability of the six CNP constituents in rat plasma samples for the PK study. Table S8. Plasma concentration time of the six target constituents after oral administration of CTE, NGTS, and CNP. Table S9. Regression equations, linear ranges, and LLOQs of the seven metabolites for the cocktail analysis. Table S10. Intra- and inter-day precisions and determination accuracies of the seven metabolites for cocktail analysis. Table S11. Extract recoveries and matrix eﬀects of seven target constituents and two ISs for the cocktail analysis. Table S12. Michaelis constant (Km) determined for the enzymatic reaction of the probe substrates and the inhibition IC50 values were measured for the positive inhibitors of seven CYP450s. Table S13. Responses (% control) of HSYA, GRb1, GRd, GRe, GRg1, and NGR1 at their Cmax levels in the rat plasma. Author Contributions: Conceptualization, P.T. and Y.J.; data curation, J.C.; funding acquisition, X.G. 3.5. Discussion In order to search for the single components contained in CNP responsible for the inhibition of CYP1A2 and other CYP450s, 18 main components from CNP were evaluated for their inhibition on CYP450s. The results showed that GRg1 (A1), GRb1 (A2), GRd (A3), 6-hydoxykaempferol-3-O-glucoside (A6), kaempferol-3-O-glucoside (A7), anhydroxysaﬄor yellow B (AHSYB, A8), hydroxysaﬄor yellow A (HSYA, A9), 6-hydroxykaempferol-3,6-di-O-glucoside (A13), quercetin (A16), kaempferol (A17), and 6-hydroxykaempferol (A18) were the main active components for the CYP450 inhibition, and GRg1 11 of 12 Pharmaceutics 2020, 12, 180 (A1), GRb1 (A2), GRd (A3), quercetin (A16), kaempferol (A17), and 6-hydroxykaempferol (A18) were the main active components for CYP1A2 inhibition. To further discuss the possibility of in vivo interaction between CTE and NGTS, the inhibitions of GRg1, GRb1, GRd, HSYA, NGR1, and GRe to CYP450s at their Cmax levels were calculated by their respective “dose–response curve”. The results (Table S13) showed GRg1, GRb1, GRd, HSYA, NGR1, and GRe could not signiﬁcantly inhibit CYP450s at their Cmax levels, which is in accordance with the results that the combination use of CTE and NGTS can only increase the system exposures of these components to some extent. 4. Conclusions and Y.J.; investigation, J.C., Y.L., M.S., H.M., Y.Q., J.W., and M.L.; methodology, J.C., X.G., Y.L., M.S., and M.Z.; project administration, Y.J.; resources, P.T. and Y.J.; supervision, Y.J.; visualization, J.C.; writing—review and editing, J.C., Y.S., and Y.J. All authors have read and agreed to the published version of the manuscript. Funding: This work was ﬁnancially supported by National Natural Science Foundation of China (no. 81573684), Beijing Municipal Science and Technology Project (no. Z181100002218028), and National Key Technology R&D Program “New Drug Innovation” of China (no. 2018ZX09711001-008-003 and 2012ZX09103201-036). 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[CrossRef] [PubMed] 2. Meng, Y.; Du, Z.; Li, Y.; Wang, L.; Gao, P.; Gao, X.; Li, C.; Zhao, M.; Jiang, Y.; Tu, P.; et al. Integration of metabolomics with pharmacodynamics to elucidate the anti-myocardial ischemia eﬀects of combination of notoginseng total saponins and saﬄower total ﬂavonoids. Front. Pharmacol. 2018, 9, 667. [CrossRef] [PubMed] 2. Meng, Y.; Du, Z.; Li, Y.; Wang, L.; Gao, P.; Gao, X.; Li, C.; Zhao, M.; Jiang, Y.; Tu, P.; et al. Integration of metabolomics with pharmacodynamics to elucidate the anti-myocardial ischemia eﬀects of combination of notoginseng total saponins and saﬄower total ﬂavonoids. Front. Pharmacol. 2018, 9, 667. [CrossRef] [PubMed] 3. Han, S.Y.; Li, H.X.; Bai, C.C.; Wang, L.; Tu, P.F. Component analysis and free radical-scavenging potential of Panax notoginseng and Carthamus tinctorius extracts. Chem. Biodivers. 2010, 7, 383–391. [CrossRef] [PubMed] 12 of 12 12 of 12 Pharmaceutics 2020, 12, 180 4. Zhou, D.; Andersson, T.B.; Grimm, S.W. 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Combinatorial metabolism notably aﬀects human systemic exposure to ginsenosides from orally administered extract of Panax notoginseng roots (Sanqi). Drug Metab. Dispos. 2013, 41, 1457–1469. [CrossRef] [PubMed] q 18. Ung, C.Y.; Li, H.; Cao, Z.W.; Li, Y.X.; Chen, Y.Z. Are herb-pairs of traditional Chinese medicine distinguishable from others? Pattern analysis and artiﬁcial intelligence classiﬁcation study of traditionally deﬁned herbal properties. J. Ethnopharmacol. 2007, 111, 371–377. [CrossRef] [PubMed] 18. Ung, C.Y.; Li, H.; Cao, Z.W.; Li, Y.X.; Chen, Y.Z. Are herb-pairs of traditional Chinese medicine distinguishable from others? Pattern analysis and artiﬁcial intelligence classiﬁcation study of traditionally deﬁned herbal properties. J. Ethnopharmacol. 2007, 111, 371–377. [CrossRef] [PubMed] 19. Song, Y.; Zhang, N.; Jiang, Y.; Li, J.; Zhao, Y.; Shi, S.; Tu, P. Simultaneous determination of aconite alkaloids and ginsenosides using online solid phase extraction hyphenated with polarity switching ultra-high performance liquid chromatography coupled with tandem mass spectrometry. RSC Adv. 2015, 5, 6419–6428. [CrossRef] 20. Rendic, S.; Guengerich, F.P. Survey of human oxidoreductases and cytochrome p450 enzymes involved in the metabolism of xenobiotic and natural chemicals Chem Res Toxicol 2015 28 38 42 [CrossRef] [PubMed] 19. Song, Y.; Zhang, N.; Jiang, Y.; Li, J.; Zhao, Y.; Shi, S.; Tu, P. Simultaneous determination of aconite alkaloids and ginsenosides using online solid phase extraction hyphenated with polarity switching ultra-high performance liquid chromatography coupled with tandem mass spectrometry. RSC Adv. 2015, 5, 6419–6428. [CrossRef] 19. Song, Y.; Zhang, N.; Jiang, Y.; Li, J.; Zhao, Y.; Shi, S.; Tu, P. Simultaneous determination of aconite alkaloids and ginsenosides using online solid phase extraction hyphenated with polarity switching ultra-high performance liquid chromatography coupled with tandem mass spectrometry. RSC Adv. 2015, 5, 6419–6428. [CrossRef] 20. Rendic, S.; Guengerich, F.P. Survey of human oxidoreductases and cytochrome p450 enzymes involved in the metabolism of xenobiotic and natural chemicals. Chem. Res. Toxicol. 2015, 28, 38–42. [CrossRef] [PubMed] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2020 by the authors. References Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W4287995434	https://zenodo.org/records/3605928/files/92%20Dalit%20of%20Odisha%20A%20Case%20Study%20of%20the%20Dombos%20in%20Rayagada%20District.pdf	English	null	Dalit of Odisha: A Case Study of the Dombos in Rayagada District	Zenodo (CERN European Organization for Nuclear Research)	2,019	cc-by	2,410	INRODUCTION from the Caste Hindus in return Gandhi accepting measures along these lines. In 2001, the quality of life of the Dalit population in India was not similar to that of the overall Indian population, on metrics such as access to health care, life expectancy, education attainability, access to drinking water and housing. In 2010 Dalits received international attention due to a portrait exhibition by Marcus Perkins that depicted Dalits. from the Caste Hindus in return Gandhi accepting measures along these lines. In 2001, the quality of life of the Dalit population in India was not similar to that of the overall Indian population, on metrics such as access to health care, life expectancy, education attainability, access to drinking water and housing. In 2010 Dalits received international attention due to a portrait exhibition by Marcus Perkins that depicted Dalits. The term “Dalit,” in Sanskrit is both a noun and an adjective. As a noun, Dalit can be used for all three genders, masculine, feminine and neuter. It has been derived from the root word “dal’’ which means to crack, split etc. The word ‘Dalit” has come to mean that things or persons come under, the category of downtrodden, scattered, crushed, destroyed etc. The term “Dalit” has also its parallel in Hebrew with the root “dal” means, weak, poor, helpless etc. Dalits have been the most degraded, downtrodden, exploited and the least educated people in our society. They have been socially, culturally, economically and politically subjugated and marginalized through three thousand years of our history and remain so, even after half-a-century of protective discrimination (as Scheduled Caste) under the aegis of the Government of India. The Schedule Caste and Schedule Tribes comprise about 16.6 % and 8.6 %, respectively of India’s population (according to the 2011 census). The Constitution of India, introduced after the country gained independence included measures to improve the socio- economic conditions of Dalits. Besides abolishing untouchability, these include the reservation system, a means of positive discrimination that created the classifications of Scheduled Castes, Scheduled Tribes and Other Backward classes (OBCs). Communities that were categorised as being one of those groups were guaranteed a percentage of the seats in the national and state legislatures, as well as in government jobs and places of education. ABSTRACT How to cite this paper: Paramananda Naik "Dalit of Odisha: A Case Study of the Dombos in Rayagada District" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-4 \| Issue-1, December 2019, pp.483-485, URL: https://www.ijtsrd.com/papers/ijtsrd29 601.pdf IJTSRD29601 The term “Dalit,” in Sanskrit is both a noun and an adjective. As a noun, Dalit can be used for all three genders, masculine, feminine and neuter. It has been derived from the root word “Dal” which means to crack, split etc. The word ‘Dalit’ has come to mean that things or persons come under, the category of downtrodden, scattered, crushed, destroyed etc. The listed 93 Scheduled Caste communities of Odisha are known as Dalit in Odishan social system. But, in fact, the untouchables among the Scheduled Castes are the Dalits. The Schedule Caste and Schedule Tribes comprise about 16.6 % and 8.6 %, respectively of India’s population (according to the 2011 census). There are total population in socially untouchables and economically poor in the lowest point of social structure in Odisha. After of independence of India, their social, political, education justice, economic status has not been changed as expected. The examination and analysis of present status of Dalits will be made through empirical study. The outcome of this study will draw a clear picture of the position of Dalits in Odisha and it will motivate government and non- government agencies to take initiative to promote Dalits. Copyright © 2019 by author(s) and International Journal of Trend in Scientific Research and Development Journal. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0) (http://creativecommons.org/licenses/by /4.0) KEYWORDS: Dalit, Scheduled Caste, Downtrodden, Scattered, destroyed INRODUCTION KEYWORDS: Dalit, Scheduled Caste, Downtrodden, Scattered, destroyed License (http:// /4.0) KEYWORDS: Dalit, Scheduled Caste, Downtrodden, Scattered, destroyed Dalit of Odisha: A Case Study of the Dombos in Rayagada District Paramananda Naik Ph.D Research Scholar, Utkal, University Vani, Vihar Bhubaneswar, Odisha, India @ IJTSRD \| Unique Paper ID – IJTSRD29601 \| Volume – 4 \| Issue – 1 \| November-December 2019 Page 483 arch and Development (IJTSRD) @ www.ijtsrd.com eISSN: 2456-6470 In Odisha, the Dom, Dombo, Duria, Pano etc belong to Schedule Caste community. The total number of 93 Castes are there (Census 2011) in Odisha. Dombo is the major community under Schedule Castes in Rayagada district. Dom / Dombo in Oriya Dom / Dombo are recognized as particularly vulnerable groups among the Schedule Castes. The Dombo is considered inferior in social status to the sanction of Antera Oriya Dom of the Caste Dom / Dombo. About 80 percent population belong to Dalit communities in Rayagada district out of the total population. Where the Schedule Caste communities are 20 percent and Schedule Tribes are around 60 percent. The livelihood status of the Schedule Caste communities is intermediaries, lenders and small traders in tribal area of this district. The women empowerment is very weak in this area. The women of the Schedule Caste communities go to sale the necessary goods of people to every village and door to door. This is the main source of livelihood of women in this a district. The relationship between Dongaria and Dombo is symbiotic. They is also living by wages-earning, excepting a few household, who depends on cultivation and agricultural labour. But major portion of their annual income comes from agricultural labour. Almost all household derive income by adopting agricultural labour as main or subsidiary occupation. Thus it is appropriate to consider agriculture labour as their main occupation. Besides wage-earning and cultivation, they also earn through other activities, like Drum-beating, small business in nearby weekly markets, rickshaw pulling and engaging themselves in trading animals, like Cow, Buffalo, Bullock, etc. Some of them also collect raw hides and supply them to tanners through middle men. In this district in recent past, the Dalit were treated low caste with the stigma of untouchability. Their lowest status in the caste hierarchy and untouchability played a significant role in arresting the progress and development of said community. Generally the Dalit live together in separate ward which is usually located at the outer area of a town of the main hamlet or village where upper Caste People live. The stigma of untouchability which is attached to them deprives them of using water from the same source used by higher Caste groups in the villages. The influential and established higher Caste groups never allow the Dalit to take water from the existing Tube-wells and wells in the village. Conclusion: Dombos, (Dalit) despite their education and a amount of economic advancement are unable to improve their status by their individual efforts most of them in spite of more than two decades of government efforts to improve their economic and social position, remain desperately poor. Semi-literate or illiterate, subject to discrimination and economic exploitation and with no realistic prospect for improvement of their condition. Relishing the significance of education and occupation as mechanisms of status determination and power the central government as well as the statue government diverted broth money and efforts to strengthen the programmer of education and occupational upliftment of the Harijans. In contemporary Odisha, Dalits have been victims of voidance and atrocities. They have been killed and their females are raped. They are victims to loot and their houses are set on fire. They are also denied minimum wages. Reference [1] Beteille, A., “Changing Pattern of Stratification in Taniore Village”, University of California USA, 1971. [2] Chatterjee, S. K., the Scheduled Castes in India. Gyan Publishing House New Delhi, 1996. [3] Chowdhury B and Mishar, H. S., Action plan for Scheduled Castes in Puri Municipality Tribal and Harijan Research cum Training Institute Bhubaneswar1982, [4] Das, Bhaskar., “Social and Economy Life of South Odisha” Calcutta 1985. [5] Ganguly, Debjani., Caste and Dalit life world : Postcolonial Perspectives,: Orient Longman , New Delhi 2008. [6] Krishna. S and Samudrala., A. K., “Dalit and Human Rights” Serils Publication New Delhi 2007. arch and Development (IJTSRD) @ www.ijtsrd.com eISSN: 2456-6470 Thus they face lot of problems in getting drinking water. Rice is staple food of these communities. They are fond of non- vegetarian dishes, but they required sufficient quantity and quality of rice. Therefore rice is there primary food and constitutes major item of their daily meals. The cooked vegetable, fish or meat are eaten as curry with rice. But quantity and quality of curry varies from house to house based on their economic condition. In the rural area, of this district the wages and employment situation is not up to the mark. They do not get ample opportunities for occupational mobility. As their economic status is low and educational attainment is not satisfactory shifting from the traditional occupation is very rare. In the Present study in the village, it is evident that quite a small number of persons engage in Drum beating and rope-making, small business. Service and labourer except agricultural activities. Objectives of the Study 1 To education the History and socio cultural life of the 3. To provide suggestion for protection against exclusion and discrimination in the present society by encouraging their effective participation in the general Economic, Social and Political processes in the country. 3. To provide suggestion for protection against exclusion and discrimination in the present society by encouraging their effective participation in the general Economic, Social and Political processes in the country. Research Methodology The study will be based on primary source materials like, Government Reports, various agreement made by Government of India etc. and Secondary sources like, books, journals, articles, Internet, Newspapers etc. The Seminar Library of the P. G .Department of History, Parija Library and Ancient Indian History, Culture and Archaeology, Department Utkal University, Bhubaneswar, Centre for Ambedkar Studies, Utkal University, the Harekrushna Mahatab State Library, Bhubaneswar, Odisha State Museum, Bhubaneswar, will be quite helpful in collecting the relevant materials. The Libraries in Hyderabad and New Delhi will be visited for the collection of data for the proposed research work. For the collection of Government documents concerned offices or libraries of the Government Department will be visited. Internet will also be an important source of information for present research. An empirical historical method will be adopted for the present research work. INRODUCTION The system has its origins in the 1932 Poona Pact between Ambedkar and Gandhi, when Ambedkar conceded his demand that the Dalits should have an electorate separate Odisha, a State in the union of India where in 51,311 villages and around 4,19,74,218 population are living (Census 2011). Rich in history and geography, the State is today one of the poorest states in the country. There are 22.85 percent of Odisha’s population belong to Tribal communities and around 17 percent population are belonging to Scheduled Caste in this State. The people are accustomed to celebrate a succession of festivals with great gusto round the year and are also reputed for producing by indigenous methods unique handicraft and excellent artefacts. Scheduled Caste population of the district constitutes 13.92 percent and among them the major Caste group is Dombo (76.12 per cent), within the Dalit community, there are many divisions into sub-castes. Dalits are dividing into leather workers, street sweepers, cobblers, agricultural works and manual “scavengers”. The latter group, considered the lowest of the low and officially estimated of dead animals, and cleaning human excreta. Approximately three-quarters of the country’s four million people who are bonded labours or Dalits. Their job rarely provided income for Dalits to feed their families or to send their children to school. As a effect, many Dalits are needy, uneducated and illiterate. @ IJTSRD \| Unique Paper ID – IJTSRD29601 \| Volume – 4 \| Issue – 1 \| November-December 2019 Page 483 l of Trend in Scientific Research and Development (IJTSRD) @ www.ijtsrd.com eISSN: 2456-6470 @ IJTSRD \| Unique Paper ID – IJTSRD29601 \| Volume – 4 \| Issue – 1 \| November-December 2019 Page 484 Objectives of the Study [7] Mallaiah, L. C. and Ratnakumari. L., “Economic Development and Dalit Agricultural Workers” Abhijeet Publication New Delhi 2007. 1. To education the History and socio-cultural life of the Dalit communities of Rayagada District. 2. To style an analytical study of the economic and Social status of Scheduled Caste in Rayagada District. @ IJTSRD \| Unique Paper ID – IJTSRD29601 \| Volume – 4 \| Issue – 1 \| November-December 2019 Page 484 International Journal of Trend in Scientific Research and Development (IJTSRD) @ www.ijtsrd.com eISSN: 2456-6470 International Journal of Trend in Scientific Research and Development (IJTSRD) @ www.ijtsrd.co [8] Misha, S, N., “Political Socialisation in Rural India”, Inter-India Delhi, 1980. [17] Rout, S, P., “Dom Exploitation in Kondh Village of Koraput District” Adivasi Vol-I No 1964-1965. [8] Misha, S, N., “Political Socialisation in Rural India”, Inter-India Delhi, 1980. [17] Rout, S, P., “Dom Exploitation in Kondh Village of Koraput District” Adivasi Vol-I No 1964-1965. [9] Mishra, P. K., “Harijans in Hindu and Tribal Structures”, Discovery Publication House New Delhi, 1972. [18] Saksena, H. S., “Safeguards for Schedule Caste and Tribal”,: Uppal Publication House, New Delhi 1981. [10] Mohanti K. K., “Social Mobility and Caste Dynamics”, Rawat Publication Jaipur 1993. [19] Senapati, N. A and N. K. Sahu Odisha District Gazetteer Koraput (Revised Report 1966) Chapter III people pp.93-97 1966. [11] Mishar N., “Exploitation and Atrocities’ on the Dalit in India”, Kalpaz Publication Delhi 2004. [20] Sethi, B. N., Lobour Out Migration Odisha, A Social Economise Study) Mahit Publication New Delhi, 2007. [12] Pradhan A, C., “The Emergence of Depressed Class”, Bhubaneswar 1986. [21] Thorat, Sukhadeo., Dalits in India Search for a Common Destiny,: Sage Publications India Pvt Ltd, New Delhi 2009. [13] Prasad, R. R., “Profile of the Scheduled Caste” in “Readings on Scheduled Caste” (National institute of Rural development Hyderabad), 2010. [22] Tripati, R. B., “Dalit Sub-Human Society”, South Asia Books, 1994. [14] Bakshi, R. K., “Dalit and Human Rights”, Akhand Publication New Delhi 2010. [23] Zakir. Abedi., “Dalit Social Empowerment in India” Arise Publishers and Distributors New Delhi 2010. [15] Ram, J., “Castes Challenge in India” Vision, New Delhi, 1980. [24] Zelliot. E., “From Untouchables to Dalit”, Manohar Publishers, New Delhi, 1992. [16] Kumar Ravindra and Mrs. Kumar Kamles. “Voice of Babashed Ambedkar” Gyan Publishing, New Delhi 2007.
W2788690918.txt	http://eprints.gla.ac.uk/162166/1/162166.pdf	en		Areca Nut Chewing and the Risk of Re-hospitalization and Mortality Among Patients With Acute Coronary Syndrome in Pakistan	Journal of preventive medicine and public health	2,018	cc-by	6,158	Journal of Preventive Medicine & Public Health Original Article J Prev Med Public Health 2018;51:71-82 • https://doi.org/10.3961/jpmph.17.189 pISSN 1975-8375 eISSN 2233-4521 Areca Nut Chewing and the Risk of Re-hospitalization and Mortality Among Patients With Acute Coronary Syndrome in Pakistan Muhammad Tariq Karim1, Sumera Inam2, Tariq Ashraf3, Nadia Shah2, Syed Omair Adil4, Kashif Shafique2,5 Research Evaluation Unit, College of Physicians and Surgeons Pakistan, Karachi, Pakistan; 2School of Public Health, Dow University of Health Sciences, Karachi, Pakistan; 3National Institute of Cardiovascular Diseases, Karachi, Pakistan; 4Department of Research, Dow University of Health Sciences, Karachi, Pakistan; 5Institute of Health and Wellbeing, Public Health, University of Glasgow, Glasgow, UK 1 Objectives: Areca nut is widely consumed in many parts of the world, especially in South and Southeast Asia, where cardiovascular disease (CVD) is also a huge burden. Among the forms of CVD, acute coronary syndrome (ACS) is a major cause of mortality and morbidity. Research has shown areca nut chewing to be associated with diabetes, hypertension, oropharyngeal and esophageal cancers, and CVD, but little is known about mortality and re-hospitalization secondary to ACS among areca nut users and non-users. Methods: A prospective cohort was studied to quantify the effect of areca nut chewing on patients with newly diagnosed ACS by categorizing the study population into exposed and non-exposed groups according to baseline chewing status. Cox proportional hazards models were used to examine the associations of areca nut chewing with the risk of re-hospitalization and 30-day mortality secondary to ACS. Results: Of the 384 ACS patients, 49.5% (n =190) were areca users. During 1-month of follow-up, 20.3% (n =78) deaths and 25.1% (n=96) re-hospitalizations occurred. A higher risk of re-hospitalization was found (adjusted hazard ratio [aHR], 2.05; 95% confidence interval [CI], 1.29 to 3.27; p=0.002) in areca users than in non-users. Moreover, patients with severe disease were at a significantly higher risk of 30-day mortality (aHR, 2.77; 95% CI, 1.67 to 4.59; p<0.001) and re-hospitalization (aHR, 2.72; 95% CI, 1.73 to 4.26; p<0.001). Conclusions: The 30-day re-hospitalization rate among ACS patients was found to be significantly higher in areca users and individuals with severe disease. These findings suggest that screening for a history of areca nut chewing may help to identify patients at a high risk for re-hospitalization due to secondary events. Key words: Areca nut, Acute coronary syndrome, Mortality, Re-hospitalization, Pakistan INTRODUCTION Received: November 17, 2017 Accepted: January 12, 2018 Corresponding author: Kashif Shafique, PhD School of Public Health, Dow University of Health Sciences, Suparco Road, KDA Scheme-33, Karachi 74200, Pakistan E-mail: k.shafique@duhs.edu.pk This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/bync/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Approximately 600 million individuals throughout the world chew areca nut, making it the fourth leading form of substance abuse worldwide, and more than a quarter of areca nut users live in South Asia [1,2]. Areca nut chewing is emerging as a public health challenge in many Western countries due to the immigration of youth from Asia and Africa to these coun- Copyright © 2018 The Korean Society for Preventive Medicine 71 Muhammad Tariq Karim, et al. tries. Areca nuts are the main component of the vast majority of commonly consumed chewing products, including betel quid (paan), gutka, and supari. Areca nut is consumed in several forms, including green/unripe, ripe, raw, boiled/roasted, or fermented, in high-consuming countries such as India (where 46.4% of males and 20% of females consume gutka), Pakistan (with areca nut consumption rates of 50.3% for males and 28.5% for females) [3], Nepal, Bangladesh, Sri Lanka, Thailand, Malaysia, Indonesia, China, Cambodia, and Papua New Guinea. Additional sweeteners and flavors such as cloves, amber, coconut, mace, sugar crystals, nutmeg, camphor; tobacco, and lime are added as flavor enhancers [2]. Several studies have shown that areca nut chewing is linked with cancer of the oral cavity, esophagus, and liver, as well as cirrhosis of the liver [4-10]. Furthermore, some evidence also suggests that areca nut chewing is linked with obesity, type 2 diabetes mellitus (DM), hypertension (HTN), dyslipidemia, metabolic syndrome, and chronic kidney disease [11-15], which are also well-known risk factors of cardiovascular disease (CVD) [16,17]. CVD is still the leading cause of mortality globally, and the South Asian population is expected to significantly contribute to global CVD morbidity and mortality in decades to come. Evidence from prospective studies suggests that areca nut chewing significantly amplifies the risk of all-cause and cardiovascular mortality [18,19]. Betel quid causes instant effects of vasoconstriction, an elevated pulse rate, and raised blood pressure [20]. Since it contains arsenic and manganese, it may increase the risk of HTN in chewers. Further, it also stimulates the sympathetic nervous system, resulting in the secretion of adrenal medullary catecholamine, which is linked to elevated homocysteine levels, another risk factor for heart disease. Moreover, periodontal disease, a known risk factor for CVD, is also a consequence of consuming betel quid [21]. A pooled analysis of 12 studies reported increased risk of developing CVD’s (relative risk [RR], 1.20, p<0.05) and of all-cause mortality (RR, 1.21, p<0.05) among areca nut chewers compared with non-chewers [22]. Among the forms of CVD, acute coronary syndrome (ACS) accounts for approximately 3.5 million hospital admissions worldwide [23]. Considerable disparities exist in the incidence and mortality of ACS from country to country. These disparities are also partly explained by differences in lifestyle habits and the prevalence of various cardiovascular risk factors [24]. Some evidence suggests that long-term areca nut chewing 72 increases the risk of ACS by 3-fold (odds ratio [OR], 3.5; 95% confidence interval [CI], 2.0 to 6.2) [25]. However, there is little published evidence regarding the relationship of long-term areca nut chewing with the prognosis of patients who develop ACS. Given that 15 to 30% of patients with ACS either are rehospitalized or die within 30 days of the acute event, it is important to understand the effects of areca nut chewing on the prognosis of these patients. Therefore, the purpose of the study was to determine the short-term (30-day) risk of re-hospitalization and mortality among ACS patients according to baseline areca nut chewing. METHODS Study Setting This study was a hospital-based prospective cohort study conducted in 2014-2015. The study was conducted at the Department of Cardiology, Civil Hospital Karachi and the National Institute of Cardiovascular Diseases. Both of these are tertiary care hospitals, serving more than 60% of CVD patients in Karachi, the largest city of Pakistan, at low cost. Cohort Selection ACS represents “a set of clinical sign and symptoms similar to acute myocardial ischemia, covering clinical conditions from unstable angina to none-ST-segment elevation myocardial infarction (NSTEMI) to ST-segment elevation myocardial infarction (STEMI)” [26]. Patients were diagnosed with ACS by a consultant cardiologist at the time of presentation with any of the following conditions: STEMI: “A history of typical chest pain for more than 20 minutes with ST elevation at the J point of ≥2 mm (0.2 mV) in at least 2 contiguous leads in males ≥1.5 mm (0.15 mV) in females in leads V2-V3, or ≥1 mm (0.1 mV) in other contiguous chest or the limb leads.” NSTEMI: “A history of typical chest pain for >20 minutes with electrocardiogram (ECG) changes such as ST depression and raised creatine kinase-MB (CKMB) enzymes and troponin levels.” Unstable angina: The presence of 1 or more of the following presentations without elevated CKMB enzymes or troponin was labeled as rest angina (usually lasting >20 minutes), new-onset angina (<2 months previously), or an increased pattern of occurrence (increasing in duration, intensity, and/ or frequency). Risk of Areca Chewing in ACS Patients Only newly admitted patients 30-70 years of age with a diagnosis of ACS at the time of admission were included in the study. Subjects with congenital heart disease, any previous or family history of ACS, or valvular or rheumatic heart disease were excluded from this study. Data Collection Tool A standardized questionnaire was used by a team of welltrained researchers to interview patients, gathering information about their demographic characteristics, lifestyle, health behaviors, and history of chewing areca nut. All information was collected by face-to-face interviews with patients within the first 48 hours of hospital admission. The socio-demographic measures included age (30-40, 4050, 50-60, and >60 years), sex (male or female), monthly income (used to define socioeconomic status [SES] as below average or average) and employment status (employed or unemployed). Health behaviors, particularly physical activity, were captured through self-reporting, and patients who reported no activity or less than 150 minutes of activity per week were labeled as physically inactive. Patients were also asked about their smoking status, and based on their responses were classified as “never smokers (those who had never smoked a cigarette), current smokers (those who reported smoking at least 100 cigarettes during their lifetime and reported smoking every day or some days at the time of participation), and ex-smokers (individuals who had quit smoking for at least 6 months)”. Physical measures included measurements of weight, height, blood pressure, and body mass index (BMI, kg/m2). BMI was categorized according to the standard World Health Organization BMI cut-offs that classify “normal weight as <23.0 kg/m2, overweight as 23.0-26.9 kg/m2, and obese as ≥27.0 kg/m2” [27]. Patients were classified as having DM if they had “a self-reported history of DM, were on anti-diabetic medications, or had abnormal fasting blood glucose levels (blood glucose concentration ≥110 mg/dL)”. HTN was defined as “a blood pressure ≥140 (systolic)/90 (diastolic) mmHg, previously diagnosed HTN, the usage of anti-hypertensive medications”. According to the guidelines of the American Heart Association, the current pathway for assessing patients who may have ACS is based on the following 4 main diagnostic tools: “clinical history, ECG results, levels of cardiac markers, and the results of stress testing. On the basis of this initial information, patients are assigned to one of 4 categories: a non-cardiac diag- nosis, chronic stable angina, possible ACS, or definite ACS”. Based on the diagnosis made by the cardiologist, we generated a variable for the initial severity of disease. Respondents with ‘unstable/mild angina’ were classified as having ‘non-severe disease’, whereas ‘possible myocardial infarction/definite’ ACS were considered to indicate ‘severe initial disease’. Exposure Definition Patients who persistently consumed areca nut on daily basis (on average 2-3 times a day) for more than 5 years were considered to be exposed [28,29], and were coded as ‘yes’ for this variable, while those who never used or had quit consuming areca for more than a year were considered to be non-exposed and coded as ‘no’ for this variable. Patients categorized as exposed were asked about the form of areca nut they used. Respondents who consumed “unripe or ripe; whole or sliced; raw, roasted, or sun-dried; boiled or soaked in water; or fermented (under mud) areca nut” were considered to consume areca nut ‘without additives’. In contrast, “mixtures of areca nut and flavoring ingredients with processed tobacco leaves, slaked lime, catechus, sweeteners, spices, piper betel, and essences” were considered to be forms ‘with additives or tobacco’ [30]. Follow-up The follow-up of ACS patients started once they were discharged from the hospital. Patients were followed regularly in the outpatient clinic, and the final outcomes were recorded after 30 days had passed from the time of discharge. The outcomes: 30-day mortality and 30-day re-hospitalization were measured separately. Mortality was defined as death within 30 days due to any cardiac problem from the index admission date, and re-hospitalization was defined as readmission to the hospital due to any cardiac problem within 30 days from the discharge date. Therefore, 2 survival times were defined: the time from entry to death from CVD (within 30 days) and the time from entry to re-hospitalization (within 30 days) due to any cardiac problems. For patients who did not return in 30 days, a telephone call was made by the researchers to evaluate their vital status and any cardiac re-hospitalization event in the 30 days after discharge. Only the first re-hospitalization within 30 days of discharge was considered a 30-day readmission. However, if a readmitted patient died within this 30-day period, then in the re-hospitalization multivariate model, that patient’s follow-up was truncated at the date of re-hospitalization. 73 Muhammad Tariq Karim, et al. Sample Size Estimation The sample size was calculated using the expected proportions of mortality in the exposed (30%) [31] and unexposed (15%) groups with a 95% confidence level and 80% power of the test. Accordingly, the minimum sample size for this study was 268, which was upsized to reduce estimation bias from loss to follow-up. The final estimated sample size was 384. Ethical Approval The study protocol was approved by the institutional review board (IRB) of Dow University of Health Sciences, Karachi, Pakistan (IRB no. IRB-514/DUHS/-14). Informed consent was obtained from all study participants prior to conducting the study. Statistical Analysis For data analysis, Stata version 11 (StataCorp., College Station, TX, USA) was used. Demographic characteristics, history of substance use, and the prevalence of comorbidities in patients with ACS were recorded. The statistical analysis included the chi-square test, as well as Cox proportional hazards regression with a significance level of 0.05 to examine the associations of areca use and other explanatory variables with 30-day mortality and re-hospitalization, which were the dependent variables used in the current study. Univariate analyses were performed using a Cox proportional hazards regression model to estimate the magnitude of the effect of areca nut chewing on 30-day mortality and re-hospitalization of ACS patients. Multivariable proportional hazards regression models were further applied, and the adjusted hazard ratio (aHR) for areca nut chewing was computed after adjusting for the confounders identified in the univariate analyses (p=0.25) separately for both 30-day mortality and re-hospitalization, including the following covariates: age, sex, BMI, smoking status, initial severity of disease, SES, exercise status, employment status, DM, and HTN. RESULTS A total of 384 patients with ACS were included in this study, among whom 38.2% (n =147) were diagnosed with severe disease. During 1-month follow-up, death occurred in 20.3% (n=78) of the patients and re-hospitalization in 25.0% (n=96). Of the patients, 49.5% (n=190) were areca nut users, among whom 78.9% (n=150) were users of areca nut with additives. 74 A significant association was observed between areca use and 30-day re-hospitalization (p<0.001). Further, significant associations were found between disease severity and 30-day mortality (p<0.001) and re-hospitalization (p<0.001). Among the patients with severe initial disease, 39.5% (n =58) were re-hospitalized and 34.0% (n =50) died within the 1-month follow-up. Of the ACS patients, 72.1% (n=277) were male and 27.9% (n=107) were female. Males were more likely to use areca nut with tobacco additives than females (p=0.008). Death within 30 days occurred in 22.0% (n=61) of the male patients and 15.9% (n=17) of the female patients, and re-hospitalization took place in 25.6% (n=71) of the male patients and 23.4% (n=25) of the female patients. However, no statistically significant association was found between sex and 30-day mortality (p=0.18) or 30-day re-hospitalization (p=0.65). Of the 286 smokers, 29.6% (n=114) were current smokers and 18.8% (n=72) were ex-smokers. The 30-day mortality rate was 32.5% (n=37) and 13.9% (n=10) in current smokers and ex-smokers, respectively, and the 30-day re-hospitalization rate was 35.1% (n=40) and 19.4% (n=14) in current smokers and ex-smokers, respectively. Respondents with a normal BMI (n =80), overweight (n =200), and obesity (n =104) had 30day mortality rates of 23.7% (n=19), 24.0% (n=48), and 10.6% (n =11), respectively, and 30-day re-hospitalization rates of 28.8% (n=23), 27.0% (n=54), and 18.3% (n=19), respectively. The chi-square test showed significant associations of current smoking with 30-day mortality (p<0.01) and re-hospitalization (p =0.01). Furthermore, BMI was also significantly (p = 0.01) associated with mortality among the ACS patients. Among the patients with DM, 12.9% (n=17) died, whereas 87.1% (n=115) remained alive over the 30-day follow-up, and 19.7% (n=26) were re-hospitalized. Similarly, among patients with HTN, 16.4% (n=32) and 23.6% (n=46) died and were rehospitalized, respectively. A significant association was observed between DM and 30-day mortality (p=0.009). There were no significant associations of mortality and re-hospitalization with age, SES, employment status, or HTN. Table 1 summarizes the basic characteristics of the study sample. Univariate Analyses In the univariate Cox proportional hazards regression analyses, areca nut users had a higher risk of 30-day re-hospitalization (hazard ratio [HR], 2.09; 95% CI, 1.37 to 3.18; p<0.001) and 30-day mortality (HR, 1.43; 95% CI, 0.91 to 2.24; p=0.11) Risk of Areca Chewing in ACS Patients Table 1. Baseline characteristics by status mortality and re-hospitalization (n=384) Characteristics Total Re-hospitalization Mortality Yes No p-value Yes No p-value1 <0.001 33 (17.0) 161 (82.9) 0.10 45 (23.7) 145 (76.3) 1 Areca use No 194 (50.5) 33 (17.0) 161 (82.9) Yes 190 (49.5) 63 (33.2) 127 (66.8) Areca type (n=190) Without additive 40 (21.1) 14 (35.0) 26 (65.0) 150 (78.9) 49 (32.7) 101 (67.3) Never smoked 198 (51.5) 42 (21.2) 156 (78.8) Current smokers 114 (29.6) 40 (35.1) 74 (64.9) 72 (18.8) 14 (19.4) 58 (80.6) No 237 (61.7) 38 (16.0) 199 (83.9) Yes 147 (38.2) 58 (39.5) 89 (60.5) 30-40 50 (13.0) 12 (24.0) 38 (76.0) 40-50 116 (30.2) 31 (26.7) 85 (73.3) 30 (25.9) 86 (74.1) 50-60 121 (31.5) 23 (19.0) 98 (80.9) 19 (15.7) 102 (84.3) >60 Sex 97 (25.3) 30 (30.9) 67 (69.1) 20 (20.6) 77 (79.4) Male 277 (72.1) 71 (25.6) 206 (74.4) 61 (22.0) 216 (77.9) Female 107 (27.9) 25 (23.4) 82 (76.6) 17 (15.9) 90 (84.1) Additive 0.78 12 (30.0) 28 (70.0) 33 (22.0) 117 (78.0) 31 (15.6) 167 (84.3) 37 (32.5) 77 (67.5) 10 (13.9) 62 (86.1) 28 (11.8) 209 (88.2) 50 (34.0) 97 (65.9) 9 (18.0) 41 (82.0) 0.29 Smoking status Ex-smokers 0.01 <0.001 Disease sverity <0.001 <0.001 Age (y) 0.23 0.65 0.26 0.18 Body mass index Normal 80 (20.8) 23 (28.8) 57 (71.2) 19 (23.7) 61 (76.3) Over weight 200 (52.1) 54 (27.0) 146 (73.0) 0.17 48 (24.0) 152 (76.0) Obese 104 (27.1) 19 (18.3) 85 (81.7) 11 (10.6) 93 (89.4) Below average 117 (30.5) 24 (20.5) 93 (79.5) 17 (14.5) 100 (85.5) Average 267 (69.5) 72 (26.9) 195 (73.0) 61 (22.8) 206 (77.2) Unemployed 159 (41.4) 33 (20.8) 126 (79.2) 25 (15.7) 134 (84.3) Employed 225 (58.6) 63 (28.0) 162 (72.0) 53 (23.5) 172 (76.4) No 343 (89.3) 91 (26.5) 252 (73.5) 71 (20.7) 272 (79.3) Yes 41 (10.7) 5 (12.2) 36 (87.8) 7 (17.1) 34 (82.9) No 252 (65.6) 70 (27.8) 182 (72.2) 61 (24.2) 191 (75.8) Yes 132 (34.4) 26 (19.7) 106 (80.3) 17 (12.9) 115 (87.1) No 189 (49.2) 50 (26.5) 139 (73.5) 46 (24.3) 143 (75.7) Yes 195 (50.8) 46 (23.6) 149 (76.4) 32 (16.4) 163 (83.6) 0.01 Socioeconomic status 0.18 0.06 Occupation 0.11 0.06 Physically active 0.04 0.59 Diabetes mellitus 0.08 0.009 Hypertension 0.52 0.05 Values are presented as number (%). 1 Chi-square test. than non-users. Moreover, patients with severe disease had a higher risk of both 30-day re-hospitalization (HR, 2.69; 95% CI, 1.79 to 4.06; p<0.001) and 30-day mortality (HR, 3.17; 95% CI, 1.99 to 5.03; p<0.001). Current smokers also had a higher risk 75 Muhammad Tariq Karim, et al. of both 30-day mortality (HR, 2.23; 95% CI, 1.38 to 3.59; p<0.001) and 30-day re-hospitalization (HR, 1.73; 95% CI, 1.12 to 2.67; p=0.01) than non-smokers. Respondents who were obese or had DM had a significantly lower risk of 30-day mortality (Table 2). Table 2. Areca nut use and risk of 30-day re-hospitalization and mortality among ACS patients: univariate analysis1 Characteristics Re-hospitalization HR (95% CI) Mortality p-value HR (95% CI) p-value Areca use No 1.00 (reference) Yes 2.09 (1.37, 3.18) 1.00 (reference) <0.001 1.43 (0.91, 2.24) 0.11 Areca type Without additive 1.00 (reference) Additive 0.98 (0.54, 1.79) 1.00 (reference) 0.97 0.74 (0.38, 1.44) 0.39 Smoking status Never smoked 1.00 (reference) Current smokers 1.73 (1.12, 2.67) 1.00 (reference) 0.01 2.23 (1.38, 3.59) <0.001 Disease severity No 1.00 (reference) Yes 2.69 (1.79, 4.06) 1.00 (reference) <0.001 3.17 (1.99, 5.03) <0.001 Age (y) 30-40 1.00 (reference) 40-50 1.16 (0.59, 2.27) 0.65 1.00 (reference) 1.47 (0.70, 3.11) 0.30 50-60 0.80 (0.40, 1.62) 0.55 0.85 (0.38, 1.86) 0.71 > 60 Sex 1.41 (0.72, 2.75) 0.31 1.17 (0.53, 2.58) 0.69 Male 1.00 (reference) Female 0.89 (0.56, 1.41) 1.00 (reference) 0.64 0.68 (0.40, 1.18) 0.17 Body mass index Normal 1.00 (reference) Over weight 0.93 (0.57, 1.52) 0.79 1.00 (reference) 1.04 (0.61, 1.77) 0.87 Obese 0.59 (0.32, 1.08) 0.09 0.42 (0.20, 0.89) 0.02 Socioeconomic status Average 1.00 (reference) Below average 0.75 (0.47, 1.19) 1.00 (reference) 0.234 0.61 (0.36, 1.05) 0.08 Occupation Un-employed 1.00 (reference) Employed 0.68 (0.45, 1.04) 1.00 (reference) 0.08 0.62 (0.39, 1.01) 0.06 Physically active No 1.00 (reference) Yes 0.41 (0.16, 1.02) 1.00 (reference) 0.057 0.76 (0.35, 1.65) 0.49 Diabetes mellitus No 1.00 (reference) Yes 0.66 (0.42, 1.03) 1.00 (reference) 0.07 0.49 (0.28, 0.84) 0.01 Hypertension No 1.00 (reference) Yes 0.87 (0.58, 1.30) 1.00 (reference) 0.51 ACS, acute coronary syndrome; HR, hazard ratios; CI, confidence interval. 1 Cut-off ≤0.25: a univariate cut-off value to shortlist variables for multivariable analyses. 76 0.64 (0.41, 1.01) 0.06 Risk of Areca Chewing in ACS Patients Multivariate Analyses Respondents who chewed areca nut had a slightly increased risk of 30-day mortality (aHR, 1.65; 95% CI, 0.99 to 2.75; p=0.05) compared with non-chewers after adjusting for sex, disease severity, SES, employment status, smoking status, DM, and HTN. However, we also observed a significant higher risk of 30-day mortality (aHR, 2.77; 95% CI, 1.67 to 4.59; p<0.001) among patients with severe disease. Females (aHR, 1.15; 95% CI, 0.54 to 2.42; p=0.71) were found to be at a higher but nonsignificant increased risk for 30-day mortality compared with males. Individuals, who had below-average SES, were unemployed, or had DM or HTN had a lower but non-significant reduced risk of 30-day mortality (Table 3). The overall findings of a significant increase in the risk of rehospitalization and a non-significant increase in the risk of Areca nut chewers had a significantly increased risk (aHR, 2.05; 95% CI, 1.29 to 3.27; p=0.002) of re-hospitalization within 30-days compared with non-chewers after adjusting for disease severity, SES, employment status, physical activity, smoking status, and DM. Patients diagnosed with severe disease had a significantly higher risk of re-hospitalization (aHR, 2.72; 95% CI, 1.73 to 4.26; p<0.001). Current smokers (aHR, 1.28; 95% CI, 0.81 to 2.04; p=0.28) had a higher but non-significantly increased risk of re-hospitalization compared with nonsmokers. Respondents who had below-average SES, were unemployed, were physically active, or had DM showed lower but non-significantly reduced risks of 30-day re-hospitalization. Table 3. Areca nut use and risk of 30-day mortality and re-hospitalization among ACS patients: multivariate analysis Characteristics Re-hospitalization1 aHR (95% CI) Mortality2 p-value aHR (95% CI) p-value Areca use No 1.00 (reference) Yes 2.05 (1.29, 3.27) 1.00 (reference) 0.002 1.65 (0.99, 2.75) 0.05 Smoking status Never smoked 1.00 (reference) Current smokers 1.28 (0.81, 2.04) 1.00 (reference) 0.28 1.61 (0.91, 2.84) 0.10 Disease severity No 1.00 (reference) Yes 2.72 (1.73, 4.26) <0.001 1.00 (reference) - - 2.77 (1.67, 4.59) <0.001 Sex Male Female 1.00 (reference) 1.15 (0.54, 2.42) 0.71 SES Average 1.00 (reference) Below average 0.87 (0.52, 1.46) 1.00 (reference) 0.62 0.75 (0.41, 1.36) 0.35 Employment status Unemployed 1.00 (reference) Employed 0.76 (0.46, 1.25) 1.00 (reference) 0.29 0.82 (0.45, 1.50) 0.52 0.15 - - Physical activity No 1.00 (reference) Yes 0.51 (0.20, 1.27) DM No 1.00 (reference) Yes 0.77 (0.46, 1.27) 0.31 1.00 (reference) - - 0.55 (0.29, 1.03) 0.06 HTN No Yes 1.00 (reference) 0.73 (0.43, 1.26) 0.27 ACS, acute coronary syndrome; aHR, adjusted hazard ratios; CI, confidence interval; SES, socioeconomic status; DM, diabetes mellitus; HTN, hypertension. 1 Adjusted for severity SES, employment status, physical activity, smoking status and DM. 2 Adjusted for severity, sex, SES, employment status, smoking status, DM and HTN. 77 Muhammad Tariq Karim, et al. Areca use: mortality Figure 1. Thirty-day mortality in acute coronary syndrome patients according to areca nut consumption stratified by DM and HTN. (A) Areca nut use and 30-day mortality risk. (B, C) Areca nut use and mortality stratified by the presence of DM. (D, E) Areca nut use and mortality stratified by the presence of HTN. DM, diabetes mellitus; HTN, hypertension. Hazard function 0.025 No areca nut consumption Areca nut consumption 0.020 0.015 0.010 0.005 0 5 10 15 20 25 A Time (d) DM: negative p-value=0.287 Hazard function Hazard function 0.025 DM: positive 0.025 0.020 0.015 0.010 p-value=0.519 0.020 0.015 0.010 0.005 0.005 0 5 10 15 20 25 B 0 5 10 Time (d) 25 20 25 C HTN: positive 0.025 p-value=0.372 p-value=0.017 0.025 Hazard function Hazard function 20 Time (d) HTN: negative 0.020 0.015 0.010 0.020 0.015 0.010 0.005 0.005 0 5 10 15 20 25 D Time (d) mortality remained consistent when the analyses were stratified based on HTN (Figure 1) and DM (Figure 2). DISCUSSION The findings of this study suggest that ACS patients who are long-term users of areca nut may have a slightly higher risk of 30-day mortality than non-users. This finding was not statistically significant when we controlled for other potential confounding factors such as smoking status, SES, the severity of disease, sex, and history of HT or DM. However, the severity of disease in ACS patients was found to be significantly related to 78 15 0 5 10 15 E Time (d) the risk of 30-day re-hospitalization and mortality. Furthermore, we observed a significantly higher risk of 30-day re-hospitalization among ACS patients who were areca nut users than among non-users. The doubled risk of re-hospitalization remained consistent when we accounted for the effects of age, sex, severity of disease, history of HTN, and other covariates. These findings are not directly comparable with any published findings, as to our knowledge no previous evidence has been reported regarding the relationship of areca nut chewing to the prognosis of ACS patients. Some studies have reported that areca nut chewers had significantly higher risks of cardiovascular and all-cause mortality Risk of Areca Chewing in ACS Patients Hazard function Figure 2. Re-hospitalization in acute coronary syndrome patients according to areca nut consumption stratified by DM and HTN. (A) Areca nut use and 30-day re-hospitalization risk. (B, C) Areca nut use and 30-day re-hospitalization stratified by the presence of DM. (D, E) Areca nut use and 30-day rehospitalization stratified by the presence of HTN. DM, diabetes mellitus; HTN, hypertension. Areca use: re-hospitalization 0.025 No areca nut consumption Areca nut consumption 0.020 0.015 0.010 0.005 0 5 10 15 20 25 A Time (d) DM: negative p-value=0.023 0.020 0.015 0.010 0.005 0 5 10 15 DM: positive 0.025 Hazard function Hazard function 0.025 20 25 p-value=0.010 0.020 0.015 0.010 0.005 B 0 5 10 Time (d) Hazard function Hazard function 0.020 0.015 0.010 0.005 5 10 25 20 25 C 15 HTN: positive 0.025 p-value=0.002 0 20 Time (d) HTN: negative 0.025 15 20 25 D Time (d) than non-users [18,19]. The increase in mortality may be due to a higher risk of developing coronary artery disease [25], atrial fibrillation [32], and other forms of CVD [22] in the first place among areca nut chewers than among non-chewers. Since areca nut chewers are at a higher risk of developing CVD’s, they may experience high rates of cardiovascular and all-cause mortality later on compared with non-chewers. Our study also highlights the role of areca nut chewing in the prognosis of ACS patients after the acute event. Our findings suggest a slight increase in mortality and a significant risk of re-hospitalization among chewers compared with non-chewers. Several potential pathways can explain the observed dif- p-value=0.044 0.020 0.015 0.010 0.005 0 5 10 15 E Time (d) ference in mortality and re-hospitalization between areca nut chewers and non-chewers. Areca nut chewing is strongly linked with established CVD risk factors, including obesity [33], HTN [21], DM, and metabolic syndrome. Furthermore, recent studies have also reported that areca nut chewing increased systemic inflammation both in young [34] and middle-aged individuals [33]. Elevated inflammation may also explain the observation of subclinical atherosclerosis manifesting as increased carotid intima media thickness among areca nut chewers compared with nonchewers [20]. In essence, the higher prevalence of CVD risk factors coupled with elevated systemic inflammation and un- 79 Muhammad Tariq Karim, et al. derlying atherosclerosis may explain the poor prognosis of areca nut chewers after ACS. However, the exact underlying biological mechanism that leads to repeated hospitalization and a slight increase in the risk of death needs further exploration. To our knowledge, this is the first clinical study to examine the relationship between areca nut chewing and the shortterm prognosis of ACS patients. The prospective design and reasonably large sample with clinically diagnosed ACS make the findings of this study fairly robust. However, certain limitations need to be highlighted. The sample of this study was recruited from 2 public sector hospitals, and the majority of the patients were from low to lower-middle socioeconomic classes, which may limit the generalizability of this study. However, the areca nut chewing habit is also remarkably common in these SESs of the population [35], especially in comparison with the upper-middle and upper socioeconomic classes. Therefore, the sample recruited for this study may nonetheless be representative of middle-aged and elderly areca nut users in our population. Secondly, we relied on self-reporting of cigarette smoking and other health risk behaviors, which may have some potential to bias our results. For instance, the higher risk of re-hospitalization observed in this study may have been due to a differential distribution of health risk behaviors between areca nut chewers and non-chewers. Secondhand smoke is a particular concern in this regard, as higher exposure to secondhand smoke among areca nut chewers may have led to increased re-hospitalization in this group. This is fairly plausible because many health risk behaviors are usually clustered together, and it may be reasonable to assume that areca nut chewers would have a higher exposure to secondhand smoke than non-chewers. We do not have any evidence to support this possibility, but if it is true, the findings of this study may have been prone to confounding bias, which remains the major limitation of this study. Nevertheless, we attempted to control for the potential confounding effects of several covariates, and the overall findings of the study remained consistent after multivariate adjustments. In conclusion, areca nut chewing significantly increased the risk of re-hospitalization among ACS patients; however, there is little evidence that it increased short-term mortality. Further evidence from large clinical studies is needed to validate our findings and to elucidate the biological mechanism underlying these observations. 80 ACKNOWLEDGEMENTS We would like to acknowledge the Department of Cardiology, Civil Hospital Karachi and the National Institute of Cardiovascular Diseases for providing their support for data collection for this study. CONFLICT OF INTEREST The authors have no conflicts of interest associated with the material presented in this paper. ORCID Muhammad Tariq Karim https://orcid.org/0000-0001-8720234X Sumera Inam https://orcid.org/0000-0002-6110-5653 Tariq Ashraf https://orcid.org/0000-0002-6680-1017 Nadia Shah https://orcid.org/0000-0002-9166-2047 Syed Omair Adil https://orcid.org/0000-0002-2370-1390 Kashif Shafique http://orcid.org/0000-0001-7151-5122 REFERENCES 1. Pankaj C. Areca nut or betel nut control is mandatory if India wants to reduce the burden of cancer especially cancer of the oral cavity. 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https://openalex.org/W2952729484	https://europepmc.org/articles/pmc6558677?pdf=render	English	null	Bridging the gap in genetics: a progressive model for primary to specialist care	BMC medical education	2,019	cc-by	8,816	Harding et al. BMC Medical Education (2019) 19:195 https://doi.org/10.1186/s12909-019-1622-y Harding et al. BMC Medical Education (2019) 19:195 https://doi.org/10.1186/s12909-019-1622-y Open Access © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: The rapid expansion of genetic knowledge, and the implications for healthcare has resulted in an increased role for Primary Care Providers (PCPs) to incorporate genetics into their daily practice. The objective of this study was to explore the self-identified needs, including educational needs, of both urban and rural Primary Care Providers (PCPs) in order to provide genetic care to their patients. Methods: Using a qualitative grounded theory approach, ten key informant interviews, and one urban and two rural PCP focus groups (FGs) (n = 19) were conducted. All PCPs practiced in Southeastern Ontario. Data was analyzed using a constant comparative method and thematic design. The data reported here represent a subset of a larger study. Results: Participants reported that PCPs have a responsibility to ensure patients receive genetic care. However, specific roles and responsibilities for that care were poorly defined. PCPs identified a need for further education and resources to enable them to provide care for individuals with genetic conditions. Based on the findings, a progressive stepped model that bridges primary and specialty genetic care was developed; the model ranged from PCPs identifying patients with genetic conditions that they could manage alone, to patients who they could manage with informal or electronic consultation to those who clearly required specialist referral. Conclusions: PCPs identified a need to integrate genetics into primary care practice but they perceived barriers including a lack of knowledge and confidence, access to timely formal and informal consultation and clearly defined roles for themselves and specialists. To address gaps in PCP confidence in providing genetic care, interventions that are directed at accessible just-in-time support and consultation have the potential to empower PCPs to manage patients’ genetic conditions. Specific attention to content, timing, and accessibility of educational interventions is critical to address the needs of both urban and rural PCPs. A progressive framework for bridging primary to specialty care through a ‘stepped’ model for providing continuing medical education, and genetic care can was developed and can be used to guide future design and delivery of educational interventions and resources. Keywords: Primary care providers, Genetics, Genetic care, Continuing medical education, Undergraduate medical education, Prevention Bridging the gap in genetics: a progressive model for primary to specialist care Brittany Harding1, Colleen Webber2, Lucia Rühland2, Nancy Dalgarno3, Christine Armour4, Richard Birtwhistle9, Glenn Brown5, June C. Carroll6, Michael Flavin7, Susan P. Phillips5 and Jennifer J. MacKenzie8,10* * Correspondence: mackej4@mcmaster.ca 8Department of Pediatrics, McMaster Children’s Hospital, 1280, Main St. West, 3N11-G, Hamilton, Ontario L8S 4K1, Canada 10Department of Medicine, Queen’s University, Kingston, Ontario, Canada Full list of author information is available at the end of the article Background genetics is increasingly important in the diagnosis and management of common conditions such as diabetes, hypertension, cancer, heart disease, and stroke; conditions that are leading causes of death in Canada [1]. With expanding clinical utility and demand for genetic care, including direct-to-consumer testing, PCPs [2–4] are increasingly called upon to integrate genetics into their practices [5–8]. Although there is a general increase in awareness about genetics, there remains concern that PCPs do not receive sufficient training in clinical genetics, that medical school genetic education curriculae do not The rapid expansion of genetics in medicine has resulted in an increased role for Primary Care Providers (PCPs), which could include family physicians, nurse practitioners and nurses, to assess and educate patients about genetic risks and realities. In this current study, however, the PCPs are family physicians. No longer limited to rare conditions, * Correspondence: mackej4@mcmaster.ca 8Department of Pediatrics, McMaster Children’s Hospital, 1280, Main St. West, 3N11-G, Hamilton, Ontario L8S 4K1, Canada 10Department of Medicine, Queen’s University, Kingston, Ontario, Canada Full list of author information is available at the end of the article * Correspondence: mackej4@mcmaster.ca 8Department of Pediatrics, McMaster Children’s Hospital, 1280, Main St. West, 3N11-G, Hamilton, Ontario L8S 4K1, Canada 10Department of Medicine, Queen’s University, Kingston, Ontario, Canada Full list of author information is available at the end of the article Harding et al. BMC Medical Education (2019) 19:195 Page 2 of 10 Page 2 of 10 Page 2 of 10 address the practicalities of primary care practice, and that continuing medical education (CME) efforts have had a limited impact [9, 10]. The ability for PCPs to include gen- etics in their practice is especially relevant given the lim- ited number of trained genetics professionals (Canadian Medical Association [Internet]), (Shuman, Personal Com- munications, August 9, 2017). resources, given the increasing role that genetics is ex- pected to play in primary care [11]. Therefore, an up to date needs assessment of primary care providers is timely with a specific emphasis on genetic education needs. The objective of this study is to explore the self- identified genetic needs of PCPs, with specific consider- ation paid to the unique needs of both urban and rural PCPs. To date, few studies have explicitly considered the impact of practice locale on the availability of genetic education, whereas this study incorporates the perspec- tives of both urban and rural practitioners. PCP perspectives of genetics have been explored over the past two decades in various countries and medical systems [3, 4, 8, 11–15]. Previous studies have demon- strated that PCPs perceived themselves to lack know- ledge and understanding of genetics [11], and the confidence and resources required to implement genetic care into clinical practice [3, 5–8, 11–14, 16, 17]. Thus, genetics in clinical practice trails behind scientific and technological advances, which has the potential to im- pede management of patients and their families. Specific issues identified included a need for more knowledge about the modes of inheritance, environmental and gen- etic factors [5, 6, 18], the role of genetics in common disorders [15], and the type of information available [14]. PCPs indicated that they would benefit from more training in (i) linking family histories to risk assessments [4–7, 15, 16, 18], (ii) communicating and counselling pa- tients about genetics (e.g. managing family dynamics and facilitating informed decision-making in a way that reduces fears and/or concerns, and helps guide patients through complex issues) [6, 14, 18–21], and (iii) know- ing when and how to refer patients to a genetic specialist [3–5, 12, 16, 18]. Evidence suggests that PCPs would benefit from a better understanding about the options for early detection of disease, what genetic tests exist, how to interpret results, and prevention, management, and treatment strategies after a diagnosis [14, 15, 18]. For health care providers to be able to respond to the rapid increase in understanding about how genetic make-up shapes health and illness, medical education needs to transition from a traditional focus on the basic science of genetics to include a more clinical perspective [22–24]. PCPs suggested that this information is best taught through integrating genetics into existing pre- service medical education [14, 18] with CME used to educate current practitioners. Optimal CME programs would be accessible, short, engaging, timely, and either free or incentive-based [5, 14]. PCPs emphasized that education should incorporate a case-based practical ap- proach, include information on clinical applications, focus on strategies to improve patient outcomes and practice (e.g., blended learning courses, online modules, PCP involvement in face-to-face or virtual genetics ap- pointments), and be relevant to day-to-day clinical prac- tice [4, 5, 7, 14, 25–30]. In spite of efforts to date, PCPs continue to express Methods A qualitative grounded theory approach was used [31]. Ten key informant interviews (n = 10) and three PCP focus groups (FGs) (n = 19) were conducted.The inter- view results were used to develop the FG protocol. The FGs explored PCPs perceptions of their current and fu- ture roles in providing genetic care, and the effectiveness of their genetic education, educational preferences, and perceived needs for improving future educational strat- egies. All interviews and FGs were audio-recorded and transcribed verbatim. Participants were de-identified to ensure confidentiality. All participants were given pseu- donyms by assigning them a number for the key inform- ant interview (I) or PCP focus group (FG). For example, informant number one was identified as (Informant 1), whereas, a PCP in FG one was identified as (FG1). The informants were also identified by their professional role (e.g., Informant 8GC refers to Informant Number 8 who is a genetic counsellor). NVivo 10 was used to store and manage the data. This study reports a subset of data from a larger study about genetics in primary care [11]. Ethics compliance was received from the Queen’s Uni- versity Health Sciences and Affiliated Teaching Hospitals Research Ethics Board (File # 6005987). Results “the kind of psychosocial support that they [can] provide to patients in a very general way… in any circumstance [it] is relevant to the kind of support that one provides to individuals with, or at risk of, an inherited disease.” (Informant 8CG) Ten key informant interviews (n = 10) and three PCP focus groups (FGs) (n = 19) were conducted, over 18 months. All participants worked in SEO. The PCPs in the interviews and FGs were between the ages of 30 and 60 years, and included men and women who had been in practice for a minimum of five years. Participants acknowledged that PCPs have a responsibil- ity to ensure patients receive genetic care. However, opin- ions were inconsistent about who would provide which type of genetic care. To ensure they provided appropriate care and referrals, PCPs believed that they required Key informant interviews y Stratified purposive sampling was used to recruit key in- formants to ensure a cross-section of perspectives and experiences. The key informants were selected by the re- search team and included one health care administrator (A), one clinical geneticist (CG), one nurse practitioner (NP), one public health medical doctor (PH/MD), two genetic counsellors (GC), and four primary care medical doctors (MD). Interview protocols were revised using an iterative approach as new themes emerged. All inter- views were conducted by a distanced Research Assistant (RA) who had no prior involvement with the genetics program. To ensure authenticity of the data, member- checking was offered to all and completed by 4/10 par- ticipants. Each interview took approximately 45 min. Analysis of the interviews informed the development of the FG script. In spite of efforts to date, PCPs continue to express concern about inadequate knowledge, confidence, and Page 3 of 10 Harding et al. BMC Medical Education (2019) 19:195 Harding et al. BMC Medical Education (2019) 19:195 Theme 1: roles and responsibilities of PCPs Overall, most participants recognized that as the de- mand for genetic testing and care increases, this demand will exceed the supply of specialists. For example, one participant stated, “I don’t know one single medical gen- eticist that is not overrun with work and I just can’t see how it’s going to get easier. I think it’s going to get much more difficult for them to facilitate their workload” (In- formant 1GC). Another participant suggested that PCPs’ key role lies in “identification [of genetic conditions] and referral when appropriate, or [patient] education about the illness” (Informant 7MD). Another informant went on to explain that where cases do not warrant a referral to the specialist, PCPs must be prepared to offer, Data analysis Interviews and focus groups were analyzed through a grounded theory methodology, using the constant com- parative method, to identify emerging patterns in the data [32–35]. Two RAs (LH, CW) used line-by-line open coding to independently analyze all transcripts. The RAs were se- lected for expertise in qualitative research analysis and had no experience in genetic research. The results were com- pared and revised until consensus about the emergent sub- themes and themes was reached. Using all data points, the codes were constantly compared to create eleven broader sub-themes of which four overarching themes emerged (see Table 1). The findings were then discussed with the re- search team until a final set of themes was created. Finally, the themes were used to provide the basis to develop a pro- gressive model for bridging primary to specialty care. The trustworthiness and consistency of the data was ensured by using member checking, distanced RAs, analysis of the same scripts by multiple RAs, input of the research team, and correlation with the literature. Focus groups Table 1 Themes and sub-themes captured in interviews and focus groups Theme Sub-Themes 1. Roles and responsibilities of PCPs Current demand exceeds supply of specialists; increased patient requests for education and testing 2. Genetic education needs General knowledge Referral issues Managing patient care 3. Genetic education strategies to meet PCP needs Medical education needs additional focus on clinical genetics Formal Informal 4. General considerations Time constraints Awareness of educational opportunities Amount of knowledge necessary/ appropriate for Primary Care Providers Rural-specific concerns Table 1 Themes and sub-themes captured in interviews and focus groups g p Participants for the three FGs were selected using pur- posive homogeneous group sampling to include only family physicians practicing in Southeastern Ontario (SEO). The Family Health Teams (FHT) in the region were contacted by the RA, and each local FHT adminis- trator extended invitations to PCPs on their team and in the area. One FG consisted of urban PCPs (n = 5), while two FGs consisted of rural PCPs (n = 14). FGs were facil- itated by a different distanced RA than the RA who held the interviews, and the Principal Investigator (PI) re- corded field notes. The RA was selected for experience in facilitating FGs and had no prior involvement with genetics or with the PI. The FGs identified additional themes. Saturation of the data was reached with three focus groups. FGs took approximately one hour. Both the semi-structured interview and FG protocols (Appendix 1 and 2) included open-ended questions that explored resource and educational needs, and preferred CME strategies for PCPs in SEO. additional education. Participants highlighted educational strategies from which they would benefit and noted con- siderations that should be made when planning educa- tional interventions. Table 1 identifies the four themes and 11 sub-themes that emerged from the data. Theme 3: genetic education strategies Participants identified a range of strategies and sugges- tions for education that they perceived as being particu- larly beneficial for them to enhance their ability to incorporate genetics into their practices. One participant states quite succinctly: “The reality of any medicine is you’re always learning new stuff. And that would be the main thing I think for family docs is finding a way to keep up with specific knowledge based aspects of genetics” (Informant 2MD). Most participants identified a need for education that would specifically guide decision-making processes about referrals. As one PCP stated, “I would have to do some research on their [patients’] behalf to figure out what my role is next; if I refer to genetics, if I refer to a specialist, and if it’s necessary” (FG1). From the perspective of somebody who sees many of the PCP referrals, one genetic counsellor indicated that, “There’s probably a lot of people that I’m not getting any referrals for, that could use the service, and part of it is that nobody knows it exists” (Inform- ant 3GC). Genetic counsellors acknowledged that making decisions about referrals was important for providing quality care: “The biggest thing I think is recognizing that a patient may benefit, and then knowing where to refer or where to get additional guidance” (Informant 1GC). The need for detailed in- formation when PCPs referred patients to genetics was highlighted in addition to deciding which patients were appropriate to refer: Some participants suggested that undergraduate medical education should incorporate genetics into an array of subject areas, refrain from discussing it only in the context of rare conditions, and ensure consistency across medical schools. The educational challenge, however, continues to include strategies to enable health care providers to re- main current in the rapidly evolving knowledge-base within genetics. Some participants suggested that under- graduate medical education provided adequate education but, due to the rapid growth in the field of genetics, know- ledge obtained in medical school quickly becomes out- dated: “So I think in medical school and residency – the training we were provided with was up-to-date at that point. But it is not up-to-date any longer, depending on when they trained” (Informant 10PH/MD). “I think that’s the biggest thing, having more information when they send me the referrals... Theme 2: genetic education needs All participants indicated that to appropriately refer and to provide genetic care, certain educational needs must be met. First, participants acknowledged the rapid growth of Page 4 of 10 Harding et al. BMC Medical Education (2019) 19:195 Harding et al. BMC Medical Education (2019) 19:195 the field and application of genetics, and recognized that they require additional knowledge if they are to stay current: the field and application of genetics, and recognized that they require additional knowledge if they are to stay current: When PCPs determined a referral was not necessary, most stated that they would benefit from increased educa- tion about how to provide genetic care within their prac- tice. It was suggested that knowledge should be, “You have to be pretty up to date on the evidence available to really do proper counselling and make recommendations especially if people are coming to you saying 'what should I do now’. Instead of giving them options, if you’re making specific recommendations then you need to be pretty comfortable with your level of knowledge and that it’s up to date.” (FG3) “enough… that you can appropriately counsel your pa- tients” (Informant 2MD), and include the “positive and negative impacts the testing can have” (Informant 10PH/ MD). Theme 3: genetic education strategies for a better ability to do triage…you can’t just send the referral form that says, please see this person.” (Informant 3GC) Participants indicated that the continual need for gen- etics education could be accomplished both formally and informally. Formal educational strategies included continuing medical education (CME), lectures, seminars, or conferences—all with a focus on case-based learning. Participants discussed a range of CME options, with one saying, “I personally like the CME format because it gets me there and it gets my full attention and there’s oppor- tunity to interact” (FG1). Others agreed that the “most useful things are the good old fashioned face to face semi- nar[s]… the best way to educate family doctors is to come and give them re-education sessions... Come to the hospi- tals and the seminars and rounds” (Informant 9PH/ MD), or perhaps “a half day conference just in genetics” (Informant 7MD). Regardless of format, some partici- pants suggested that, “case-based learning actually would be the most helpful. Because… when you see a case and you learn about it, that’s when you remember it” (FG3). In acknowledging the value of genetic specialty expert- ise, one PCP stated: “I’ve been always very pleased when we refer someone for genetic counselling… and some expert spends time with the patient, gives them a ton of information and sends me a three-page letter at which I’m always amazed…. I know… [a] moderately small amount…. It would be nice to have something in the middle…. I sometimes think, this problem that [the patient is] con- cerned about is too small for the full genetic interview with a real expert, but I don’t know enough to give them something between the full consultation and my limited knowledge.” (FG2) Page 5 of 10 Harding et al. BMC Medical Education (2019) 19:195 Harding et al. BMC Medical Education (2019) 19:195 Harding et al. BMC Medical Education (2019) 19:195 something that should trigger a genetics referral or another test… the EMR [asks] ‘have you considered ____ because you’ve entered three people with breast cancer in this person’s family history’… it prompts.” (Informant 2MD) something that should trigger a genetics referral or another test… the EMR [asks] ‘have you considered ____ because you’ve entered three people with breast cancer in this person’s family history’… it prompts.” (Informant 2MD) In addition to formal education sessions, participants also valued informal learning opportunities. Theme 4: general considerations They found value in electronic resources that could be quickly accessed when searching for information in specific cases: “Websites where they can easily obtain the information and access what the next best steps should be.” (Informant 5MD) “Websites where they can easily obtain the information and access what the next best steps should be.” (Informant 5MD) “FAQ on genetics set up for family doctors… [with] answers to] basic questions,‘Should I refer this person for breast cancer screening or whatever else? Yes or no.’ A little blurb on the evidence. And then a little thing down below saying – if you’re still having problems contact us. And you [could] fill your question and fire it off.” (FG2) “Remembering the details is not all that important as long as you remember that there is some aid. There is a piece of paper that [includes what] I need to know… about genetics that will give me some information about what to do with patients with genetic and sensitive issues. That’s the kind of superficial knowledge I think we need.” (FG1) Some participants discussed the benefits of an auto- mated notification in the electronic medical record (EMR) which highlighted the need for genetic testing such that, Theme 3: genetic education strategies PCPs re- ported that e-mail or telephone conversations with col- leagues with genetic expertise could help educate them, and guide their decisions in practice: “Having a person to bounce an idea off without having to make a formal refer- ral that can be helpful” (Informant 2MD). As well, mul- tiple participants stated that mentorship from someone with expertise would be educationally beneficial. One par- ticipant recalled the value of hallway consultations with other physicians and suggested that facilitating this type of informal contact would contribute to improved genetic care, particularly in complex cases: Theme 4: general considerations g With respect to formal and informal methods of edu- cation, participants asked that those responsible for planning educational interventions apply certain con- siderations. First, as one participant indicated, practi- tioners are not always “aware of a lot of CME opportunities… [he is] not aware of a lot of outreach education” (Informant 2MD), hence effective market- ing strategies are key to increasing participation in genetic education offered to PCPs. Second, some par- ticipants drew attention to time constraints, pointing out that, “You’d have these hallway consultations with somebody … it was just a fantastic – he was the best resource. He was around most days and you’d run into him and you had this specific clinical scenario involving this individual patient. And you can’t really find the answer in books because this patient had a peculiar set of co-morbidities that made their answer really the only answer. And I think that whole idea of the ability to access some sensible expert to give you timely advice in a fairly, a user friendly way would be a superb tool. Not just for genetics but for everything– call this world medicine, e-curb site consult.” (FG2) “In this day and age, when everyone’s busy – it makes a lot of sense to do these webinars and all this kind of stuff. I find I don’t participate in them nearly as much because there’s always something else pulling your attention.” (FG1) Rural practitioners acknowledged specific challenges that distance could pose for receiving adequate genetics CME. One participant indicated that, “there is easier ac- cess to in-services and education presentations at the urban centres as opposed to the rural hospitals or the rural centres” (Informant 6A) while another stated that “an [urban] physician that has access to ongoing rounds at a major university centre may have a different level of exposure to genetics” (Informant 1GC). Finally, one participant reminded educators that the scope of PCP practice should be considered and that it should be ac- knowledged that PCPs cannot be expected to be genetic experts. Like this respondent, many participants discussed the importance of electronic resources. Discussion Using this model, genetic care can be targeted to the patient and may range from providing education and reassurance, to performing genetic test- ing with or without specialist support, to referring pa- tients for management by genetic specialists (Fig. 1). Step 1 includes reassuring the patients and requires minimal knowledge of genetics or confidence in dis- cussing it by the PCP. Step 2 involves the individual PCP becoming comfortable educating and/or ordering genetic testing for a patient. This requires an in- creased level of knowledge about genetic care and the various tests possible. In Step 3, the PCP requires educational interventions that allow them to become effective in managing more complex patient cases that do not require a full consult. However, managing these patients does require PCPs to seek support from an expert. The final step, describes PCPs who are knowledgeable and confident enough about genet- ics to realize when a patient should be referred for a full genetics consult. This stepped model provides a method for PCPs to be more involved in the genetic care of their patients and clearly define when collab- oration is required with a genetics specialist. a framework to make the roles of different care pro- viders more explicit. PCPs view their role as providing the care they feel capable of and determining which pa- tients warrant a more detailed assessment. However, PCPs have often received little formal education in gen- etic medicine and have had limited exposure to the iden- tification and management of patients with genetic conditions. Therefore, while PCPs feel a responsibility to provide genetic care to their patients, many discussed a lack of knowledge, confidence, and resources to do so and, therefore, are either not comfortable or limit the degree to which they integrate genetics into their daily clinical practice. In spite of efforts at CME, these find- ings are consistent with prior literature [3–6, 12, 14, 16, 18, 20, 26]. Many PCPs expressed a concern that they were un- sure when referral was appropriate, and expressed a need for education or a guide which can be used to help support decision-making surrounding referrals to genetics. PCPs want a contact they can e-mail or call to informally discuss a case to determine whether to refer or manage a patient themselves. Discussion “If you entered,‘three people in the family who had breast cancer’ for example… not only [are you] entering the data in to figure out the risk... but inadvertently… you don’t even realize… that might be The rapid expansion and increasing clinical utility of genetics in medicine requires that PCPs be prepared to provide genetic care for their patients. PCPs de- scribe that their role is determined by patient needs Harding et al. BMC Medical Education (2019) 19:195 Page 6 of 10 Page 6 of 10 and the complexity of the condition, as well as phys- ician and logistical factors. With various aspects of genetic care being provided for by both PCPs and specialists, there is ambiguity surrounding the genetic care expectations and requirements of PCPs [15]. A framework for bridging primary and specialty genetic care through a progressive stepped model emerged from the data. Using this model, genetic care can be targeted to the patient and may range from providing education and reassurance, to performing genetic test- ing with or without specialist support, to referring pa- tients for management by genetic specialists (Fig. 1). Step 1 includes reassuring the patients and requires minimal knowledge of genetics or confidence in dis- cussing it by the PCP. Step 2 involves the individual PCP becoming comfortable educating and/or ordering genetic testing for a patient. This requires an in- creased level of knowledge about genetic care and the various tests possible. In Step 3, the PCP requires educational interventions that allow them to become effective in managing more complex patient cases that do not require a full consult. However, managing these patients does require PCPs to seek support from an expert. The final step, describes PCPs who are knowledgeable and confident enough about genet- ics to realize when a patient should be referred for a full genetics consult. This stepped model provides a method for PCPs to be more involved in the genetic care of their patients and clearly define when collab- oration is required with a genetics specialist. and the complexity of the condition, as well as phys- ician and logistical factors. With various aspects of genetic care being provided for by both PCPs and specialists, there is ambiguity surrounding the genetic care expectations and requirements of PCPs [15]. A framework for bridging primary and specialty genetic care through a progressive stepped model emerged from the data. Conclusions In summary, while PCPs identify a need to include genetics in their practices, they perceive that a lack of knowledge and resources is one of the major fac- tors that impedes their ability to provide quality pa- tient care in genetics. To address gaps in PCP knowledge, a diverse set of educational opportunities and interventions should be made available in order to meet the varying needs of different PCPs and in- clude specific attention to content, timing, and accessibility. Regardless of method, we found that participants highlight that when considering education specifically intended for PCPs, program developers and organizers must consider time constraints, the level of the learner, and understand that PCPs cannot be expected to be as knowledgeable as genetic experts [7], thus set realistic limits on what PCPs are expected to know. Timely access to information would also assist PCPs in effectively embedding genetic care into their practices [5]. Education should be relevant to daily practice and include information on clinical applica- tion [5, 14, 26]. These PCP-focused educational as- pects are supported in the literature, however, our results suggest that PCPs must be made explicitly aware of educational opportunities. Improved access to genetic education would allow other healthcare providers, such as nurses and nurse practitioners, to participate in assessing genetic risk factors that may include taking family histories and providing patients with education around their concerns. The findings from this research are pertinent to the development of future educational opportunities and interventions. While educational interventions aimed at improving PCPs’ knowledge of genetic medicine may increase knowledge and perceived competence in genetic medicine, they are not always accompanied by a change in practice or referral patterns [39–42]. The progressive stepped model of bridging primary and specialty care developed from this study has the po- tential to assist in bridging the gap between primary care and genetics expert settings. Supporting PCPs in the care of patients they are uncertain about can be addressed with just-in-time strategies including timely access to experts. With the current study and the previous literature in mind, future research should not only assess the educational merit of opportunities and interventions, but should also seek to assess to what extent, and in which situations PCPs support the integration of genetics into their practices. Discussion We recommend a more explicit and accessible role for e-mail and e- consults as a corridor consultation method given that systems change is needed if PCPs are going to play a larger role in genetic care. For example, an approach to this e-consult model could be in the form of a sin- gle email address to which PCPs can direct their questions. Once the email is received by a centralized location, it is forwarded to an on-call genetics special- ist who would respond within a specified timeframe. The specialist would assist the PCP in determining which step(s) would be most appropriate be it to The advantage of this model is that it provides a devel- opmental and sequential continuum of primary to ter- tiary care for increasingly complex cases. It also provides Fig. 1 A progressive stepped model bridging primary and specialty care Fig. 1 A progressive stepped model bridging primary and specialty care Fig. 1 A progressive stepped model bridging primary and specialty care Harding et al. BMC Medical Education (2019) 19:195 Page 7 of 10 Harding et al. BMC Medical Education (2019) 19:195 Page 7 of 10 informal means of education [14]. Lack of regular con- tact with larger academic centres, leads to fewer infor- mal learning opportunities for rural PCPs compared to their urban counterparts [37, 38]. As programs are de- veloped it is essential to include options (such as just-in- time e-mail access to an expert) that increase CME ac- cessibility and informal interactions regardless of geo- graphic location. assist in reassuring the patient, provide education, offer expert advice about the specific tests needed, or expedite a referral, as described in our ‘stepped model’. An electronic forum would also address some of the barriers outlined by rural PCPs. The challenge in implementing this approach to genetic care lies in PCP access to genetics colleagues/experts in a timely manner. In addition, in some jurisdictions, access to funding for genetic tests requires a geneticist consult. g g q g To improve PCP confidence in meeting patients’ genetics needs, participants made specific recommen- dations. PCPs’ suggestion of improving undergraduate medical education in genetics through an integration across disciplines is consistent with other reports [14, 18]. Incorporation of genetics into Family Medicine residency programs was also considered important. Limitations of study Thi d y This study was conducted in SE Ontario with a small number of PCPs and is, therefore, limited in terms of generalizability at a national and international level. However, we do believe that the progressive stepped model for bridging primary to specialty care can be adapted to specific contexts within a healthcare system and inform future CME interventions. Conclusions To optimize the future of genetic care, ongoing support is needed to facilitate greater informal as well as for- mal collaboration, including the sharing of knowledge and skills between PCPs and genetic specialists. Discussion Consistent with CME literature in general, PCPs dis- cussed the need to improve formal CME opportun- ities and to utilize a case-based approach that connects theory to practice [14, 25]. In addition, par- ticipants also emphasized the value of informal learn- ing that occurs through regular contact with other professionals, with a goal to avoid unnecessary refer- rals. For example, “just-in-time” consultations with experts or methods wherein a librarian is employed to help PCPs locate necessary diagnostic information, have been shown to improve the speed and quality of PCP decision making, subsequently having the poten- tial to improve patient access to care [36]. Considerations for education of rural practitioners Rural PCPs identify some specific concerns regarding ac- cess to education. Most importantly, they highlight that distance is a significant barrier to both formal and Page 8 of 10 Page 8 of 10 Harding et al. BMC Medical Education (2019) 19:195 2. How do you view your role in the genetic care of a patient? 2. How do you view your role in the genetic care of a patient? 5. Do you know the laboratories that do the genetic testing for the tests you order? And doyou know how they’re paid for it? i. What are your thoughts about the purpose of genetic testing? In what situations would it be useful? ii. Do physicians have a gate-keeper role to play with respect to genetic counselling/testing? iii. Is this an area of interest to you? i. What are your thoughts about the purpose of genetic testing? In what situations would it be useful? 6. Do you know what requisition you use to order the test? 7. What cases would you say are referred or not? ii. Do physicians have a gate-keeper role to play with respect to genetic counselling/testing? iii. Is this an area of interest to you? 8. What would you say is the role of the primary care providers with regard to genetics? 9. Do you think that primary care providers should provide more or less genetic testing? 3. Consensus among key informant interviews was that genetics will play an increasing role in primary care practice. There were also concerns about barriers to genetic care in practice. What barriers do you foresee? 10. Would you say that the role of primary care providers would be different in a rural vs urban setting? i. What ideas do you have to address these barriers? 12. Do you think genetics is important in primary care now? ii. Are there potential problems that may arise with patients being more aware of genetics and personalized genetic testing? 4. What competencies should family physicians have in genetic care? g 5. What would facilitate the incorporation of genetics into primary care? 16. Do you think that there are genetic resources that would help to family physicians, like diagnostic testing or counselling resources? i. In your experience who has provided genetics educational support and resources for your practice? 17. Do you that leaders in your region are keeping you up-to-date? ii. Who do you think should be providing this support? 18. Is there genetic diagnostic, testing, or counselling resources that you think would be helpful to family physicians? iii. What strategies/solutions have you used to increase integration of genetics into your practice? 19. Are there genetic tools that you think would be helpful for family physicians? 20. Focus Group Protocol Focus Group Protocol 1. When you think of genetics in primary care what do you think of? 1. There is broad coverage of genetics in the popular press and medical journals. It seems genetics is having an increasingly important role in medicine. y 2. Have you treated patients with genetics concerns or illness? 3. How often would you say you have patients in your practice with genetic issues? i. When you think about genetics in your daily practice what do you think of? i. Would you typically mange the patient, once the genetic diagnosis is made or would you refer them on to another physician to do this? y ii. What role does genetics play in your practice? iii. Can you describe some experiences from your practice? 4. How often do you order genetic tests? And what tests would you actually order? Funding h k i. Do you foresee family physicians having a greater role in genetic counselling and testing? This work was funded by the Clinical Teachers’ Association of Queen’s University (CTAQ) Endowment Fund. The role of the CTAQ was solely financial support, they were not involved in any aspect of the research. ii. Is there a value in genetic counselling in primary care? Authors’ information Michael Flavin MDB BCh is an Emeritus Professor of Pediatrics in the Department of Pediatrics, Faculty of Health Sciences, Queen’s University, Kingston, Ontario, CA. 7. There are ongoing advances in the understanding of the uses of genetics in medicine. How do you anticipate that your use of genetics in practice will change over time? Jennifer J. MacKenzie MD MEd is a Pediatrician and Clinical Geneticist, Professor in the Department of Pediatrics at McMaster University, Hamilton, Ontario, CA, and an Adjunct Associate Professor in the Department of Medicine at Queen’s University, Kingston, Ontario, CA. Medicine at Queen’s University, Kingston, Ontario, CA. Abbreviations l h d A: Health care administrator; CG: Clinical geneticist; CME: Continuing medical education; EMR: Electronic medical record; FG: Focus group; GC: Genetic counsellor; MD: Medical doctor; NP: Nurse practitioner; PCP: Primary care provider; PH/MD: Public health medical doctor; PI: Principal investigator; RA: Research assistant; SEO: Southeastern ontario CME? Information sessions? Emails? Webpage? Acknowledgements We would like to thank Jyoti Kotecha for co-ordinating the initial part of this project, and to Jane Yealland for conducting the focus groups. A special thanks goes to all the participants for making this work possible. vii. Which method have you found to be the most useful? viii.Which would you like more access to? Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. iii. What impact do you anticipate that the integration of genetics into primary care will have on health resources? Ethics approval and consent to participate This study was approved by the Queen’s University and Affiliated Teaching Hospitals Health Sciences Research Ethics Board (File No. 6005987). Written, informed consent was obtained from all individual participants included in the study. iv. How will family physicians manage the increasing number of conditions that can be tested for and potentially treated? Authors’ contributions ix. What screening tools would helpful – cancer? Cardiac? EMR/Hard Copy? JM, CA, RB, GB, MF, JC, SP, and CW made substantial intellectual contributions to the research concept and design for the work. JM, LR, CW, BH and ND analyzed and interpreted the data. All listed authors contributed to writing the manuscript and providing final approval for the submitted manuscript. All authors read and approved the final manuscript. x. What is your perspective on using an EMR based genetic screening tool? Authors’ information 6. One of our goals is to understand the unique challenges of incorporating genetics into PCP in a rural setting. How would you describe these challenges? Brittany Harding BSc MEd is a research assistant with the Office of Professional Development and Educational Scholarship in the Faculty of Health Sciences, Queen’s University, Kingston, Ontario, CA. Colleen Webber PhD is a research associate at the Ottawa Hospital Research Institute, Ottawa, Ontario, CA. Lucia Rühland, MSc is a research project manager in the Department of Rehabilitation Therapy at Queen’s University, Kingston, Ontario, CA. Nancy Dalgarno MEd PhD OCT is the Director of Education Scholarship for the Office of Professional Development and Educational Scholarship in the Faculty of Health Sciences, Queen’s University, Kingston, Ontario, CA. Christine Armour MSc, MD is a clinical geneticist at CHEO and the University of Ottawa. i. How are the challenges in a rural practice different compared to an urban setting? ii. Does an urban setting also entail unique challenges? Faculty of Health Sciences, Queen’s University, Kingston, Ontario, CA. Christine Armour MSc, MD is a clinical geneticist at CHEO and the University of Ottawa. iii. Are the priorities different in a rural setting? For patients? For physicians? Richard Birtwhistle MD is a Family Physician, Clinical Epidemiologist and Professor in the Department of Public Health Sciences at Queen’s University, Kingston, Ontario, CA. Richard Birtwhistle MD is a Family Physician, Clinical Epidemiologist and Professor in the Department of Public Health Sciences at Queen’s University, Kingston, Ontario, CA. iv. If there is a proportionately low referral rate from rural areas– what are the potential causes? If services aren’t available does that mean no testing? Glenn Brown MD MPH is Associate Professor in the Department of Family Medicine, Faculty of Health Sciences, Queen’s University, Kingston, Ontario, CA. Glenn Brown MD MPH is Associate Professor in the Department of Family Medicine, Faculty of Health Sciences, Queen’s University, Kingston, Ontario, CA. June C Carroll MD is a Professor and Clinician Scientist with the Department of Family & Community Medicine at the University of Toronto, Toronto, Ontario, CA. v. What strategies or resources could be successful to integrate genetics into rural care? (e.g., Different models - just ordering tests, curb side consultations) Susan P. 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Genet Med. 1999;1:56–6 Author details 1 f 1Department of Pediatrics, McMaster University, Hamilton, Ontario, Canada. 2Queen’s University, 99 University Avenue, Kingston, Ontario K7L 3N6, Canada. 3Botterell Hall, Queen’s University, 18 Stuart Street, Kingston, Ontario K7L 3N6, Canada. 4Children’s Hospital of Eastern Ontario, 401 Smyth Road, Ottawa, Ontario K1H 8L1, Canada. 5Centre for Studies in Primary Care, Queen’s University, 220 Bagot Street, P.O.#8888, Kingston, Ontario K7L 5E9, Canada. 6Department of Family and Community Medicine, Granovsky Gluskin Family Medicine Centre, Mount Sinai Hospital, University of Toronto, 60 Murray St., 4th Floor, Box 25, Toronto, Ontario M5T 3L9, Canada. 7Department of Pediatrics, Faculty of Health Sciences, Queen’s University, Kingston, Ontario, Canada. 8Department of Pediatrics, McMaster Children’s Hospital, 1280, Main St. West, 3N11-G, Hamilton, Ontario L8S 4K1, Canada. 9Department of Public Health, Kingston, Ontario, Canada. 10Department of Medicine, Queen’s University, Kingston, Ontario, Canada. 21. 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Australas Psychiatry. 2005;13:302–6. 26. Fetters MD, Doukas DJ, Luan Dinh Phan K. Family physicians’ perspectives on genetics and the human genome project. Clin Genet. 1999;56:28–34. Received: 3 July 2018 Accepted: 22 May 2019 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 16. Emery J, Watson E, Rose P, Andermann A. A systematic review of the literature exploring the role of primary care in genetic services. Fam Pract. 1999;16:426–45. 16. Emery J, Watson E, Rose P, Andermann A. A systematic review of the literature exploring the role of primary care in genetic services. Fam Pract. 1999;16:426–45. 17. Weir M, Morin K, Ries N, Castle D. Canadian health care professionals’ knowledge, attitudes and perceptions of nutritional genomics. Br J Nutr. 2010;104:1112–9. 18. 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https://openalex.org/W2053173317	https://journals.ispan.edu.pl/index.php/abs/article/download/abs.2014.021/503	Polish	null	Ewa Golachowska, "Jak mówić do Pana Boga? Wielojęzyczność katolików na Białorusi na przełomie XX i XXI wieku". Instytut Slawistyki PAN, Wydawnictwo Agade, Warszawa 2012, ss. 189	Acta Baltico-Slavica	2,014	cc-by	3,865	This is an Open Access article distributed under the terms of the Creative Commons Attribution 3.0 PL License (creativecommons.org/licenses/by/3.0/pl/), which permits redistribution, commercial and non ‑commercial, provided that the article is properly cited. © The Author(s) 2014. Publisher: Institute of Slavic Studies PAS & The Slavic Foundation [Wydawca: Instytut Slawistyki PAN & Fundacja Slawistyczna] Acta Baltico‑Slavica, 38 SOW, Warszawa 2014 Acta Baltico‑Slavica, 38 SOW, Warszawa 2014 DOI: 10.11649/abs.2014.021 Małgorzata Ostrówka Instytut Slawistyki PAN Warszawa mostrowka@poczta.onet.pl Ewa Golachowska, Jak mówić do Pana Boga? Wielojęzyczność katolików na Białorusi na przełomie XX i XXI wieku. Instytut Slawistyki PAN, Wydawnictwo Agade, Warszawa 2012, ss. 189 Ewa Golachowska, Jak mówić do Pana Boga? Wielojęzyczność katolików na Białorusi na przełomie XX i XXI wieku. Instytut Slawistyki PAN, Wydawnictwo Agade, Warszawa 2012, ss. 189 Temat związków religii, języka i tożsamości narodowej na terenie dawnego Wiel kiego Księstwa Litewskiego od dłuższego czasu znajdował się w kręgu zainteresowań uczennic profesor Elżbiety Smułkowej. W koleżeńskich rozmowach brały udział dr hab. Anna Engelking, dr hab. Anna Zielińska, dr hab. Zofia Sawaniewska-Mochowa i oczywiście dr hab. Ewa Golachowska. Ta ostania podjęła się przeprowadzenia badań terenowych i na Białorusi, a potem opisania ich w książce Jak mówić do Pana Boga. Wielojęzyczność katolików na Białorusi na przełomie XX i XXI wieku. ję y p Tereny byłych Kresów Wschodnich od wieków cechowała wieloetniczność, a tym samym wielojęzyczność i wielokulturowość. Używanie kilku języków przez jedną osobę jest do dziś dość powszechne i ma najczęściej charakter dyglosyjny. Dla osób wyznania rzymskokatolickiego najwyższe miejsce w hierarchii zajmował, i dla wielu nadal zajmuje, język polski jako język sfery sacrum. Tak było do lat 90. XX w., kiedy katolicyzm na ogół utożsamiano z polskością. Pod koniec lat 80. XX w. na Białorusi nastąpiły zmiany polityczne, które zapoczątkowały ewolucję w zachowaniach językowych katolików. Ewa Golachowska postanowiła opisać dystrybucję oraz status polszczyzny i innych języków używanych przez katolików po upływie 20 lat od pierwszych badań. Efektem tych badań jest praca Jak mówić do Pana Boga? Małgorzata Ostrówka Rec.: Ewa Golachowska, Jak mówić do Pana Boga? Wielojęzyczność katolików… Książka Ewy Golachowskiej podzielona jest na dwie części – część pierwsza nosi tytuł „Wielojęzyczność ludności katolickiej na Białorusi. Raport z badań terenowych” i składa się z trzech rozdziałów zakończonych wnioskami. Część druga zatytułowana „Wielojęzyczność ludności katolickiej na Białorusi w relacjach świadków historii” zawiera zapisy żywego języka katolików różnych pokoleń mieszkających obecnie na Białorusi. Na końcu zamieszczono listę cytowanych informatorów, streszczenia w językach bia łoruskim i angielskim, bibliografię, mapkę z położeniem miejscowościami, w których przeprowadzono badania oraz fotografie dokumentujące badania terenowe. p p gi ją Część pierwsza rozpoczyna się „Wprowadzeniem” (s. 13–23), w którym Autorka opisała cel badań i zastosowane metody pracy terenowej. Badania terenowe prowadziła wśród ludności katolickiej na Białorusi Zachodniej i Wschodniej. Eksploracja miała przy nieść odpowiedź na pytanie: „Czy coraz powszechniejsze używanie w liturgii Kościoła katolickiego języka białoruskiego zmienia status i zakres funkcjonowania polszczyzny w środowiskach katolickich na Białorusi? Jeśli tak, to jaki ma to wpływ na identyfikację narodową katolików mieszkających w tym kraju?” Autorka objęła badaniami wszystkie pokolenia katolików. Otwarte i niestandaryzowane wywiady oraz obserwacja uczestni cząca pozwoliły jej wejść w środowisko informatorów, pozyskać ich zaufanie oraz ocenić faktyczną dystrybucję funkcjonalną poszczególnych języków. Ewa Golachowska, Jak mówić do Pana Boga? Wielojęzyczność katolików na Białorusi na przełomie XX i XXI wieku. Instytut Slawistyki PAN, Wydawnictwo Agade, Warszawa 2012, ss. 189 Godny podkreślenia jest fakt, że dla zapewnienia informatorom anonimowości i komfortu w relacji z eksploratorem, nie wszystkie rozmowy były nagrywane. Ważnym elementem badań było uczestniczenie Autorki w życiu religijnym – w mszach świętych, nabożeństwach, religijnych spotkaniach młodzieży, a nawet w pieszej pielgrzymce do Ostrej Bramy. Okazywało się to z jednej strony bardzo pomocne, lecz z drugiej budziło jej obawę o obiektywność spostrzeżeń oraz wywoływało problemy natury etycznej, ponieważ będąc osobą wierzącą, wykorzystywała ten fakt jako narzędzie w nawiązywaniu więzi z rozmówcami. Mimo iż postrzegano ją jako rzeczniczkę utrzymywania języka polskiego w Kościele na Białorusi, umiała spojrzeć niejako „z góry” na całokształt problemów i wyciągnąć własne wnioski, często różne od sugerowanych przez informatorów. Stąd widoczny w pracy intelektualny dystans i brak wartościowania w opisie zagadnień dotyczących związku języka i religii wśród katoli ków na Białorusi. Ten brak oceny przejawia się w używaniu określenia „katolicy”, a nie „Polacy”, ponieważ, Jej zdaniem, „jest ono pojemniejsze i obejmuje zarówno katolików utożsamiających się z polskością, jak i takich, którzy mówią tylko o polskich korzeniach lub mają podwójną identyfikację – polską i białoruską – albo, co jest szczególnie częste w młodym pokoleniu, mają jednoznacznie określoną białoruską identyfikację narodową” (s. 14). Podobnie rzecz ma się ze sformułowaniami „utrata języka polskiego” i „eliminacja polszczyzny z kościołów”, które Autorka traktuje raczej jako przeobrażenia językowe, zachodzące w sferze religii wśród katolików na Białorusi. Podrozdział „Problemy językowe Kościoła katolickiego na Białorusi w XX wieku w literaturze przedmiotu” (s. 17–23) omawia problemy związane z funkcjonowaniem języka/ów w Kościele katolickim na Białorusi (w zasadzie podobne na całym obszarze 347 orzata Ostrówka Rec.: Ewa Golachowska, Jak mówić do Pana Boga? Wielojęzyczność katolików… dawnego Wielkiego Księstwa Litewskiego) w świetle badań socjolingwistycznych i etno logicznych, m.in. Elżbiety Smułkowej, Iwony Kabzińskiej, Anny Engelking, Justyny Straczuk, Zofii Sawaniewskiej-Mochowej i Anny Zielińskiej, Kojego Mority, Mirosława Jankowiaka, księdza Romana Dzwonkowskiego, Olega Gorbaniuka i Julii Gorbaniuk. Fenomenem charakterystycznym dla badanego terenu na przestrzeni lat było utożsa mianie religii z identyfikacją narodową – w powszechnym użyciu były określenia Polak -katolik, Ruski-prawosławny; wiara katolicka, czyli polska i prawosławna – ruska lub białoruska. Język stawał się jej wyróżnikiem, niezależnie od tego, jakim kodem wierzący posługiwali się w kręgu rodzinnym czy sąsiedzkim. Ważny był też utrwalony przez tradycję podział na języki niskie i wysokie. Wysoki (polski) rozbrzmiewa w kościele, a niski (prosty) używany jest przy pracy w gospodarstwie. Rozpoczęty w latach 90. XX w. Ewa Golachowska, Jak mówić do Pana Boga? Wielojęzyczność katolików na Białorusi na przełomie XX i XXI wieku. Instytut Slawistyki PAN, Wydawnictwo Agade, Warszawa 2012, ss. 189 proces wprowadzania do liturgii języka białoruskiego (niskiego) spotkał się ze sprzeciwem wiernych, którzy odczuli to jako zamach na własną tożsamość. W rozdziale I „Funkcjonowanie polszczyzny w środowiskach katolickich na Biało rusi” (s. 25–51 ) Autorka przedstawia procesy socjolingwistyczne, które zaszły na Białorusi w ostatnim dwudziestoleciu. Wyzwanie, które podjęła, było niezwykle trudne, ponieważ teren jest bardzo zróżnicowany zarówno geograficznie (podział na Białoruś Wschodnią i Zachodnią), jak i społecznie (chłopi, potomkowie dawnej szlachty, młoda inteligencja) oraz pokoleniowo. Już wcześniej prowadzone badania wykazały, że przedwojenna granica polsko-radziecka pozostawiła trwałe ślady widoczne do dziś. Najważniejszym z nich jest obecność, szczególnie na wsi, dawnej polszczyzny, tej, która jest dziedzictwem dawnego Wielkiego Księstwa Litewskiego w części przed II wojną światową należącej do Rzeczy pospolitej i prawie całkowity jej zanik w części wschodniej, gdzie ludność polska została poddana surowym represjom ze strony władz komunistycznych. Przykładem jest tzw. Kojdanowszczyzna/Dzierżyńszczyzna – polski okręg autonomiczny utworzony w 1932 r. tuż przy granicy z Polską, który został zlikwidowany w roku 1938, a ludność w ciągu jednej nocy wywieziona. W latach 1936–1938 wszelkie przejawy patriotyzmu polskiego trakto wano jako nacjonalizm, dochodziło do sytuacji, gdy rozstrzeliwano mężczyzn, którzy mieli na imię Edward, Stanisław lub Władysław (s. 27). Właśnie w tym okresie rozpoczęło się usuwanie języka polskiego z komunikacji w rodzinach, przerywając tym samym tradycję polskości. Rzadko był to dobrowolny wybór – najczęściej decydowała o tym obawa o życie najbliższych. Stąd zakres używania polszczyzny na wschodzie i zachodzie Białorusi, co ewidentnie pokazały badania Autorki, różni się diametralnie. O ile na zachodzie mogła go jeszcze usłyszeć w niektórych rodzinach, w sąsiedztwie i bardzo często w kościele, o tyle na Białorusi Wschodniej tylko czasami w kościele – tam, gdzie proboszczowie prowadzą własną politykę językową lub też wyraźnie życzyli sobie tego wierni (najczęściej przybysze z Grodzieńszczyzny). Jednak i w tych kościołach często tylko stałe teksty liturgiczne czytane są po polsku, a kazania, śpiewy i ogłoszenia – po białorusku. W tym języku odbywa się także katechizacja. W większych miastach Białorusi Wschodniej organizowane są kursy języka polskiego, które cieszą się dużą popularnością. Zainteresowanie językiem polskim 348 orzata Ostrówka Rec.: Ewa Golachowska, Jak mówić do Pana Boga? Wielojęzyczność katolików… nie ma związku z poczuciem polskich korzeni, raczej podyktowane jest względami eko nomicznymi – staje się on językiem biznesu, otwiera drogę na Zachód. Ważnym powo dem nauki jest też perspektywa otrzymania Karty Polaka, która umożliwia uzyskiwanie długoterminowych wiz i podejmowanie pracy w Polsce. Ewa Golachowska, Jak mówić do Pana Boga? Wielojęzyczność katolików na Białorusi na przełomie XX i XXI wieku. Instytut Slawistyki PAN, Wydawnictwo Agade, Warszawa 2012, ss. 189 Rzadziej spotyka się deklaracje o polskim pochodzeniu – „jestem Polką”, „czuję się Polakiem”. Czasami pada stwierdzenie „bo polski to mój język ojczysty”. Na s. 37–38 Autorka wyjaśnia specyfikę tego pojęcia: „W warunkach białoruskich nie można [go] w żaden sposób łączyć z pojęciowym apa ratem socjolingwistyki […]. Jest natomiast kategorią administracyjną obecną w spisach ludności”, a przede wszystkim, cytując T. M. Mikulicza, przychyla się do jego definicji, zgodnie z którą „język ojczysty jest kategorią etnopsycholingwistyczną, która odzwierciedla emocjonalny stosunek do języka, orientację etniczną człowieka” (s. 38). Ewa Golachowska podkreśla, że na Białorusi, w odróżnieniu od polskiego obszaru etnicznego, definiowanie języka ojczystego zawsze odbywa się w kontekście wielojęzyczności. Na zachodnich terenach Białorusi, które od zawsze cechowała wielojęzyczność – funkcjonowały język polski, gwara białoruska (nazywana potocznie językiem prostym), w niektórych sferach gwary litewskie, których użycie miało charakter dyglosyjny. Cechą charakterystyczną społeczności polskiej na Grodzieńszczyźnie był jej podział na wsie chłopskie i okolice szlacheckie. O ile w okresie międzywojennym w sferach publicznych wszystkie grupy społeczne używały języka polskiego (wtedy urzędowego), o tyle w sferze rodzinnej i sąsiedzkiej chłopi posługiwali się najczęściej gwarą białoruską, mimo iż w kościele i jego obrębie oraz na tzw. wieczorkach rozmawiano po polsku. Nawet szlachta w kontaktach z mieszkańcami wsi używała języka prostego. Po wojnie sytuacja uległa zmianie – językiem urzędowym stał się rosyjski i to on osiągał coraz większy prestiż, gdyż zapewniał awans społeczny. Mowa prosta nosiła piętno wiejskości, aby uniknąć ośmieszenia, należało posługiwać się językiem rosyjskim. Stąd masowe przechodzenie całych rodzin na ten język. Zakres użycia polszczyzny został ograniczony – dziś posługuje się nią najstarsze pokolenie w sferze sacrum. Młode pokolenie często nawet w tej sferze nie posługuje się polszczyzną. Jednak Autorka zaobserwowała w trakcie badań, że duża grupa osób uczących się języka polskiego ma polską autoidentyfikację. Mimo to język polski jako środek komunikacji wewnątrzrodzinnej zanika. Jest używany wyłącznie na lekcjach języka polskiego i w kontaktach z przyjezdnymi z Polski. Na co dzień nie używają go nawet osoby, które zdobyły wyższe wykształcenie w Polsce. W związku z tym nie tworzy się nowy regionalny wariant polszczyzny z cechami systemowymi, jest to raczej zbiór idiolektów. Cechy różne od języka ogólnopolskiego (także te, które opisują badacze polszczyzny północnokresowej) występują w tych idiolektach niere gularnie i zależą od kompetencji mówiącego. W podrozdziale „Symboliczne znaczenie języka białoruskiego” (s. 47–51) Ewa Golachowska przedstawia funkcjonowanie języków białoruskiego, rosyjskiego, polskiego i trasianki w środowisku katolików na Białorusi. Ewa Golachowska, Jak mówić do Pana Boga? Wielojęzyczność katolików na Białorusi na przełomie XX i XXI wieku. Instytut Slawistyki PAN, Wydawnictwo Agade, Warszawa 2012, ss. 189 O ile rosyjski i trasianka pełnią funkcje komunikatywne i dla większości informatorów są emocjonalnie obojętne, o tyle język 349 orzata Ostrówka Rec.: Ewa Golachowska, Jak mówić do Pana Boga? Wielojęzyczność katolików… Małgorzata Ostrówka Rec.: Ewa Golachowska, Jak mówić do Pana Boga? Wielojęzyczność katolików… białoruski ma znaczenie głównie symboliczne. Zmiana jego statusu rozpoczęła się na przełomie lat 80. i 90. XX w. od odbudowy białoruskiego szkolnictwa i wprowadzenia języka białoruskiego do środków masowego przekazu. Proces ten został zahamowany w roku 1995, kiedy językami państwowymi stały się dwa języki – rosyjski i białoruski. Nastąpiła wówczas znaczna rusyfikacja i zmniejszenie roli języka białoruskiego. Jednakże pięć lat odrodzenia zaowocowało zwiększeniem jego prestiżu, szczególnie w odmia nie literackiej. Obecnie jest on postrzegany jako język inteligencji, elity intelektualnej i artystycznej. W pracach badaczy sytuacji językowej na Białorusi, które Autorka cytuje, podkreśla się jego funkcję symboliczną, o wiele ważniejszą od komunikatywnej. Badaczka przedstawia też modele funkcjonowania języków polskiego i białoruskiego: „mówimy po rosyjsku, ale to polski jest naszym językiem ojczystym” i „mówimy po rosyjsku, ale to białoruski jest naszym językiem ojczystym”, choć zakres ich użycia znacznie się różni – polszczyzna ograniczona jest do kilku sfer (religii, domu, stosunków towarzyskich i sąsiedztwa), a białoruszczyzna ma o wiele większy zasięg. Rozdział II nosi tytuł „Wielojęzyczność sfery sacrum” (s. 53–77), autorka omawia w nim funkcję poszczególnych języków obsługujących sferę sacrum, którą dzieli na sferę katechezy, kazań, liturgii i modlitwy. Osobno rozważa funkcje języka rosyjskiego w Kościele. Na wstępie podrozdziału „Języki w sferze sacrum i ich funkcja komunika tywna” przytacza definicje „języka religii” R. Pankiewicza, Ireny Bajerowej, Małgorzaty Nowak oraz stanowiska (św. Augustyn, B. Nadolski, Sobór Watykański II) dotyczące języka sakralnego. Jedno z nich nie uznaje za konieczne rozumienie go – teksty litur giczne i modlitewne są od wieków sformalizowane i ich skuteczność nie zależy od stopnia zrozumienia przez osobę wypowiadającą. Drugie głosi, iż Słowo musi być zrozumiałe dla „wszystkich ludów, jakie są pod niebem”. Badaczka zwraca uwagę, że wśród katoli ków na Białorusi, zastosowanie postanowień Soboru o sprawowaniu liturgii w języku, którym wierni posługują się na co dzień, jest utrudnione ze względu na wszechobecną wielojęzyczność oraz dyglosję – wyraźne oddzielenie języka sacrum od profanum. Sytuację komplikuje istniejący do dziś podział, nawet w obrębie tej samej parafii, na polskojęzyczne okolice szlacheckie i katolickie wsie chłopskie, w których językiem komu nikacji jest gwara białoruska (tzw. Ewa Golachowska, Jak mówić do Pana Boga? Wielojęzyczność katolików na Białorusi na przełomie XX i XXI wieku. Instytut Slawistyki PAN, Wydawnictwo Agade, Warszawa 2012, ss. 189 język prosty), a językiem modlitwy język polski; ich mieszkańcy mają też polską identyfikację narodową (dotyczy to głównie najstarszego pokolenia). W tych społecznościach polszczyzna ma wysoką rangę i wartość symboliczną. W rezultacie na Grodzieńszczyźnie, gdzie dominuje wielojęzyczność polsko-białoruska, a polszczyzna bywa jeszcze obecna w życiu codziennym, liturgia sprawowana jest po polsku. W części wschodniej, np. na Mohylewszczyźnie, gdzie przeważa wielojęzyczność białorusko-rosyjska, do sfery religijnej wprowadzany jest białoruski. Zmiany te są nie tylko wynikiem zaleceń soborowych, ale też mają na celu stworzenie wspólnot katolickich, stojących ponad podziałami narodowymi. Sprzyja temu zmiana pokoleniowa – wymiera pokolenie przyzwyczajone do starych podziałów religijnych, narodowych i językowych. Z obserwacji Ewy Golachowskiej wynika, że nadanie językowi codziennemu wysokiej 350 orzata Ostrówka Rec.: Ewa Golachowska, Jak mówić do Pana Boga? Wielojęzyczność katolików… rangi języka sakralnego niweluje tak mocno zakorzenioną na Białorusi dyglosję, która przejawia się tam w wyraźnym oddzieleniu funkcji poszczególnych języków – białoruski/ prosty wiąże się z utylitaryzmem i pragmatyzmem – polski to język wysoki, a jego wartość jest symboliczna. W kolejnym podrozdziale Autorka analizuje użycie języków polskiego i białorus kiego w poszczególnych mikrosferach: katechizacja, homiletyka, modlitwa osobista, liturgia mszy świętej i nabożeństw pomocniczych oraz pieśni religijne. Jest ono zwią zane z kompetencją językową katolików, funkcjami komunikatywnymi tych języków oraz możliwością wyboru języka w określonej mikrosferze. Wybór języka jest możliwy praktycznie tylko w dziedzinie modlitwy osobistej, w pozostałych jest on uzależniony od duchowieństwa. To proboszcz decyduje o języku nabożeństw. Jeśli chodzi o kate chizację, to jej język również zależy od proboszcza lub preferencji rodziców. Większość księży uważa też, że na Białorusi nie powinno się już przekazywać treści religijnych po polsku. W rezultacie w nauczaniu prawd wiary bierze się pod uwagę kompetencję komunikatywną dzieci. W podsumowaniu tej mikrosfery Autorka prognozuje całkowite wycofanie języka polskiego z Kościoła katolickiego na skutek przerwania jego przekazu w rodzinie oraz w religijnym wychowaniu dzieci – nauczając pacierza i głównych prawd wiary po polsku, rodzice lub dziadkowie dawali im też podstawy języka. Brak nawet podstaw jego znajomości oraz scedowanie nauki religii na katechetów, powoduje, że edukacja religijna odbywa się w języku zrozumiałym dla większości, co z kolei wpływa na wybór kodu dla innych praktyk religijnych. Wierni nie mają też wpływu na język homilii, który zależy od kompetencji duchow nych – starsi księża, pochodzący z Polski wygłaszają je zazwyczaj po polsku lub po biało rusku z interferencją rosyjską, księża młodzi wykształceni w latach 90. i później władają literackim białoruskim i głoszą kazania w tym języku. Ewa Golachowska, Jak mówić do Pana Boga? Wielojęzyczność katolików na Białorusi na przełomie XX i XXI wieku. Instytut Slawistyki PAN, Wydawnictwo Agade, Warszawa 2012, ss. 189 Spotyka się też, szczególnie na mszach dla dzieci, stosowanie kilku języków dla lepszego zrozumienia treści. Czasami szczególnie ważny fragment nauki wygłaszanej po polsku, powtarzany jest po rosyjsku. Język liturgii, którego wybór w zasadzie nie zależy od wiernych, budzi wiele kontro wersji wśród katolików na Białorusi. Mają one charakter pokoleniowy – zdecydowanymi przeciwnikami białorutenizacji Kościoła są osoby starszego i średniego pokolenia, pocho dzące z drobnej szlachty lub inteligencji o różnym stopniu wykształcenia, które bronią jego polskości. Zwolennikami języka białoruskiego w liturgii są osoby młode, najczęściej wykształcone w szkołach białoruskich w latach 90. XX w. Osoby głęboko wierzące i zaan gażowane w życie religijne nad język przedkładają godne uczestnictwo w nabożeństwach. Konkludując, Autorka potwierdza wcześniejszą opinię prof. E. Smułkowej, iż spór o język liturgii w Kościele katolickim na Białorusi ma „szerszy kontekst polityczny” a nie religijny. W sferze modlitwy prywatnej wierny ma swobodę wyboru języka. Korzystają z tego ludzie młodzi, którzy naukę religii pobierali poza domem, zwykle w języku białoruskim, a polskie pacierze znają z domu. Starsi pozostają wierni polszczyźnie, w której od dziecka zwracają się do Boga. 351 Małgorzata Ostrówka Rec.: Ewa Golachowska, Jak mówić do Pana Boga? Wielojęzyczność katolików… Rozdział II kończy się omówieniem funkcji języka rosyjskiego w sferze sacrum. Wprawdzie nie jest on językiem liturgii, ale jego rola jest znaczna ze względu na powszechną znajomość. Po rosyjsku często czytane są ogłoszenia parafialne, odbywa się przygotowywanie ministrantów do służby przy ołtarzu, jego elementy mogą pojawiać się w kazaniu polsko- lub białoruskojęzycznym, może też być językiem spontanicznej modlitwy ludzi młodych.i Rozdział III monografii Ewy Golachowskiej nosi tytuł „Uwagi na temat tożsamości narodowej katolików na Białorusi na początku XXI wieku”. Opierając się na definicjach tożsamości A. Kłoskowskiej, E. Smułkowej, M. Melchior oraz A. Engelking, Autorka próbuje odpowiedzieć na pytania: „Czy zmiana języka sfery sacrum katolików na Biało rusi będzie miała wpływ na ich identyfikację narodową?” oraz „Jak współcześnie należy rozumieć określenie «Polak» na Białorusi?”. W tym celu przedmiotem swoich wywiadów uczyniła przede wszystkim kwestie wyboru języka liturgii, modlitwy oraz zależności tych wyborów od tożsamości narodowej. Dla osób starszych, których tożsamość była już wielokrotnie analizowana i opisywana, religia katolicka pełni funkcję identyfikacji społeczno-kulturowej w wieloetnicznej społeczności i bardzo często niesie też treści patriotyczne. Bycie Polakiem wynikało z urodzenia się w katolickiej rodzinie, chrztu w Kościele katolickim i modlenia się po polsku. Dlatego też Badaczka skupiła uwagę na pokoleniu młodym – urodzonym w latach 70. Ewa Golachowska, Jak mówić do Pana Boga? Wielojęzyczność katolików na Białorusi na przełomie XX i XXI wieku. Instytut Slawistyki PAN, Wydawnictwo Agade, Warszawa 2012, ss. 189 i później, które dorastało, kształciło się i kształtowało swoje postawy w warunkach diametralnie różnych od swoich rodziców i dziadków. Rozmówców wybierała spośród osób wierzących, zaangażowanych w życie Kościoła na Białorusi, których inicjacja religijna miała miejsce jeszcze przed pieriestrojką. Każdy z nich spędził dłuższy czas w Polsce. To, co ich różni, to sposób dochodzenia do wiary – tu ważne jest pochodzenie: rodzina chłopska i utożsamianie katolicyzmu z pol skością, rodzina mieszana, często obojętna religijnie i patriotycznie i wreszcie rodzina szlachecka, gdzie wiarę i polskość przekazuje się dzieciom do dziś. Na podstawie pogłębionych wywiadów, których obszerne fragmenty są w tym rozdziale przytoczone, Ewa Golachowska wysnuwa wniosek o polifonicznej tożsamości młodych katolików na Białorusi, którą za J. Fishmanem nazywa dietnią. Oznacza to, że elementy polskie przenikają się u nich z białoruskimi – mogą oni mieć polską przeszłość rodzinną i wybierać białoruską teraźniejszość, mogą modlić się po polsku i być zwo lennikami białoruskiej opozycji, chodzić na mszę w języku polskim, a deklarować się jako Białorusini, a przy tym wszystkim słuchać rosyjskiej muzyki rozrywkowej i czytać rosyjską literaturę. Ta złożoność tożsamości młodych katolików na Białorusi wynika właśnie z wielokulturowości, będącej dziedzictwem Wielkiego Księstwa Litewskiego. Z tym wiąże się także pewna oryginalność Kościoła katolickiego na Białorusi – przenikają się w nim elementy polskie i białoruskie. Zmiany polityczne, jakie zaszły na Białorusi pod koniec lat 80. i na początku 90., pociągnęły za sobą zmianę sytuacji Kościoła kato lickiego. Nałożyły się na to przemiany w modelu religijności. Dla młodego pokolenia sprawy tożsamości, praktyk religijnych, ich języka zależą od własnych wyborów. 352 Małgorzata Ostrówka Rec.: Ewa Golachowska, Jak mówić do Pana Boga? Wielojęzyczność katolików… W części II „Wielojęzyczność ludności katolickiej na Białorusi w relacjach świadków historii” (s. 79–162) Autorka zamieściła zapisy rozmów – żywego języka katolików wszystkich pokoleń z terenów Białorusi Zachodniej i Wschodniej. W ten sposób udokumentowała ważne i dramatyczne wydarzenia z historii Kościoła katolickiego oraz losy jego wiernych, a także tamtejszą polszczyznę, która odchodzi wraz z jej użyt kownikami. Teksty uporządkowane według języków (polskie i wschodniosłowiańskie) i miejscowości zawierają krótkie uwagi o języku mówiących oraz wyodrębnione na marginesach autorskie tytuły poszczególnych fragmentów, np. „Ciężki los wywie zionych na Syberię”, „Córka wroga narodu”. Taki układ ułatwia czytelnikowi odbiór i szukanie interesujących go zagadnień. Teksty polskie zapisane są w transkrypcji półfonetycznej (aby pokazać pewne charakterystyczne cechy – półpalatalność c’, dz’, s’, z’ , miękkość l czy rozłożoną wymowę samogłosek nosowych przed szczelinowymi), teksty białoruskie i rosyjskie zapisane są ortograficznie. Ewa Golachowska, Jak mówić do Pana Boga? Wielojęzyczność katolików na Białorusi na przełomie XX i XXI wieku. Instytut Slawistyki PAN, Wydawnictwo Agade, Warszawa 2012, ss. 189 Stanowią one cenne źródło do badań językoznawczych, socjologicznych, historycznych i in. j y y j g y y y Książka Ewy Goalchowskiej Jak mówić do Pana Boga? jest ważną pozycją w litera turze poświęconej problematyce współzależności wyznania, języka praktyk religijnych i tożsamości katolików wszystkich pokoleń na Białorusi. Napisana została w oparciu o materiał zebrany podczas badań terenowych, zawiera zatem aktualne dane na temat wielokulturowości i wielojęzyczności a także tożsamości wyznawców religii rzymskoka tolickiej w tym kraju. Pogłębione wywiady, wnikliwa obserwacja uczestnicząca Badaczki dają gwarancję rzetelności badań i trafności wyciąganych wniosków. Godny podkreślenia jest jej dystans do przedstawianych problemów – jakże trudnych i budzących wiele emocji, gdyż związane są ze sferą sacrum. Opisuje je i interpretuje bez wartościowania – szerzenie się języka białoruskiego w liturgii Kościoła katolickiego, zmiany tożsamości narodowej traktuje jako przeobrażenia będące skutkiem historycznych przemian. Ukazuje przy tym różnorodność i dynamiczność postaw katolików, szczególnie młodych, wśród których nie ma antagonizmu między polszczyzną a językiem białoruskim oraz narodowością polską i białoruską. Współczesny Kościół jest nośnikiem wyłącznie wzorców religijnych, a nie jak dotychczas także narodowych, jest w nim miejsce dla Polaka i dla Białorusina. Przestaje funkcjonować stereotyp Polaka-katolika, język białoruski zdobywa wysoką rangę, nabiera wartości symbolicznej. Praca Ewy Golachowskiej jest swego rodzaju raportem z badań, może być także cennym źródłem do badań dla przedstawicieli innych nauk humanistycznych i spo łecznych. Bibliografia Golachowska, E. (2012). Jak mówić do Pana Boga?: wielojęzyczność katolików na Białorusi na przełomie XX i XXI wieku. Warszawa: Instytut Slawistyki PAN; Wydawnictwo Agade. Bibliografia 353 ata Ostrówka Rec.: Ewa Golachowska, Jak mówić do Pana Boga? Wielojęzyczność katolików… Małgorzata Ostrówka Małgorzata Ostrówka Rec.: Ewa Golachowska, Jak mówić do Pana Boga? Wielojęzyczność katolików… Keywords: Belarus; Polish language; Belarussian language; Russian language Summary The subject of the review is a book of Eve Golachowski How to talk to God? The book is a report from the field. The author moves on the issue of distribution of functional languages spoken by Catholics in Belarus (Polish, Belarusian, Russian) in cross-generational. The book also includes texts that are a record of living speech of Catholics of all generations of Belarusian sites. They document important and dramatic events in the history of the Catholic Church in Belarus and the fate of his faithful, as well as the local Polish language, which goes along with its users. Keywords: Belarus; Polish language; Belarussian language; Russian language 354 354
https://openalex.org/W4254181948	https://osf.io/b654q/download	Indonesian	null	Pengaruh Gaya Kepemimpinan dan Stres Kerja Terhadap Kepuasan Kerja Karyawan	null	2,017	cc-by	6,883	ABSTRAK Penelitian ini bertujuan untuk menguji dan menganalisis pengaruh gaya kepemimpinan dan stres kerja terhadap kepuasan kerja karyawan pada PT. Utama Duta Harapan Makassar. Pengumpulan data menggunakan data primer yang di peroleh dari hasil penyebaran kuesioner. Populasi dalam penelitian ini adalah seluruh karyawan pada PT. Utama Duta Harapan Makassar yang berjumlah 110 orang karyawan tetap, Penentuan sampel dilakukan dengan menggunakan teknik stratified random sampling dengan menggunakan formulasi Slovin pada presisi sebesar 10% sehingga diperoleh jumlah sampel sebanyak 50 responden. Pengujian hipotesis dilakukan dengan menggunakan teknik Structural Equation Modelling (SEM) dengan bantuan software WarphPLS Ver.5.0. Hasil penelitian ini membuktikan bahwa gaya kepemimpinan yang ditunjukkan pimpinan berpengaruh positif dan signifikan terhadap kepuasan kerja, sedangkan pada pengujian pengaruh stress kerja terhadap kepuasan kerja ditemukan bahwa stress kerja yang di miliki karyawan tergolong rendah namun tidak memberikan pengaruh yang nyata terhadap peningkatan kepuasan kerja. Kata kunci : Gaya Kepemimpinan, Stres Kerja, Kepuasan Kerja Hamsinah1), Herman Sjahruddin2), Mustafa Gani3) Ref.inha@yahoo.com 1)Mahasiswa Program Studi Manajemen pada Sekolah Tinggi Ilmu Ekonomi Bongaya Makassar 2, 3) Dosen Program Studi Manajemen pada Sekolah Tinggi Ilmu Ekonomi Bongaya Makassar JURNAL ORGANISASI DAN MANAJEMEN Issue 2 (Oktober, 2017) Latar Belakang g Tingginya intensitas persaingan yang dialami PT. Utama Duta Harapan dalam pemasaran produk elektronik seperti merek Miyako, Rinnai, dan Shimizu, mengakibatkan perusahaan perlu memperhatikan sumber daya manusianya melalui peningkatan kepuasan kerja karyawan. Pengelolaan sumber daya manusia dalam organisasi bukan hal yang mudah karena melibatkan berbagai elemen dalam sebuah organisasi, yaitu karyawan, pimpinan, maupun sistem itu sendiri.Perpaduan antara ketiga hal tersebut diharapkan mampu memunculkan kepuasan kerja karyawan.Kepuasan kerja mempunyai pengaruh yang cukup besar terhadap produktivitas organisasi baik secara langsung maupun tidak langsung.Kepuasan kerja merupakan suatu keadaann yang berkaitan dengan reaksi emosional dari persepsi seseorang yang telah mendapatkan kebutuhan dan permintaan yang diinginkannya dari pekerjaan yang dia lakukan . kepuasan kerja merupakan predikator kuat membangun komitmen karyawan diikuti dengan kepemimpinan (Soegihartono, 2012; dalam Pradifta, 2014). Hasil pengamatan lapangan menunjukkan bahwa tingkat kepuasan kerja karyawan cenderung rendah, hal ini diperlihatkan melalui rendahnya disiplin kerja karyawan yang ditunjukkan dengan rendahnya kepatuhan karyawan pada aturan kerja, datang dan pulang tidak sesuai waktu kerja serta pemberian insentif yang tidak dilakukan secara adil. Kondisi tersebut, berdasarkan hasil wawancara dengan karyawan disebabkan karena pemimpin dalam melaksanakan tugas dan tanggung jawabnya memiliki kepedulian yang rendah kepada karyawan dan suasana kerja yang tidak kondusif (sering terjadi konflik antara karyawan bahkan dengan pimpinan). Faktor- faktor yang mempengaruhi kepuasan kerja (indikator) yaitu kesempatan untuk maju, keamanan kerja, gaji, pangkat, faktor instrinsik dari pekerjaan, kondisi kerja, aspek sosial dalam pekerjaan, fasilitas kesejahteraan. Pada penelitian ini, tinggi ataupun rendahnya kepuasan kerja karyawan disebabkan karena faktor instrinsik dari pekerjaan yang ditunjukkan melalui gaya kepemimpinan dan faktor kondisi kerja yang ditunjukkan dengan beban kerja yang berlebihan (stres kerja) (Sutrisno, 2009:83, dalam Tukimin, 2014). Teori Keseimbangan (Equity Theory) yang dikemukakan Gibson, (1996); dalam Sinollah (2011) mengemukakan bahwa puas atau tidaknya karyawan merupakan hasil dari perbandingan antara input - outcome dirinya dengan input - outcome karyawan lain (comparison person). Jadi jika perbandingan tersebut dirasakan seimbang (equity) maka karyawan tersebut akan merasa puas. Tetapi, apabila terjadi tidak seimbang dapat menyebabkan dua kemungkinan yaitu over compensation inequity (ketidak seimbangan yang menguntungkan dirinya) dan sebaliknya, under compensation inequity (ketidakseimbangan yang menguntungkan karyawan lain yang menjadi pembanding). ABSTRACT This Study aimed to examine and analyze the interact Leadership Style and Work Stress of employees job satisfaction in PT. Utama Duta Harapan Makassar. Data collecting by using primary data obtained from the result questionnaires spread. Population in this study is all the employees in PT. Utama Duta Harapan Makassar amount 110 employees remain, samples stipulation using stratified random sampling by using Slovin formulation technic on 10% precision so that obtainable 50 respondents. Hypothesis testing using Structural Equation Modelling (SEM) technic with WarphPLS Ver.5.0. The result of this study prove that Leadership Style indicated by leader take positive effects and significant to Employess job satisfaction, while on testing the influence of work stress to job satisfaction found that work stress of employees is low but it is not giving the real effect to increase employees job satisfaction. Keywords: Leadership style, work stress, employees job satisfaction Halaman 62 Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q Halaman 62 Halaman 62 Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q JURNAL ORGANISASI DAN MANAJEMEN Issue 2 (Oktober, 2017) Latar Belakang Bahwa kepuasan kerja disebabkan karena adanya kesesuaian antara harapan dan kenyataan.Teori Keseimbangan (Equity Theory) seperti yang dikemukakan menjelaskan bahwa perasaan puas atau tidaknya karyawan ditentukan antara keseimbangan/kesesuaian antara harapan dan kenyataan (input – outcome) yang diperoleh karyawan dari pimpinan serta keseimbangan/kesesuaian antara harapan dan kenyataan (input – outcome) dari beban pekerjaan yang merupakan tugas dan tanggung jawab karyawan. Keseimbangan yang dimaksud dalam penelitian ini yaitu keseimbangan atau kesesuaian antara harapan dan kenyataan (input – outcome) dari gaya kepemimpinan dengan harapan dan kenyataan (input – outcome) dari pelaksanaan kerja (stress kerja). Halaman 63 Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q Halaman 63 Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q JURNAL ORGANISASI DAN MANAJEMEN Issue 2 (Oktober, 2017) Berdasarkan teori tersebut dapat dijelaskan bahwa peningkatan kepuasan kerja karyawan pada suatu organisasi tidak bisa dilepaskan dari peranan pemimpin dalam organisasi tersebut, serta jumlah (kuantitas) pekerjaan (beban kerja) yang jika diberikan secara proporsional akan mengakibatkan perasaan puas dari karyawan dalam bekerja yang kemudian menjadi kunci utama atau memainkan peran penting dan strategis dalam kelangsungan hidup suatu perusahaan. Gaya kepemimpinan ialah pola perilaku yang akan ditunjukkan oleh pemimpin dalam mempengaruhi orang lain atau karyawan. Pola perilaku tersebut dapat dipengaruhi oleh beberapa faktor seperti, seperti nilai-nilai, asumsi, persepsi, harapan maupun sikap yang ada dalam diri pemimpin (Ardana dkk, 2011: 181, dalam satyawati,dkk., 2014). Penelitian mengenai hubungan gaya kepemimpinan terhadap kepuasan kerja mengacu pada penelitian yang dilakukan Fitriansyah, dkk., (2013); Purnomo, dkk., (2011); Pradifta, dkk., (2014) bahwa gaya kepemimpinan transformasional dan transaksional berpengaruh positif dan signifikan terhadap kepuasan kerja, dan penelitian ini ditunjukkan pada penelitian Simanungkalit, dkk., (2013); Losiana lomanto, dkk., (2012); bahwa gaya kepemimpinan berpengaruh negatif dan signifikan terhadap kepuasan kerja. Faktor lainnya yang mempengaruhi kepuasan kerja karyawan adalah stress kerja. Stres kerja merupakan istilah umum yang dapat diartikan sebagai tekanan hidup yang dirasakan terlalu sulit bagi seseorang. Stres akan terjadi jika seorang individu tidak mampu memahami keterbatasannya akan suatu hal. Ketidakmampuan ini nantinya akan menimbulkan rasa frustasi, gelisah, serta rasa bersalah yang merupakan awal dari permulaan stres tersebut. Stres kerja adalah suatu keadaan yang dialami individu dalam bekerja seperti, menghadapi sebuah peluang, kendala, atau tuntutan yang hasilnya dianggap tidak pasti namun penting (Robbins, 2007:793); dalam Noor, dkk., 2016). Pandangan lainnya menyatakan bahwa stres kerja adalah suatu kondisi ketegangan yang menciptakan adanya ketidakseimbangan fisik dan psikis, yang mempengaruhi emosi, proses berpikir dan kondisi seorang karyawan (Rivai, 2010:108; dalam Afrizal dkk., 2014). Penelitian mengenai hubungan stress kerja terhadap kepuasan kerja mengacu pada penelitian yang di lakukan Dhania, dkk., (2010); Dewi, dkk., (2015); Pratama, dkk., (2015); Bahwa stress kerja berpengaruh negative dan signifikan terhadap kepuasan kerja. 1. Konsep Gaya Kepemimpinan p y p p Kepemimpinan secara harfian berasal dari kata pimpin. Kata pimpin mengandung pengertian mengarahkan, membina atau mengatur, menuntun dan juga menunjukkan ataupun mempengaruhi. Pemimpin mempunyai tanggung jawab baik secara fisik maupun spiritual terhadap keberhasilan aktivitas kerja dari yang dipimpin, sehingga menjadi pemimpin itu tidak mudah dan tidak akan setiap orang mempunyai kesamaan di dalam menjalankan ke-pemimpinannya. Kepemimpinan didefinisikan sebagai upaya mempengaruhi pengikut melalui proses komunikasi untuk mencapai tujuan tertentu. Definisi di atas menunjukkan bahwa kepemimpinan melibatkan penggunaan pengaruh. Unsur kedua, menyangkut pentingnya proses komunikasi, kejelasan dan ketepatan komunikasi mempengaruhi perilaku dan prestasi bawahan. Unsur terakhir yaitu pencapaian tujuan.Pemimpin yang efektif mungkin harus berurusan dengan tujuan individu, kelompok, dan organisasi. Para ahli biasanya memberikan definisi dengan cara yang bervariasi mengenai kepemimpinan. Halaman 64 Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q Halaman 64 Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q JURNAL ORGANISASI DAN MANAJEMEN Issue 2 (Oktober, 2017) Kepemimpinan adalah kegiatan untuk mempengaruhi orang lain atau seni untuk mempengaruhi perilaku manusia, baik secara perseorangan atau kelompok (Thoha, 2012:8). Pandangan lainnya mengemukakan bahwa kepemimpinan adalah kemampuan untuk mempengaruhi suatu kelompok untuk pencapaian suatu visi atau tujuan (Robbins, 2011:410), Kepemimpinan adalah proses mempengaruhi dalam menentukan tujuan organisasi, memotivasi perilaku pengikutnya untuk mencapai tujuan dan mempengaruhi kelompok dan budaya (Rivai, 2004:2). Berdasarkan pada definisi yang telah dikemukakan beberapa ahli, maka dapat disimpulkan bahwa kepemimpinan adalah penggunaan pengaruh bukan paksaan untuk memotivasi anggota organisasi serta memahami manfaat kerja bersama orang lain dan dapat mengartikulasikan visi, mewujudkan nilai dan menciptakan lingkungan untuk mencapai tujuan yang telah ditetapkan. Selain itu kepemimpinan dapat menyatu dan menstimulus serta memotivasi pengikutnya guna mencapai sesuatu yang telah ditargetkan. Setiap pemimpin mempunyai gaya kepemimpinan yang berbeda antara satu dengan lainnya, dan tidak mesti suatu gaya kepemimpinan lebih baik atau lebih buruk daripada gaya kepemimpinan lainnya. 2. Gaya Kepemimpinan Gaya seseorang dalam memimpin, membimbing dan mempengaruhi atau mengontrol pikiran, perasaan atau tingkah laku orang lain dimaksudkan dalam rangka mencapai suatu tujuan tertentu. Menurut Hasibuan (2007:70) gaya kepemimpinan adalah suatu cara pemimpin untuk mempengaruhi bawahannya, agar mau bekerja sama secara produktif untuk mencapai tujuan organisasi. Sedangkan menurut Suwatno dan Priansa (2011:155) : “Gaya kepemimpinan yaitu perilaku yang disukai oleh pemimpin dalam proses mengarahkan dan mempengaruhi pengikut “.Dan Menurut Tjiptono (2006:161) gaya kepemimpinan adalah suatu cara yang digunakan pemimpin dalam berinteraksi dengan bawahannya. Sementara itu, pendapat lain menyebutkan bahwa gaya kepemimpinan adalah pola tingkah laku (kata-kata dan tindakan-tindakan) dari seorang pemimpin yang dirasakan oleh orang lain (Hersey, 2004:29). 3. Gaya Kepemimpinan Transformasional Kepemimpinan transformasional menunjuk pada proses membangun komitmen terhadap sasaran organisasi dan memberi kepercayaan kepada para pengikut untuk mencapai sasaran-sasaran tersebut. Teori transformasional mempelajari juga bagaimana para pemimpin mengubah budaya dan struktur organisasi agar lebih konsisten dengan strategi-strategi manajemen untuk mencapai sasaran organisasional. Kepemimpinan transformasional merupakan kemampuan pemimpin mengubah lingkungan kerja, motivasi kerja, dan pola kerja, dan nilai-nilai kerja yang dipersepsikan bawahan sehingga mereka lebih mampu mengoptimalkan kinerja untuk mencapai tujuan organisasi. Berarti, sebuah proses transformasional terjadi dalam hubungan kepemimpinan manakala pemimpin membangun kesadaran bawahan akan pentingnya nilai kerja, memperluas dan meningkatkan kebutuhan melampaui minat pribadi serta mendorong perubahan tersebut ke arah kepentingan bersama termasuk kepentingan organisasi (Bass dan Avolio, 2003). Pemimpin transformasional harus mempunyai kemampuan untuk menyamakan visi masa depan dengan bawahannya, serta mempertinggi kebutuhan bawahan pada tingkat yang lebih tinggi dari pada apa yang mereka butuhkan. pemimpin transformasional harus mampu membujuk para bawahannya Halaman 65 Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q 4. Gaya Kepemimpinan Transaksional Kepemimpinan transaksional merupakan salah satu gaya kepemimpinan yang intinya menekankan transaksi di antara pemimpin dan bawahan. Kepemimpinan transaksional memungkinkan pemimpin memotivasi dan mempengaruhi bawahan dengan cara mempertukarkan reward dengan kinerja tertentu. Artinya, dalam sebuah transaksi bawahan dijanjikan untuk diberi reward bila bawahan mampu menyelesaikan tugasnya sesuai dengan kesepakatan yang telah dibuat bersama Mustopadidjaja, (2008).Argumentasi terhadapreward mendorong Bass & Avolio (2003) untuk mendefinisikan kepemimpinan transaksional sebagai bentuk hubungan yang mempertukarkan jabatan atau tugas tertentu jika bawahan mampu menyelesaikan dengan baik tugas tersebut. Bass dan Avolio (2003) yang mengemukakan bahwa hubungan pemimpin transaksional dengan karyawan tercermin dari tiga hal yakni: (1) pemimpin mengetahui apa yang diinginkan karyawan dan menjelaskan apa yang akan mereka dapatkan apabila kerjanya sesuai dengan harapan; (2) pemimpin menukar usaha- usaha yang dilakukan oleh karyawan dengan imbalan, (3) Pemimpin responsif terhadap kepentingan pribadi karyawan selama kepentingan tersebut sebanding dengan nilai pekerjaan yang telah dilakukan karyawan. JURNAL ORGANISASI DAN MANAJEMEN Issue 2 (Oktober, 2017) Issue 2 (Oktober, 2017) melaksanakan tugas mereka melebihi kepentingan mereka sendiri demi kepentingan organisasi yang lebih besar. Pengembangan konsep kepemimpinan (transformasional dan transaksional) dengan berlandaskan pada pendapat Maslow mengenai hierarki kebutuhan manusia. Keterkaitan tersebut dapat dipahami dengan gagasan bahwa kebutuhan karyawan yang lebih rendah, seperti kebutuhan fisiologis dan rasa aman hanya dapat dipenuhi melalui praktek gaya kepemimpinan transaksional (Bass dan Avolio (2003). Sejauhmana pemimpin dikatakan sebagai pemimpin transformasional, dapat diukur dalam hubungan dengan pengaruh pemimpin tersebut berhadapan karyawan. Oleh karena itu, Bass & Avolio (2003), mengemukakan ada tiga cara seorang pemimpin transformasional memotivasi karyawannya, yaitu dengan: (1) mendorong karyawan untuk lebih menyadari arti penting hasil usaha, (2) mendorong karyawan untuk mendahulukan kepentingan kelompok, (3) meningkatkan kebutuhan karyawan yang tinggi seperti harga diri dan aktualisasi diri. melaksanakan tugas mereka melebihi kepentingan mereka sendiri demi kepentingan organisasi yang lebih besar. Pengembangan konsep kepemimpinan (transformasional dan transaksional) dengan berlandaskan pada pendapat Maslow mengenai hierarki kebutuhan manusia. Keterkaitan tersebut dapat dipahami dengan gagasan bahwa kebutuhan karyawan yang lebih rendah, seperti kebutuhan fisiologis dan rasa aman hanya dapat dipenuhi melalui praktek gaya kepemimpinan transaksional (Bass dan Avolio (2003). Sejauhmana pemimpin dikatakan sebagai pemimpin transformasional, dapat diukur dalam hubungan dengan pengaruh pemimpin tersebut berhadapan karyawan. Oleh karena itu, Bass & Avolio (2003), mengemukakan ada tiga cara seorang pemimpin transformasional memotivasi karyawannya, yaitu dengan: (1) mendorong karyawan untuk lebih menyadari arti penting hasil usaha, (2) mendorong karyawan untuk mendahulukan kepentingan kelompok, (3) meningkatkan kebutuhan karyawan yang tinggi seperti harga diri dan aktualisasi diri. Dengan cara demikian, antara pimpinan dan bawahan terjadi kesamaan persepsi sehingga mereka dapat mengoptimalkan usaha ke arah tujuan yang ingin dicapai organisasi. Melalui cara ini, diharapkan akan tumbuh kepercayaan, kebanggaan, komitmen, rasa hormat, dan loyal kepada atasan sehingga mereka mampu mengoptimalkan usaha dan kinerja mereka lebih baik dari biasanya. Ringkasnya, pemimpin transformasional berupaya melakukan transforming of visionary menjadi visi bersama sehingga mereka (bawahan plus pemimpin) bekerja untuk mewujudkan visi menjadi kenyataan. JURNAL ORGANISASI DAN MANAJEMEN Issue 2 (Oktober, 2017) Issue 2 (Oktober, 2017) Stres adalah kondisi ketegangan yang mempengaruhi emosi, proses berpikir sehingga menjadi gugup, merasakan kekhawatiran kronis, mudah marah, agresif dan tidak dapat rileks (Hasibuan, 2003:203). Pendapat lain menyatakan bahwa stres kerja terjadi jika seseorang tidak dapat memenuhi tuntutan atau kebutuhan dari pekerjaannya (Losyik, 2007:4). Pendapat selanjutnya Stress menurut robbins (2007:793) adalah suatu keadaan yang dialami oleh individu dalam menghadapi sebuah peluang, kendala, atau tuntutan yang hasilnya dianggap tidak pasti namun penting. Individu pada umumnya menganggap bahwa stres merupakan suatu kondisi yang mengarah kepada timbulnya penyakit fisik maupun mental atau mengarah ke perilaku yang tidak wajar (distress).Akibat lain yang ditimbulkan stres yang bersifat positif disebut sebagai eustress yang merupakan kekuatan yang positif dimana stres kadangkala dapat diperlukan untuk menghasilkan prestasi yang tinggi.(Selye, 1976; dalam Munandar, 2008:378) Cerminan dalam mengetahui tingkat stres yang dialami individu (karyawan) dalam bekerja dapat dilihat berdasarkan pada beberapa(Rahmawati, 2009, dalam Dewi dan Suwandana, 2016)adalah : (1) Tuntutan tugas; pada umumnya karyawan berpendapat bahwa pekerjaan yang dilakukan melebihi kapasitas waktu yang dimiliki sehingga karyawan merasa dikejar waktu dalam menyelesaikan pekerjaan, (2) Tuntutan peran, pada umumnya karyawan berpendapat bahwa peraturan yang cukup fleksibel dalam menjalankan tugas akan turut mendukung karyawan selama bekerja sehingga konflik peran yang dirasakan dari tugas yang dibebankan oleh atasan masih dapat diatasi, (3) tuntutan hubungan antarpribadi, pada umumnya karyawan berpendapat bahwa konflik yang terjadi dengan rekan kerja hanya sebatas pada permasalahan yang berkaitan dengan pekerjaan bukan karena permasalahan pribadi sehingga hubungan yang baik antar karyawan tetap terjaga, (4) Struktur organisasi, pada umumnya karyawan berpendapat bahwa struktur organisasi yang jelas akan menggambarkan alur komunikasi yang jelas sehingga karyawan mengetahui dari mana informasi diperoleh untuk menjalankan pekerjaannya, (5) Kepemimpinan organisasi, pada umumnya, karyawan berpendapat bahwa atasan memberikan pekerjaan berdasarkan deskripsi pekerjaan yang sudah ditetapkan, (6) Tahap hidup organisasi, perusahaan yang berada pada tahap mapan dan sedang melakukan pengembangan, pada umumnya karyawan akan berusaha bekerja keras menghadapi berbagai tuntutan tugas sebab pemberhentian karyawan menjadi pemicu kecemasannya. 5. Konsep Stres Kerja Tinggi rendahnya kinerja karyawan sangat dipengaruhi tingkat stres yang dimiliki karyawan tersebut.Stres kerjamerupakan suatu kondisi ketegangan yang mempengaruhi emosi, proses berpikir dan kondisi seseorang, tingkat stres yang tinggi dapat mempengaruhi kemampuan seseorang untuk menghadapi lingkungan kerjanya dan padaakhirnya mengganggu pelaksanaan tugas-tugasnya sehingga berdampak negatif terhadap prestasi kerjanya atau kinerjanya (Handoko, 2008). Halaman 66 Halaman 66 Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q 6. Konsep kepuasan kerja Kepuasan kerja pada dasarnya merupakan sesuatu yang bersifat individual.Setiap individu memiliki tingkat kepuasan yang berbeda-beda sesuai dengan sistem nilai yang berlaku pada dirinya.Makin tinggi penilaian terhadap kegiatan dirasakan sesuai dengan keinginan individu, maka makin tinggi kepuasannya terhadap kegiatan tersebut.Jadi, secara garis besar kepuasan kerja dapat diartikan sebagai hal yang menyenangkan atau yang tidak menyenangkan yang mana pegawai memandang pekerjannya. Kepuasan kerja didefinisikan sebagai keadaan yang menyenangkan atau emosi positif yang dihasilkan dari penilaian pekerjaan atau pengalaman kerja seseorang.Kepuasan kerja dihasilkan dari persepsi karyawan mengenai seberapa baik pekerjaan mereka menyediakan hal yang dipandang penting. Halaman 67 Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q Halaman 67 Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q Halaman 67 Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q Metode Penelitian Penelitian ini termasuk penelitian metode deskriptif kuantitatif yang bertujuan untuk menjelaskan suatu fenomena empiris yang disertai data statistik dan pola hubungan antara variabel. Pada penelitian ini variabel terikat kepuasan kerja (Y) dipengaruhi oleh variabel bebas gaya kepemimpinan (X1) dan stress kerja (X2). Penentuan sampel dilakukan dengan menggunakan teknik stratified random sampling dengan menggunakan formulasi Slovin pada presisi sebesar 10% sehingga diperoleh jumlah sampel sebanyak 50 responden. Penelitian ini menggunakan analisis SEM (Structural equation modeling) dibantu dengan WaphPLS Ver. 5.0. JURNAL ORGANISASI DAN MANAJEMEN Issue 2 (Oktober, 2017) Terdapat Lima aspek kepuasan kerja diukur dengan Job Descriptive Index(JDI), (Luthans, 2006, dalam Prabowo, 2016).yaitu; (1) Pekerjaan itu sendiri, dalam hal ini dimana perusahaan memberikan tugas yang menarik, tantangan dalam pekerjaan, tanggung jawab terhadap pekerjaan dan karakteristik pekerjaan serta kompleksitas pekerjaan menghubungkan antara kepribadian dengan kepuasan kerja, (2) Gaji, pemberian gaji yang sesuai dengan kontribusi karyawan, ketepatan waktu dalam pembayaran gaji, dan sistem pemberian gaji di dalam perusahaan merupakan salah satu faktor penentu kepuasan kerja karyawan, (3) Promosi, yaitu kesempatan untuk maju dalam organisasi merupakan salah satu penentu kepuasan kerja, (4) pengawasan, yaitu kemampuan penyedia untuk memberikan bantuan teknis dan dukungan perilaku serta hubungan yang baik akan menjadi faktor penentu kepuasan kerja karyawan dalam perusahaan, (5) Rekan kerja, yaitu rekan kerja yang kooperatif merupakan sumber kepuasan kerja yang paling sederhana pada karyawan secara individu terutama rekan kerja yang bertindak sebagai sumber dukungan, kenyamanan dan nasihat. Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q JURNAL ORGANISASI DAN MANAJEMEN X25 0.086 0.926 0.014 0.099 <0.001 X26 -0.245 0.919 0.02 0.099 <0.001 Y11 0.467 0 0.843 0.102 <0.001 Y12 -0.322 -0.082 0.759 0.106 <0.001 Y13 -0.958 0.008 0.543 0.115 <0.001 Y14 0.482 -0.022 0.852 0.102 <0.001 Y15 -0.053 0.104 0.737 0.107 <0.001 Berdasarkan hasil pengolahan data diatas maka dapat dijelaskan sebagai berikut : Pada model 1 diatas, hasil pengolahan data tersebut menjelaskan bahwa dari 2 dimensi gaya kepemimpinan, yaitu X11, X12, kedua dimensi tersebut memenuhi standar Convergent Validity dengan nilai >0.70 dan P-value juga telah memenuhi syarat yaitu memiliki nilai sebesar <0,001 (< 0,05). Untuk stres kerja menunjukkan bahwa terdapat 1 indikator yang tidak memenuhi standar Convergent Validity yaitu X21<0.70,sehingga terdapat alasan yang cukup kuat untuk mengeluarkannya dari model dan kemudian melanjutkannya dengan model 2. Mengacu pada 5 indikator kepuasan kerja, yaitu Y11, Y12, Y13,Y14,Y15. dari ke 5 indikator terdapat 1 indikator yang tidak memenuhi standar Convergent Validity yaitu Y13dengan nilai <0.70,sehingga indikator tersebut harus di keluarkan dari model.Berdasarkan hasil tersebut (Tabel 4.6) ditemukan bahwa masih terdapat indikator dari variabel kepuasan kerja (Y)yang tidak memenuhi standar Convergent validity(uji validitas) yaitu, Y13dengan nilai sebesar 0,543<0.70, sehingga terdapat alasan yang cukup kuat untuk mengeluarkannya dari model dan kemudian melanjutkannya dengan model 2. 1. Evaluasi model pengukuran (Validitas Konvergen) Convergent validity dari model pengukuran dapat dilihat dari korelasi antara skor indikator dengan skor konstruknya (loading factor) dengan kriteria nilai loading factor dari setiap indikator lebih besar dari 0,70 dapat dikatakan valid. Selanjutnya untuk nilai p-value apabila < 0,05 dianggap signifikan. Pada beberapa kasus, syarat loading di atas 0,70 sering tidak terpenuhi khususnya untuk kuesioner yang baru dikembangkan. Oleh karena itu,loading antara 0,40-0,70 harus tetap dipertimbangkan untuk dipertahankan. Selanjutnya dijelaskan pula bahwa, indikator dengan loading< 0,40 dihapus dari model. Penghapusan indikator dengan loading antara 0,40-0,70 dilakukan apabila indikator tersebut dapat meningkatkan AVE dan composite reliability diatas nilai batasannya. Nilai batasan untuk AVE 0,50 dan composite reliability adalah 0,50 ((Machfud dan Dwi, 2013: 66 dalam Arista, 2015). Hasil pengolahan Convergent Validity dapat dilihat pada tabel berikut ini: Halaman 68 Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q Tabel 1 Hasil output combined loadings and cross-loading Model 1 KPMN STRS KRJ KPSN SE P value X11 0.922 -0.110 -0.062 0.099 <0.001 X12 0.922 0.110 0.062 0.099 <0.001 X21 -0.11 -0.439 -0.168 0.119 <0.001 X22 0.121 0.935 -0.031 0.099 <0.001 X23 0.121 0.935 -0.031 0.099 <0.001 X24 -0.165 0.789 -0.06 0.104 <0.001 Tabel 1 Hasil output combined loadings and cross-loading Model 1 KPMN STRS KRJ KPSN SE P value X11 0.922 -0.110 -0.062 0.099 <0.001 X12 0.922 0.110 0.062 0.099 <0.001 X21 -0.11 -0.439 -0.168 0.119 <0.001 X22 0.121 0.935 -0.031 0.099 <0.001 X23 0.121 0.935 -0.031 0.099 <0.001 X24 -0.165 0.789 -0.06 0.104 <0.001 JURNAL ORGANISASI DAN MANAJEMEN Issue 2 (Oktober, 2017) X25 0.086 0.926 0.014 0.099 <0.001 X26 -0.245 0.919 0.02 0.099 <0.001 Y11 0.467 0 0.843 0.102 <0.001 Y12 -0.322 -0.082 0.759 0.106 <0.001 Y13 -0.958 0.008 0.543 0.115 <0.001 Y14 0.482 -0.022 0.852 0.102 <0.001 Y15 -0.053 0.104 0.737 0.107 <0.001 2. Uji Composite reliability (uji reliabilitas kontruk) Penelitian ini menggunakan tiga variabel laten (variabel yang tidak terukur) yaitu variabel gaya kepemimpinan (X1) dan stress kerja (X2) serta kepuasan kerja (Y). Suatu variabel yang dipandang mampu (handal) dalam menjelaskan data dari variabel tersebut, pengujiannya dapat dilihat pada nilai composite reliability dan Cronbach’s Alpha> 0,60, untuk itu dapat diperlihatkan pada tabel berikut: Tabel 2 Latent variable coefficients (composite reliability) (model 1) MODEL 1 GP S KRJ KPS R-Squared 0.454 Composite reliab. 0.919 0.905 0.866 Cronbach’s Alpha 0.823 0.807 0.805 Avg. Var. Extrac. 0.850 0.711 0.570 Full Collin. VIF 1.843 1.042 1.789 Q-Squared 0.518 Tabel 2 Latent variable coefficients (composite reliability) (model 1) Nilai composite reliability untuk variabel gaya kepemimpinan sebesar 0,919> 0,60 sedangkan untuk variabel stress kerja sebesar 0,905> 0,60 dan yang terakhir pada variabel kepuasan kerja sebesar 0,866> 0,60. Selanjutnya untuk Cronbach’s Alpha pada variabel gaya kepemimpinan sebesar 0,823> 0,60 sedangkan untuk variabel stress kerja sebesar 0,807> 0,60 dan yang terakhir pada variabel kepuasan kerja sebesar 0,805> 0,60. Untuk nilai Average Variances Extracted (AVE)nilai variasi rata-rata pada variabel gaya kepemmpinan sebesar 0,850> 0,50 sedangkan untuk variabel stress kerja sebesar 0,711< 0,50 dan yang terakhir pada variabel kepuasan kerja sebesar 0,570> 0,50. Berdasarkan hasil tersebut maka nilai yang Halaman 69 Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q JURNAL ORGANISASI DAN MANAJEMEN Issue 2 (Oktober, 2017) Issue 2 (Oktober, 2017) belum memenuhi kriteria pengujian ditemukan pada nilai variasi rata-rata (AVE) untuk variabelkepuasan kerja, sehingga terdapat cukup alasan yang kuat untuk mengujinya kembali pada model 2. 3. Evaluasi Model Struktural Pengukuran (Outer Model) Model 2 Pengolahan selanjutnya pada model 2 yaitu evaluasi outer model dilakukan melalui 3 kriteria yaitu convergent validity, discriminant validity dan composite reliability. Berikut ini adalah hasil pengolahan data: 3.1. Convergent Validity (Validitas Konvergen) Convergent validity dari model pengukuran dapat dilihat dari korelasi antara skor indikator dengan skor konstruknya (loading factor) dengan kriteria nilai loading factordari setiap indikator lebih besar dari 0,70 dapat dikatakan valid. Selanjutnya untuk nilai p-value apabila < 0,05dianggap signifikan. Pada beberapa kasus, syarat loading di atas 0,70 sering tidak terpenuhi khususnya untuk kuesioner yang baru dikembangkan. Oleh karena itu, loading antara 0,40-0,70 harus tetap dipertimbangkan untuk dipertahankan. Selanjutnya dijelaskan pula bahwa, indikator dengan loading< 0,40 dihapus dari model. Penghapusan indikator dengan loading antara 0,40-0,70 dilakukan apabila indikator tersebut dapat meningkatkan AVE dan composite reliability diatas nilai batasannya. Nilai batasan untuk AVE 0,50 dan composite reliability adalah 0,50 (Machfud dan Dwi, 2013: 66 dalam Arista, 2015) Tabel 3 Hasil output combined loadings and cross-loading Model 2 KPMN STRS KRJ KPSN SE P value X11 0.922 -0.097 -0.136 0.099 <0.001 X12 0.922 0.097 0.136 0.099 <0.001 X22 0.037 0.946 0.086 0.098 <0.001 X23 0.037 0.946 0.086 0.098 <0.001 X24 0.026 0.791 -0.226 0.104 <0.001 X25 0.06 0.917 0.059 0.099 <0.001 X26 -0.157 0.926 -0.041 0.099 <0.001 Y11 0.086 -0.022 0.912 0.100 <0.001 Y12 -0.054 -0.024 0.667 0.109 <0.001 Y14 0.175 -0.033 0.912 0.100 <0.001 Y15 -0.275 0.09 0.735 0.107 <0.001 Hasil pada tabel diatas, menunjukkan hasil pengujian Convergent validity untuk model 2, dimana pada model 1 sebelumnya terdapat beberapa indikator pada variabel stress kerja dan kepuasan kerja yang tidak memenuhi standar Convergent validitysehingga dilakukan pengujian model 2. Pada pengujian model 2 diatas, semua indikator yang tidak memenuhi standar Convergent validity telah dikeluarkan dari model, sehingga pengujian Convergent validity pada model 2 telah memenuhi standar Convergent Validity dengan nilai >0.70 dan P-value juga telah memenuhi syarat yaitu memiliki nilai sebesar <0,001 (< 0,05) untuk seluruh indikator dari variabel stress kerja dan kepuasan kerja. 3.2. Uji Composite reliability (uji realibilitas kontruk) 3.2. Uji Composite reliability (uji realibilitas kontruk) Halaman 70 Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q JURNAL ORGANISASI DAN MANAJEMEN Issue 2 (Oktober, 2017) Issue 2 (Oktober, 2017) Penelitian ini menggunakan tiga variabel laten (variabel yang tidak terukur) yaitu variabel gaya kepemimpinan (X1) dan stres kerja (X2) serta kepuasan kerja (Y). Suatu variabel yang dipandang mampu (handal) dalam menjelaskan data dari variabel tersebut, pengujiannya dapat dilihat pada nilai composite reliability dan Cronbach’s Alpha< 0,60, untuk itu dapat diperlihatkan pada tabel berikut: Tabel 4 Hasil output latent variable coefficients (model 2) MODEL 2 GP S KRJ KPS R-Squared 0.595 Composite reliab. 0.919 0.958 0.885 Cronbach’s Alpha 0.823 0.945 0.823 Avg. Var. Extrac. 0.850 0.822 0.662 Full Collin. VIF 2.128 1.048 2.076 Q-Squared 0.591 Tabel 4 Hasil output latent variable coefficients (model 2) Nilai composite reliability untuk variabel gaya kepemimpinan sebesar 0,919> 0,60 sedangkan untuk variabel stres kerja sebesar 0,958> 0,60 dan yang terakhir pada variabel kepuasan kerja sebesar 0,885> 0,60. Selanjutnya untuk Cronbach’s Alpha pada variabel gaya kepemimpinan sebesar 0,823> 0,60 sedangkan untuk variabel stres kerja sebesar 0,945> 0,60 dan yang terakhir pada variabel kepuasan kerja sebesar 0,823> 0,60. Untuk nilai Average Variances Extracted (AVE)/ nilai variasi rata-rata pada variabel gaya kepemimpinan sebesar 0,850> 0,50 sedangkan untuk variabel stress kerja sebesar 0,822< 0,50 dan yang terakhir pada variabel kepuasan kerja sebesar 0,662> 0,50. Berdasarkan hasil tersebut maka keseluruhan nilai memenuhi kriteria pengujian, sehingga terdapat cukup alasan yang kuat untuk mengujinya kembali pada model 2 dan memiliki alasan yang kuat untuk dianalisis lebih lanjut dimodel 2. Tabel 5 Nilai AVE (Average Variance Extracted) Variabel Laten Nilai AVE Kriteria GP 0,850 > 0,50 SK 0,822 > 0,50 KP 0,662 > 0,50 Berdasarkan hasil tersebut ketiga konstruk telah memenuhi convergent validity.Gaya kepemimpinan dengan nilai 0,850> 0,50, Stress kerja dengan nilai 0,822 juga telah memenuhi nilai > 0,50 dan Kepuasan kerja memiliki nilai 0,662> 0,50. Kesimpulannya keseluruhan variabel telah memenuhi kriteria convergent validity. Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q JURNAL ORGANISASI DAN MANAJEMEN Tabel 6 Nilai loading konstruk laten indikator ke konstruk lainnya Indikator Loading Nilai Loading ke konstruk lainnya GP SK KP X11 (0.922) -0.097 -0.136 X12 (0.922) 0.097 0.136 X22 (0.946) 0.037 0.086 X23 (0.946) 0.037 0.086 X24 (0.791) 0.026 -0.226 X25 (0.917) 0.06 0.059 X26 (0.926) -0.157 -0.041 Y11 (0.912) 0.086 -0.022 Y12 (0.667) -0.054 -0.024 Y14 (0.912) 0.175 -0.033 Y15 (0.735) -0.275 0.09 Keseluruhan indikator telah memenuhi kriteria validitas diskriminan.Variabel gaya kepemimpinan yang memiliki 2 dimensi yang dilambangkan dengan X11, X12,. Untuk dimensi X11 memiliki nilai loading 0,922 yang nilai loading-nya lebih besar dari loading ke konstruk lain yaitu -0,097 dan -0,136 dan begitu juga untuk dimensi X12memiliki nilai loading yang lebih besar dari nilai loading ke konstruk lain. Variabel stress kerja memiliki 5 indikator yang dilambangkan dengan X22, X23, X24 dan X25 ,X26. Untuk indikator X22memiliki nilai loading 0,946yang nilai loading-nya lebih besar dari loading ke konstruk lain yaitu 0,037 dan -0,086 dan ke 4 indikator stress kerja lainnya juga memiliki nilai loading yang lebih besar dari nilai loading ke konstruk lain. 3.3. Discriminant Validity (Validitas Diskriminan) Discriminant validity dinilai dari cross loading pengukuran dengan konstruk. Dapat dilihat dengan melihat loading konstruk laten, yang akan memprediksi indikatornya lebih baik daripada konstruk lainnya. Jika korelasi konstruk dengan pokok pengukuran (setiap indikator) lebih besar daripada ukuran konstruk lainnya maka validitas diskriminan terpenuhi. Halaman 71 Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q Halaman 71 Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q 4. Evaluasi Model Struktural (Inner Model) Tahap selanjutnya adalah melakukan evaluasi struktural (inner model) yang meliputi uji kecocokan model (model fit), path coefficient, dan R2. Pada uji kecocokan model terdapat 3 indeks pengujian, yaitu average path coefficient (APC), average R- squared (ARS) dan average varians factor (AVIF) dengan kriteria APC dan ARS diterima dengan syarat p – value< 0,05 dan AVIF lebih kecil dari 5 (Mahfud Sholihin dan Dwi Ratmono, 2013: 61). Tabel 7 Hasil output model fit indices Indeks P-Value Kriteria APC 0,392 P<0,001 P < 0,05 ARS 0,595 P<0,001 P < 0,05 AVIF 1,054 Acceptable if< 5 AVIF < 5 Hasil output di atas, menjelaskan bahwa APC memiliki indeks sebesar 0,392 dengan nilai p – value< 0,001. Sedangkan ARS memiliki indeks sebesar 0,595 dengan p – value< 0,001.Berdasarkan kriteria, APC sudah memenuhi kriteria karena memiliki nilai p < 0,001.Begitu pula dengan nilai p dari ARS yaitu p < 0,001.Nilai AVIF yang harus < 5 sudah terpenuhi karena berdasarkan data tersebut AVIF nilainya 1,054.Dengan demikian, maka inner model dapat diterima. Halaman 72 Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q 5. Hasil Uji Hipotesis Halaman 72 Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q 5. Hasil Uji Hipotesis 5. Hasil Uji Hipotesis 5. Hasil Uji Hipotesis Halaman 72 Halaman 72 Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q JURNAL ORGANISASI DAN MANAJEMEN Issue 2 (Oktober, 2017) 0,012dengan p – value 0,465 nilai effect size for path 0,002. Setelah melakukan uji hipotesis di atas, berikut ini tabel yang merangkum uji hipotesis-hipotesis tersebut : Tabel 9 Hasil uji hipotesis Hipotesis Independen Dependen p-values Keputusan H1 kepuasan kerja Gaya Kepemimpinan <0,001 Diterima H2 kepuasan kerja Stres Kerja <0,465 Ditolak H3 kepuasan kerja Gaya Kepemimpinan dan Stres Kerja Nilai 0,772 > dari nilai -0,012 Diterima Tabel 9 Hasil uji hipotesis Hipotesis Independen Dependen p-values Keputusan H1 kepuasan kerja Gaya Kepemimpinan <0,001 Diterima H2 kepuasan kerja Stres Kerja <0,465 Ditolak H3 kepuasan kerja Gaya Kepemimpinan dan Stres Kerja Nilai 0,772 > dari nilai -0,012 Diterima 5.1. Uji hipotesis 1 diterima, artinya variabel gaya kepemimpinan memiliki pengaruh positif dan signifikan terhadap kepuasan kerja pada PT. Utama Duta Harapan. Hal ini ditunjukkan dengan nilai beta (β) sebesar 0,772 dengan nilai p-value<0.001. 5.2. Uji hipotesis 2 ditolak, artinya variabel stress kerja memiliki pengaruh negatif dansignifikan terhadap kepuasan kerja pada PT. Utama Duta Harapan Makassar. Hal ini ditunjukkan dengan nilai beta (β) sebesar -0,012 dengan nilai p-value 0,465. 5.3. Uji hipotesis 3 diterima, artinya variabel gaya kepemimpinan dominan memiliki pengaruh positif dan signifikan terhadap kepuasan kerja pada PT. Utama Duta Harapan Makassar. Hal ini ditunjukkan dengan variabel gaya kepemimpinan memiliki nilai lebih tinggi yaitu nilai beta (β) sebesar 0,772 dengan nilai p-value<0.001 dibandingkan dengan variabel stress kerja dengan nilai beta (β) sebesar -0,012 dan nilai p-value 0,465. 6. Uji besaran pengaruh variabel independen terhadap variabel dependen Pengujian besaran pengaruh variabel independen terhadap variabel dependen dapat ditunjukkan pada tabel berikut . Tabel 10 Ringkasan model (model summary) Pengukuran GP SK KP R-squared 0.595 Adj. R-squared 0.578 Composite reliab. 0.919 0.958 0.885 5.1. Uji hipotesis 1 diterima, artinya variabel gaya kepemimpinan memiliki pengaruh positif dan signifikan terhadap kepuasan kerja pada PT. Utama Duta Harapan. Hal ini ditunjukkan dengan nilai beta (β) sebesar 0,772 dengan nilai p-value<0 001 5.3. Uji hipotesis 3 diterima, artinya variabel gaya kepemimpinan dominan memiliki pengaruh positif dan signifikan terhadap kepuasan kerja pada PT. Utama Duta Harapan Makassar. Hal ini ditunjukkan dengan variabel gaya kepemimpinan memiliki nilai lebih tinggi yaitu nilai beta (β) sebesar 0,772 dengan nilai p-value<0.001 dibandingkan dengan variabel stress kerja dengan nilai beta (β) sebesar -0,012 dan nilai p-value 0,465. JURNAL ORGANISASI DAN MANAJEMEN Issue 2 (Oktober, 2017) Issue 2 (Oktober, 2017) Hasil korelasi antar konstruk diukur dengan melihat path coefficients dan tingkat signifikansinya yang kemudian dibandingkan dengan hipotesis penelitian yang terdapat di bab dua. Tingkat signifikansi yang dipakai dalam penelitian ini adalah sebesar 5%. Berikut ini hipotesis yang dimaksudkan untuk membuktikan kebenaran dugaan penelitian yang terdiri dari tiga hipotesis, yaitu: p y g kebenaran dugaan penelitian yang terdiri dari tiga hipotesis, yaitu: H1 = Gaya Kepemimpinan berpengaruh positif dan signifikan terhadap kepuasan kerja Pada PT. Utama Duta Harapan Makassar. H2 = Stres kerja berpengaruh negatif dan signifikan terhadap kepuasan kerja Pada PT. Utama Duta Harapan Makassar. H2 = Stres kerja berpengaruh negatif dan signifikan terhadap kepuasan kerja Pada PT. Utama Duta Harapan Makassar. H3 = Gaya Kepemimpinan merupakan variabel yang dominan pengaruhnya terhadap kepuasan kerja Pada PT. Utama Duta Harapan Makassar. H3 = Gaya Kepemimpinan merupakan variabel yang dominan pengaruhnya terhadap kepuasan kerja Pada PT. Utama Duta Harapan Makassar. Gambar 1 Hasil penelitian Keterangan : X1 : Gaya Kepemimpinan X2 :Stres Kerja Y : Kepuasan Kerja Tabel 8 Direct Effects Kriteria Variabel GP SK KP Path coefficients GP - - - SK - - - KP 0,772 -0,012 - p-values GP - - - SK - - - KP <0,001 0,465 - Effect size for path GP - - - SK - - - KP 0,593 0,002 - Hasil output di atas, menjelaskan bahwa path coefficients untuk variabel gaya kepemimpinan terhadap kepuasan kerja memiliki indeks sebesar 0,772 dengan nilai p – value< 0,001 dan nilai effect size for path0,593 sedangkan untuk path Gambar 1 Hasil penelitian Gambar 1 Hasil penelitian : Gaya Kepemimpinan :Stres Kerja : Kepuasan Kerja p j Tabel 8 Direct Effects Kriteria Variabel GP SK KP Path coefficients GP - - - SK - - - KP 0,772 -0,012 - p-values GP - - - SK - - - KP <0,001 0,465 - Effect size for path GP - - - SK - - - KP 0,593 0,002 - Hasil output di atas, menjelaskan bahwa path coefficients untuk variabel gaya kepemimpinan terhadap kepuasan kerja memiliki indeks sebesar 0,772 dengan nilai p – value< 0,001 dan nilai effect size for path0,593 sedangkan untuk path coefficientsvariabel stress kerja terhadap kepuasan kerja memiliki indeks sebesar - Halaman 73 Halaman 73 Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q 6. Uji besaran pengaruh variabel independen terhadap variabel dependen Pengujian besaran pengaruh variabel independen terhadap variabel dependen dapat ditunjukkan pada tabel berikut . Tabel 10 Ringkasan model (model summary) Pengukuran GP SK KP R-squared 0.595 Adj. R-squared 0.578 Composite reliab. 0.919 0.958 0.885 Cronbach’s Alpha 0.823 0.945 0.823 Avg. Var. Extrac. 0.850 0.882 0.662 Berdasarkan tabel diatas besaran pengaruh nilai R-squared pada variabel gaya kepemimpinan dan variabel stres kerja terhadap kepuasan kerja adalah 0,595 (59,5%) dan sisanya 40,5% dipengaruhi oleh variabel lain yang tidak dianalisis dalam penelitian ini. 7. Interpretasi hasil penelitian Penelitian ini membahas pengaruh gaya kepemimpinan terhadap kepuasan kerja, pengaruh stres kerja terhadap kepuasan kerja dan gaya kepemimpinan dominan berpengaruh terhadap kepuasan kerja pada PT. Utama Duta Harapan Makassar. Halaman 74 Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q Halaman 74 Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q 7.2. Pengaruh Stres Kerja Terhadap Kepuasan Kerja e ga u St es e ja e adap epuasa e ja Berdasarkan hasil penelitian, ditemukan bahwa Stres kerja memiliki pengaruh terhadap kepuasan kerja. Hasil ini sama dengan teori atau temuan dalam penelitian sebelumnya oleh Dhania, d. R. (2010). Bahwa stress kerja signifikan mempengaruhi kepuasan kerja karyawan. Dalam penelitian ini dapat dilihat dari nilai koefisien beta yang menunjukkan bahwa stress kerja secara signifikan pengaruh terhadap kepuasan kerja yaitu dengan nilai beta (β) sebesar -0,012. Hal ini menunjukkan bahwa variabel stress kerja tidak mampu memengaruhi kepuasan kerja karyawan. Penyebab negatif dan signifikannya pengaruh stress kerja karyawan diakibatkan karena karyawan sangat cemas dalam menghadapi berbagai tuntutan tugas yang memicu kinerjanya . Kondisi tersebut didukung dengan perusahaan senantiasa memberhentikan karyawannya yang tidak dapat menghadapi tuntutan tugasnya pada PT. Utama Duta Harapan Makassar. 7.1. Pengaruh Gaya Kepemimpinan Terhadap Kepuasan Kerja g y p p p p j Berdasarkan hasil penelitian, ditemukan bahwa gaya kepemimpinan memiliki pengaruh terhadap kepuasan kerja. Hasil ini sama dengan teori atau temuan dalam penelitian sebelumnya oleh Fitriansyah,R. (2013). memberikan bukti bahwa gaya kepemimpinan berpengaruh positif dan signifikan terhadap kepuasan kerja. Dalam penelitian ini dapat dilihat dari nilai koefisien beta yang menunjukkan bahwa semakin baik gaya kepemimpinan maka semakin tinggi kepuasan kerja yaitu dengan nilai beta (β) sebesar 0,772. Hal ini menunjukkan bahwa variabel gaya kepemimpinan dapat memengaruhi peningkatan kepuasan kerja kearah yang lebih tinggi. Penyebab positif dan signifikannya pengaruh gaya kepemimpinan terhadap kepuasan kerja diakibatkan karena pimpinan selalu memberikan kesempatan kepada karyawan untuk bertanya tentang permasalahan kerja yang memang dianggap benar-benar penting sehingga berdampak pada tingginya kepuasan kerja karyawan dalam menyelesaikan pekerjaan tersebut. Kondisi tersebut didukung dengan pimpinan senantiasa memberikan bimbingan kepada karyawan dalam menyelesaikan pekerjaannya dengan standar yang telah di tetapkan pada PT. Utama Duta Harapan Makassar. JURNAL ORGANISASI DAN MANAJEMEN Issue 2 (Oktober, 2017) 7.3. Pengaruh Dominan Berdasarkan hasil penelitian, ditemukan bahwa pengaruh gaya kepemimpinan lebih dominan dibandingkan pengaruh stres kerja terhadap kepuasan kerja. Hasil tersebut dalam penelitian ini dapat dilihat dari nilai koefisien beta yang ditunjukkan gaya kepemimpinan lebih tinggi yaitu dengan nilai beta (β) sebesar 0,772 dan nilai koefisien beta yang ditunjukkan oleh stress kerja yaitu dengan nilai -0,012. Hal ini menunjukkan bahwa variabel gaya kepemimpinan dapat mempengaruhi peningkatan kepuasan kerja kearah yang lebih tinggi dibandingkan variabelkepuasan kerja. Penyebab positif dan signifikannya dominan pengaruh gaya kepemimpinan terhadap kepuasan kerja karyawan diakibatkan karena pimpinan selalu memberikan kesempatan kepada karyawan untuk bertanya tentang permasalahan kerja yang memang dianggap benar-benar penting sehingga berdampak pada tingginya kepuasan kerja karyawan dalam menyelesaikan pekerjaan tersebut. Kondisi tersebut didukung dengan pimpinan senantiasa memberikan bimbingan kepada karyawan dalam menyelesaikan pekerjaannya dengan standar yang telah di tetapkan pada PT. Utama Duta Harapan Makassar. Halaman 75 Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q Kesimpulan Gaya kepemimpinan dapat memengaruhi peningkatan kepuasan kerja kearah yang lebih tinggi. Penyebab positif dan signifikannya pengaruh gaya kepemimpinan terhadap kepuasan kerja diakibatkan karena pimpinan selalu memberikan kesempatan kepada karyawan untuk bertanya tentang permasalahan kerja yang memang dianggap benar-benar penting sehingga berdampak pada tingginya kepuasan kerjakaryawan dalam menyelesaikan pekerjaan Stress kerja yang rendah tidak mampu memengaruhi peningkatan kepuasan kerja. Kondisi tersebut diakibatkan karena karyawan sangat cemas dalam menghadapi berbagai tuntutan tugas yang memicu kinerjanya dan perusahaan senantiasa memberhentikan karyawannya yang tidak dapat menghadapi tuntutan tugasnya Gaya kepemimpinan dominan berpengaruh terhadap kepuasan kerja. Kondisi tersebut disebabkan karena pimpinan selalu memberikan kesempatan kepada karyawan untuk bertanya tentang permasalahan kerja yang memang dianggap benar-benar penting sehingga berdampak padatingginya kepuasan kerja karyawan. Penelitan ini merekomendasikan kepada peneliti selanjutnya untuk menambah beberapa variabel independen lainnya agar variabel tersebut mampu menjelaskan variabel dependen lebih besar dari penelitian sebelumnya sehingga mendapatkan hasil yang lebih akurat. Dalam hal waktu, hendaklah peneliti selanjutnya dapat memiliki waktu yang cukup untuk melakukan penelitian sehingga bisa mencapai hasil yang maksimal. Kepada PT. Utama Duta Harapan untuk lebih meningkatkan dan tetap menjaga kepemimpinannya agar lebih baik sehingga karyawan tetap merasa puas dalam bekerja, serta dapat memperbaiki system kerja yang baik agar stress karyawan dalam bekerja dapat berkurang dan teratasi. JURNAL ORGANISASI DAN MANAJEMEN Issue 2 (Oktober, 2017) DAFTAR PUSTAKA Afrizal, P. R. (2014). Pengaruh konflik kerja dan stress kerja terhadap kepuasan kerja (studi pada karyawan PT. TASPEN (PERSERO) cabang Malang).Jurnal Administrasi Bisnis, 8(1). Brahmasari, I. A., & Suprayetno, A. (2009). Pengaruh Motivasi Kerja, Kepemimpinan dan Budaya Organisasi Terhadap Kepuasan Kerja Karyawan serta Dampaknya pada Kinerja Perusahaan (Studi kasus pada PT. Pei Hai International Wiratama Indonesia). Jurnal Manajemen dan Kewirausahaan, 10(2), pp-124. Dewi, N. K. N. C., &Subudi, I. Pengaruh Kepemimpinan Transformasional Terhadap Kepuasan Kerja dan Turnover Intention Pada CV. Gita Karya Persada Denpasar. E-Jurnal Manajemen Universitas Udayana, 4(12). Faqihudin, M., & Gunistiyo, M. S. (2009). Pengaruh Stres Kerja Terhadap Kepuasan Kerja Dan Intensi Meninggalkan Organisasi Pada Bank-Bank Milik Negara Di Kota Tegal. Sosekhum, 5(7). Fitriansyah, R.(2013). Pengaruh Gaya Kepemimpinan Transformasional Dan Transaksional Terhadap Kepuasan KerjaKaryawan (Studi Pada Agen Financial Consultant PT. Axa Financial Indonesia Sales Office Malang).Jurnal Administrasi Bisnis, 4(2). Ghozali, Imam. (2013). Aplikasi Analisis Multivariate Dengan Program SPSS (Edisi ketujuh). Semarang :UniversitasDiponegoro Halaman 76 Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q JURNAL ORGANISASI DAN MANAJEMEN Issue 2 (Oktober, 2017) Noor, N. N., Rahardjo, K., & Ruhana, I. (2016). Pengaruh Stres Kerja Dan Kepuasan Kerja Terhadap Kinerja Karyawan (Studi Pada Karyawan PT Jasa Raharja (Persero) Cabang Jawa Timur di Surabaya). Jurnal Administrasi Bisnis, 31(1), 9-15. Potale, R., & Uhing, Y. (2015). Pengaruh Kompensasi dan Stres Kerja Terhadap Kepuasan Kerja Karyawan Pada PT. Bank Sulut Cabang Utama Manado. Jurnal Riset Ekonomi, Manajemen, Bisnis Dan Akuntansi, 3(1). Pradifta, N. B. H., & Sudibia, G. A. Pengaruh Kepemimpinan Transformasional dan Kepuasan Kerja Terhadap Komitmen Organisasional (Studi Pada Aerowisata Sanur Beach Hotel). E-Jurnal Manajemen Universitas Udayana, 3(9). Simanungkalit, Y. M. V., & Setyaningsih, E. (2013). Pengaruh Gaya KepemimpinanTerhadap Kepuasan Kerja Karyawan Pada PT. Lion Mentari Airlines.UG Jurnal, 7(6). Sjahruddin, H. 1 E-Library STIE YPBUP Bongaya 2016. Sjahruddin, H., Vivi, M., & Ahmad, A. (2014).Pengaruh Stres dan Lingkungan Kerja Non Fisik Terhadap Kinerja Karyawan Pada PT. Bumi Jasa Utama (Kallatransport) Makassar. E-Library STIE YPBUP Bongaya. Tunjungsari, P. (2011). Pengaruh Stress Kerja Terhadap Kepuasan Kerja Karyawan Pada Kantor Pusat PT. Pos Indonesia (Persero) Bandung. Jurnal Universitas Komputer Indonesia, 1(1), 1 Halaman 77 Author : Hamsinah dkk. (Oktober, 2017). 62 – 77 https://dx.doi.org/10.17605/OSF.IO/B654Q
https://openalex.org/W2216061085	https://www.scielo.br/j/ambiagua/a/HJtgY79KvsTgxzfwVh9JDNR/?lang=en&format=pdf	English	null	Chemical and microbiological analysis of public school water in Uberaba Municipality	Revista Ambiente & Água	2,015	cc-by	5,738	Sérgio Marcos Sanches1; Jailda Maria Muniz2; Célia Passos3; Eny Maria Vieira4 1Instituto Federal do Triângulo Mineiro (IFTM), Campus Ituiutaba, Ituiutaba, MG, Brasil 2Instituto Federal do Triângulo Mineiro (IFTM), Campus Uberaba, Uberaba, MG, Brasil 3Universidade Federal de Uberlândia(UFU) Campus Pontal, Ituiutaba, MG, Brasil 4Universidade de São Paulo (USP), São Carlos, SP, Brasil Corresponding author: e-mail: sergiosanches@iftm.edu.br, jailda@iftm.edu.br, sellyap@qui.pontal.ufu.br, eny@iqsc.usp.br ABSTRACT This study evaluated the quality of water consumed by schoolchildren in the city of Uberaba, relying upon chemical analyzes to determine the levels of free-residual chlorine and levels of chromium, copper, manganese, lead and cadmium. Microbiological analysis was also performed in order to determine total coliforms and Escherichia coli, using the values established by Ordinance n0. 2914 of 2011 of the Ministry of Health as parameters for safe drinking water. Water samples were analyzed from the drinking fountains and kitchen faucets of eight public schools that serve children aged 0-5 years. Sampling was conducted quarterly from December 2011 to September 2012, resulting in four collections. The results revealed the presence of Escherichia coli and total coliforms above the valued permitted by legislation in more than 50% of the samples. It was also observed that concentrations of free-residual chlorine were below the minimum value required by law in nearly half of the samples analyzed. In relation to the concentration of metals, some samples had water contents of copper, cadmium, chromium, manganese and lead above the permissible levels. Statistical tests revealed that when analyzing the period of sampling, only the values for the concentrations of free-residual chlorine, chromium and lead showed no significant difference (p> 0.05). The results show the need for corrective actions at the water supply point for the school population, in addition to monitoring and controlling the quality of water for human consumption. Keywords: chemical analysis, drinking water, microbiological analyzes. Ambiente & Água - An Interdisciplinary Journal of Applied Science ISSN 1980-993X – doi:10.4136/1980-993X www.ambi-agua.net E-mail: ambi.agua@gmail.com Ambiente & Água - An Interdisciplinary Journal of Applied Science ISSN 1980-993X – doi:10.4136/1980-993X www.ambi-agua.net E-mail: ambi.agua@gmail.com Ambiente & Água - An Interdisciplinary Journal of Applied Science ISSN 1980-993X – doi:10.4136/1980-993X www.ambi-agua.net E-mail: ambi.agua@gmail.com 1. INTRODUCTION Drinking water is vital to the survival of all living organisms and the functioning of ecosystems, communities and economies. However, in the last decade, water quality worldwide is being threatened due to population increases, agricultural and industrial activities (Khan et al., 2013; Baird, 2002). These changes resulted in climate change, directly affecting the global hydrological cycle. A supply of good quality water that is adequate, reliable and affordable to the population is closely linked to human health. However, according to the Environmental Company of São Paulo (2008), part of the available fresh water on the planet is in some stage of contamination (Cetesb, 2008). According to the World Health Organization - WHO, at present, 1.2 billion people do not have drinking water, and 80% of diseases and 30% of deaths worldwide are caused by contaminated water (Hênio, 2011). Already, as a result of poor water quality, the study by the UN shows that at least 1.8 billion children under five years old have died to date (Giraldi, 2010). In several educational institutions, waterborne diseases have become common. This may be related to contamination in water tanks or infiltration in pipes. Therefore, the monitoring and further analysis of the quality of waters originating from faucets and drinking fountains for school consumption becomes important. RESUMO Neste estudo a qualidade da água consumida por escolares na cidade de Uberaba-MG foi avaliada levando-se em consideração os resultados das análises químicas para determinação dos teores de cloro residual livre e níveis de cromo, cobre, manganês, chumbo e cádmio, além Rev. Ambient. Água vol. 10 n. 3 Taubaté – Jul. / Sep. 2015 531 Chemical and microbiological analysis of public school water … das análises microbiológicas para determinação de coliformes totais e Escherichia coli, tomando-se como parâmetro para água potável, os valores estabelecidos pela Portaria nº 2.914 de 2011 do Ministério da Saúde (Brasil, 2011). Foram analisadas amostras de água provenientes de bebedouros e torneiras da cozinha de oito instituições de ensino infantil da rede pública municipal que atendem crianças com a faixa etária de 0 - 5 anos. As amostragens foram realizadas em períodos trimestrais compreendidos entre dezembro de 2011 e setembro de 2012, resultando em quatro coletas. Os resultados obtidos revelaram a presença de Escherichia coli e coliformes totais, acima dos valores permissíveis pela legislação em mais de 50% das amostras analisadas. Foi verificado ainda, teor de cloro residual livre abaixo do valor mínimo exigido pela legislação vigente em quase metade das amostras analisadas. No que se refere à concentração de metais, algumas amostras de água apresentaram teores de cobre, cádmio, cromo, manganês e chumbo acima do permitido. Os testes estatísticos revelaram que, quando analisado o período de realização das amostragens, apenas os valores referentes à concentração de cloro residual livre, cromo e chumbo não apresentaram diferença significativa (p>0,05). Os resultados obtidos revelam a necessidade de ações corretivas nos pontos de fornecimento de água para a população escolar, além do monitoramento e controle da qualidade da água para consumo humano, tanto pelos gestores escolares como pelas autoridades sanitárias. Palavras-chave: água potável, análises microbiológicas, análises químicas. Rev. Ambient. Água vol. 10 n. 3 Taubaté – Jul. / Sep. 2015 2.1. Collection of water samples for chemical analysis 2.1. Collection of water samples for chemical analysis Initially, the collection site (faucet or drinking fountain) was held open, permitting the outflow of water for at least three minutes. Polypropylene bottles were used previously, for acclimating local water, in order to minimize possible interference with the collection and storage of water samples for chemical analysis. Rev. Ambient. Água vol. 10 n. 3 Taubaté – Jul. / Sep. 2015 532 Sérgio Marcos Sanches et al. 2.2. Collection of water for microbiological analysis The collection site was sanitized with 70% ethanol and then flamed to avoid interference with the results due to external contamination. Then, the water samples used for microbiological analyzes were collected from the schools’ drinking fountains and kitchen faucets in amber bottles, previously autoclaved, containing 0.1 mL of sodium thiosulfate to 10% for every 100 mL of water collected. 2.3. Analysis of free residual chlorine The determination of free-residual chlorine was taken by the direct method using a hand photometer checker chlorine-free, ranging from 0.00 to 2.50 mg L-1 - Model HI 701 at the collection site. 2.6. Analysis of results As a reference guide for analysis of the results, we used the 2914 Ordinance of the Ministry of Health, which establishes the procedures and responsibilities relating to the control and supervision of water quality for human consumption and its potability standards and other provisions (Brasil, 2011). 2.5. Analysis of bacteria Laboratory analyses of water samples collected for the assessment of total and thermos-tolerant coliforms were made by the Technical Membrane Filtering in accordance with the procedures of "Standard Methods for the Examination of Water and Wastewater'' and of the Company of Environmental Sanitation Technology (APHA et al., 2005; Cetesb, 2006). Samples were filtered through a suitable membrane filter and sterile with porosity (porosity 0.45 µm and 47 mm in diameter). The filtration was performed on a device contained in a filter funnel with lid, membrane and receiver flask. 2.4. Metals analysis The samples passed through the digestion process by Method 3005A of the Environmental Protection Agency for the analysis of metals (USEPA, 1992; Nikaido et al., 2010). Fifty mL aliquots of a water sample tube were added to the digester block to which was added 2 mL of HNO3 and 5 mL of HCl. The temperature was then raised to 110ºC and maintained until the sample volume was reduced to 15 mL under reflux. After cooling, the samples were transferred to a 50 mL volumetric flask, and the volume was then supplemented with Mille-Q water. The sample was filtered and kept at 4 0 C until further analysis. p p y Analyses of BP, Man, Cr, Cu and Cod metals were performed at the Institute of Chemistry of São Carlos - IQSC / USP of São Carlos, in an inductively coupled plasma spectrometer (ICP-OES). Rev. Ambient. Água vol. 10 n. 3 Taubaté – Jul. / Sep. 2015 2.7. Statistics For the statistical analysis of the final results, the resulting values for all analyses were copied into a database in Microsoft Excel 2003 version program and transferred to the software ASSISTAT 7.6-beta version 2008 for testing normality and homogeneity of data. The results obtained for each of the variables analyzed showed no homogeneity among themselves checked when the period to measurements, so these values were subjected to non-parametric Kruskal-Wallis test (ANOVA), ASSISTAT software - 7.6-beta version in 2008 with level significance of p> 0.05. Rev. Ambient. Água vol. 10 n. 3 Taubaté – Jul. / Sep. 2015 533 Chemical and microbiological analysis of public school water … 3.1. Free residual chlorine The values of free residual chlorine obtained in the analysis of water sampled from the fountains of the schools in the four stages of data collection ranged from 0.00 to 1.04 mg L-1, while levels of free residual chlorine in water samples from faucets of the eight schools ranged from 0.00 to 1.00 mg L-1, as showed in Table 1. Table 1. Values of free residual chlorine obtained from the analyses of water samples used for consumption by schoolchildren in eight schools in Uberaba, Minas Gerais. Table 1. Values of free residual chlorine obtained from the analyses of water samples used for consumption by schoolchildren in eight schools in Uberaba, Minas Gerais. Table 1. Values of free residual chlorine obtained from the analyses of water samples used for consumption by schoolchildren in eight schools in Uberaba, Minas Gerais. Educational Institutions Sampling sites Apparent color (uH) 1st collection (12/14/11) 2nd collection (03/19/12) 3rd collection (06/25/12) 4th collection (09/26/12) A Fountain 0.80 0.08 0.83 0.00 Faucet 0.09 0.10 0.14 0.00 B Fountain 0.30 0.60 0.73 0.30 Faucet 0.09 0.60 0.84 0.00 C Fountain 1.00 0.70 1.04 0.30 Faucet 0.80 0.70 0.96 0.30 D Fountain 0.10 0.04 0.14 0.00 Faucet 0.09 0.14 0.38 0.00 E Fountain 0.80 0.52 0.54 0.10 Faucet 0.80 0.61 0.84 0.10 F Fountain 0.10 0.16 0.14 0.00 Faucet 1.00 0.64 0.65 0.10 G Fountain 1.00 0.51 0.84 0.30 Faucet 1.00 0.63 0.11 0.30 H Fountain 0.22 0.05 0.30 0.30 Faucet 0.16 0.07 0.37 0.05 The results obtained in this study revealed that 100% of the water samples collected in water fountains and kitchen faucets conformed to the standards required by Ordinance no. 2914 of 2011 of the Ministry of Health, 2.0 mg L-1, for free residual chlorine variable (Brasil, 2011). However, with respect to the legal minimum value of 0.2 mg L-1 regarding the concentration of free residual chlorine, 40.62% of the samples from the kitchen taps and 31.25% of samples from the water fountains failed to meet the recommended standard. Also, regarding the concentration of free residual chlorine, no significant difference (p> 0.05) was found between the sampling periods for the water fountains and kitchen faucets of the eight schools selected for the study; therefore, no statistical analysis was performed for this variable. Rev. Ambient. Água vol. 10 n. 3 Taubaté – Jul. / Sep. 2015 3.2. Metals Among the various chemical contaminants, the study of heavy metals has been considered a priority in health promotion programs worldwide (Nikaido et al., 2010). According to Baird (2002), the term “heavy metal” refers to a class of chemical elements; many of them are harmful to humans. Rev. Ambient. Água vol. 10 n. 3 Taubaté – Jul. / Sep. 2015 534 Sérgio Marcos Sanches et al. Humans can be exposed to metals from several sources, and water is a main pathway of contamination, so there is a natural concern about the good quality water from the spring to the point of consumption (Chakrabarty and Sarma, 2010). This study examined the levels of lead (Pb), manganese (Mn), chromium (Cr), copper (Cu) and cadmium (Cd) present in the water samples analyzed. The results revealed copper concentrations above those permitted by legislation in 3.125% of the samples analyzed. In 51.56% of the samples analyzed, the levels of cadmium exceeded the recommendations of the Ministry of Health. Further, 12.5%, 7.82% and 45.31% of the samples did not conform to the recommendations of Ordinance 2914 / MS for concentrations of Mn, Pb and Cr, respectively (Brasil, 2011). Figure 1 shows the average rating of Mn present in the water samples from drinking fountains and kitchen faucets from the schools selected for this research over the four collection periods. The values exceed the maximum allowable value (MAV) of 0.1 mg L-1 for drinking water. Figure 1. Variation of Mn concentrations in water samples from drinking fountains and kitchen faucets from eight institutions of child education throughout the four collections undertaken from December 2011 to September 2012. Figure 1. Variation of Mn concentrations in water samples from drinking fountains and kitchen faucets from eight institutions of child education throughout the four collections undertaken from December 2011 to September 2012. It was found that, in relation to samples of water from the drinking fountains and kitchen faucets from the eight schools selected for the survey, the values for the concentration of manganese in the first collection differed statistically from the other collections, and presented a higher average value. The third and fourth collections did not differ significantly from each other; however, the first and the second collections found smaller average concentrations of Mn than those previously mentioned. Rev. Ambient. Água vol. 10 n. 3 Taubaté – Jul. / Sep. 2015 3.2. Metals In relation to the variation of the concentrations of Cu present in water samples from drinking fountains and kitchen faucets from the eight schools, it was observed that, when analyzing water samples from drinking fountains, there was no significant difference with respect to the sampling period. However, when analyzing water samples from the taps in the kitchen, it was observed that the copper concentrations differed among them (p <0.05). g p Figure 2 shows the medium values of the concentration of Cu present in water samples from kitchen taps from the eight schools selected for the research, beyond the maximum allowed value (2 mg L-1) established by current legislation for drinking water. When analyzing the average variation in Cd concentration in water samples from drinking fountains and kitchen faucets from the eight schools, there was a significant variation in the concentrations of the metal cited previously, during the four collections undertaken for the two collection points mentioned. For the water samples from the drinking fountains of the eight schools, it was also observed that the fourth collection showed a mean value higher than the other samples, while the first collection presented lower mean concentration of Cd. Rev. Ambient. Água vol. 10 n. 3 Taubaté – Jul. / Sep. 2015 535 Chemical and microbiological analysis of public school water … When analyzing water samples from the kitchen faucets of primary schools selected for the survey, it was observed that the third sample showed a significant difference in the variation of Cd concentrations from the other samples, with an average value higher than the other collections. On the other hand, the first and the second collections did not differ significantly from each other, but differed from the other collections, presenting a lower average concentration of Cd. Figure 2. Variation of Cu concentration in water samples from faucets from eight schools during the four collections. Figure 2. Variation of Cu concentration in water samples from faucets from eight schools during the four collections. Figure 3 shows the values of the variations of Cd concentrations in water samples from the drinking fountains and kitchen faucets of the eight schools selected for the research during four collections, beyond the default value of 0.005 mg L-1 recommended by the Ministry of Health for drinking water. Figure 3. Rev. Ambient. Água vol. 10 n. 3 Taubaté – Jul. / Sep. 2015 3.2. Metals Variation of Cd concentration in water samples from drinking fountains and kitchen faucets from the eight schools studied during the four collections. Figure 3. Variation of Cd concentration in water samples from drinking fountains and kitchen faucets from the eight schools studied during the four collections. The concentrations of Pb and Cr in water samples from drinking fountains and taps showed no significant difference throughout the different sampling periods. Therefore, no statistical analysis of the results is necessary. 536 Sérgio Marcos Sanches et al. 3.3. Coliforms The presence of coliforms in water indicates contamination, pointing to a potential risk of the presence of pathogenic organisms. The absence of coliforms is evidence of bacteriologically safe drinking water, since coliforms are more resistant in water than the pathogenic bacteria of intestinal origin (Oliveira et al., 2012). 0 Ordinance n0. 518 of the Ministry Health of 2004 suggests that, when verifying the presence of total coliforms even in the absence of fecal coliforms, it is necessary to take immediate corrective and preventive action, such as cleaning water tanks and cisterns. The Ordinance of the Ministry of Health n0. 2914 of 2011 considers the maximum value allowed for Escherichia coli and total coliforms in water for human consumption in systems that analyze 40 or more samples per month to be the absence of Escherichia coli and total coliforms in 95% of the 100 mL samples examined in the month. In systems that analyze less than 40 samples per month, only one sample can have a positive monthly result (Brasil, 2011). According to the values presented in Table 2, it was observed that, of the 32 samples (100%) of water from drinking fountains, 23 (71.875%) did not meet the standard established Rev. Ambient. Água vol. 10 n. 3 Taubaté – Jul. / Sep. 2015 3.4. Total coliforms The values of total coliforms obtained from the analyses of water samples collected in the drinking fountains of the eight schools selected for the research were in the range of 0 to 57 MPN/100 mL, while the values of total coliforms for samples coming from the kitchen faucets were in the range of 0 to 68 MPN / 100 mL, as shown in Table 2. Table 2. Results of analyses of water samples from drinking fountains and kitchen faucets from eight schools selected for research regarding the presence of total coliforms. Educational institutions Sampling sites Total Coliforms (MPN/100mL) 1st sampling (12/14/11) 2nd sampling (03/19/12) 3rd sampling (06/25/12) 4th sampling (09/26/12) A Fountain 22 1 2 2 Faucet 14 1 3 4 B Fountain 4 1 3 1 Faucet 0 0 4 0 C Fountain 4 0 0 0 Faucet 1 1 5 0 D Fountain 10 8 36 0 Faucet 1 2 23 0 E Fountain 57 27 5 1 Faucet 0 24 10 0 F Fountain 54 1 4 0 Faucet 15 1 3 0 G Fountain 3 2 10 0 Faucet 2 0 8 68 H Fountain 0 0 13 0 Faucet 0 0 42 1 MPN/100mL most probable number in 100 mL. Educational institutions Sampling sites Total Coliforms (MPN/100mL) 1st sampling (12/14/11) 2nd sampling (03/19/12) 3rd sampling (06/25/12) 4th sampling (09/26/12) A Fountain 22 1 2 2 Faucet 14 1 3 4 B Fountain 4 1 3 1 Faucet 0 0 4 0 C Fountain 4 0 0 0 Faucet 1 1 5 0 D Fountain 10 8 36 0 Faucet 1 2 23 0 E Fountain 57 27 5 1 Faucet 0 24 10 0 F Fountain 54 1 4 0 Faucet 15 1 3 0 G Fountain 3 2 10 0 Faucet 2 0 8 68 H Fountain 0 0 13 0 Faucet 0 0 42 1 MPN/100mL most probable number in 100 mL. Total Coliforms (MPN/100mL) According to the values presented in Table 2, it was observed that, of the 32 samples (100%) of water from drinking fountains, 23 (71.875%) did not meet the standard established Rev. Ambient. Água vol. 10 n. 3 Taubaté – Jul. / Sep. 2015 Chemical and microbiological analysis of public school water … 537 by Ordinance No. 2914 of the Ministry of Health, 2011. 3.4. Total coliforms Of the 32 water samples collected from the kitchen faucets, 65.625% (21 samples) did not comply with legislation, with values above those established by current legislation for drinking water. by Ordinance No. 2914 of the Ministry of Health, 2011. Of the 32 water samples collected from the kitchen faucets, 65.625% (21 samples) did not comply with legislation, with values above those established by current legislation for drinking water. It was observed that the water samples taken during the four collections showed significant differences (p <0.05) among themselves regarding the presence of total coliforms. It was also observed that the samples of water from drinking fountains during the fourth collection had a lower average value than the other collections. Also, in relation to water samples from the drinking fountains, the first collection presented a higher average value for the presence of total coliforms, differing from the other collections. The water samples from the third collection differed significantly from the other collections, showing a higher average only compared to the second collection’s value with regard to the presence of total coliforms. When analyzing samples of water from the kitchen faucets, it was found that all of the samples differed among themselves during the sampling period. It was observed that the samples from the third collection showed higher average of total coliforms than the other collections. The second collection’s samples showed an average lower than all of the other collections. The first collection also differed from the other collections in that the most probable number of coliforms presented a higher average value only in relation to the second collection. It was also found that water samples from the fourth collection had a lower average value only when compared to the third collection. Figure 4 is illustrated the variation in the number of total coliforms present in the water samples from drinking fountains and kitchen faucets in schools selected for the survey during the four collection periods. Figure 4. Variation of total coliforms in samples of water from drinking fountains and kitchen faucets from eight schools during the four collections. Figure 4. Variation of total coliforms in samples of water from drinking fountains and kitchen faucets from eight schools during the four collections. Rev. Ambient. Água vol. 10 n. 3 Taubaté – Jul. / Sep. 2015 3.5. Escherichia coli Ordinance no. 2914 of the Ministry of Health, 2011, establishes the absence of bacteria of the thermo tolerant coliforms group, formerly fecal coliforms as a potability standard for water intended for human consumption. Waterborne diseases are transmitted through the ingestion of water contaminated with pathogenic microorganisms eliminated in the feces of humans and / or animals. One can safely say that waterborne diseases are one of the most serious threats to the child population, since this group of diseases is among the five leading causes of death in individuals between one to four years-old. (Germano and Germano, 2001). y ( ) The results of the analySES of water samples from drinking fountains and faucets in schools selected for the research are shown in Table 3. Rev. Ambient. Água vol. 10 n. 3 Taubaté – Jul. / Sep. 2015 538 Sérgio Marcos Sanches et al. As shown in Table 3, of the 32 water samples (100%) from the fountains, 22 (68.75%) did not conform to the standards recommended by Ordinance n0 2914 of 2011 of the Ministry of Health for drinking water. In relation to the 32 samples of water from the faucets, 18 (56.250%) did not conform to the parameters established by the current law. The Kruskal-Wallis Test was performed with a 5% significance level with the objective of identifying a significant difference in relation to the presence of Escherichia coli in different water sampling periods. The samples from the kitchen faucets showed no significant difference in the presence of E. coli during the different sampling periods. Table 3. Results of analySES of water samples from drinking fountains and faucets of eight schools selected for the survey, in relation to the number of Escherichia coli. Educational institutions Sampling sites Escherichia coli (NMP/100mL) 1st collection (12/14/11) 2nd collection (03/19/12) 3rd collection (06/25/12) 4th collection (09/26/12) A Fountain 20 1 2 4 Faucet 14 1 2 4 B Fountain 2 1 3 1 Faucet 1 0 5 0 C Fountain 0 0 0 0 Faucet 0 1 4 0 D Fountain 9 3 14 1 Faucet 0 2 0 3 E Fountain 3 11 3 1 Faucet 2 25 0 0 F Fountain 6 1 4 0 Faucet 74 1 3 0 G Fountain 10 0 0 0 Faucet 0 0 0 68 *MPN/100mL most probable number in 100 mL. Table 3. Rev. Ambient. Água vol. 10 n. 3 Taubaté – Jul. / Sep. 2015 3.5. Escherichia coli Results of analySES of water samples from drinking fountains and faucets of eight schools selected for the survey, in relation to the number of Escherichia coli. However, the water samples collected in the schools’ drinking fountains differed significantly with regard to the presence of Escherichia coli depending upon the collection period. It was also observed that the water samples from the first collection differed significantly from the other samples taken and showed a higher average value of the most probable number of E. coli when compared with the values from other collections. The water samples from the fourth collection showed significant differences in relation to the other collections, showing a lower average value with regard to the presence of E. coli. The second collection differed significantly from the other collections, showing a higher average value than the fourth collection and lower than all the other collections in relation to the most probable number of fecal coliforms in the 100 mL of water sample analyzed. The third collection showed a significant difference (p <0.05) compared to the other samples with regard to the presence of Escherichia coli coliforms in the water samples, showing higher average value only in relation to the fourth collection. Figure 5 shows the variation in the Rev. Ambient. Água vol. 10 n. 3 Taubaté – Jul. / Sep. 2015 Chemical and microbiological analysis of public school water … 539 number of thermo tolerant coliforms in water samples collected in drinking fountains of the schools during the different sampling periods. Figure. 5. Variation of thermo tolerant coliforms present in water samples from drinking fountains of eight schools selected for research during the four collections. Figure. 5. Variation of thermo tolerant coliforms present in water samples from drinking fountains of eight schools selected for research during the four collections. Rev. Ambient. Água vol. 10 n. 3 Taubaté – Jul. / Sep. 2015 4. CONCLUSION Public schools use water, among other purposes, for cleaning, personal hygiene and for drinking. To ensure the maintenance of quality and purity of water used in schools, it is necessary that it be stored in clean containers that are disinfected at least twice a year. In several educational institutions, the index of waterborne diseases has become increasingly common, in addition to physical changes related to the water’s odor, color and taste. This may be due to contamination in water tanks or infiltration in pipes. For this reason, it becomes important to monitor and further analyze the quality of waters from faucets and drinking fountains and consumed by the school community. The results of this study show that, in relation to the concentration of free residual chlorine, all the samples were below the maximum value allowed by law (2.0 mg L-1). However, 40.625% of water samples did not meet the minimum required value of 0.2 mg L-1. The most worrisome results were found at School A, where all of the samples collected did not meet the minimum values recommended by Ordinance 2914, 2011, Ministry of Health. When analyzing metal concentration, 68.75% of water samples from the first collection (12/14/2011) showed levels of chromium above those permitted by Ordinance n0. 2914 of the Ministry of Health, 2011. With regard to the second collection (03/19/12), 50% of the samples drawn showed concentrations of chromium and manganese above the permissible level and 25% had lead content above that recommended for drinking water. During the third collection (06/25/12), 31.25% of the water samples were outside of the established standard for chromium concentrations. The cadmium results were even more worrisome, as all of the samples had values above those recommended by the Ministry of Health. Of the water samples from the fourth collection (09/26/12), 50% did not meet the permitted standards for concentrations of Cr and all of the samples were above the maximum value allowed for concentrations of Cd. All of the water samples originating from institution C were at odds with the current legislation regarding the maximum concentrations of chromium permissible for drinking water. Rev. Ambient. Água vol. 10 n. 3 Taubaté – Jul. / Sep. 2015 540 Sérgio Marcos Sanches et al. 5. ACKNOWLEDGMENT CAPES for their support for the implementation of this work. CAPES for their support for the implementation of this work. CAPES for their support for the implementation of this work. 4. CONCLUSION Regarding the microbiological quality of water used by the schools, the presence of total coliforms was observed in 68.75% of the water samples analyzed, although all samples of water from the drinking fountains and faucets in school A showed unacceptable levels of total coliforms that did not conform to the recommendations of the Ministry of Health. Another important fact was observed in relation to the variation of the most probable number of total coliforms in samples analyzed during the four collections: 75% of the samples from the first collection showed the presence of total coliforms. This value changed to 93.75% in the second collection, decreased to 68.75% in the third collection, and reached 37.5% in the fourth. This decrease in the value of the number of total coliforms can be explained by the fact that the fourth collection occurred on 09/26/2012, just after the cleaning of the water reservoirs which occurred during a school recess period (July/2012). Regarding the presence of Escherichia coli, 65.625% of the samples did not conform to the recommendations of the legislation for drinking water. In the first collection (12/14/11) and the third collection (06/25/12), E. coli were identified in 68.75% of the water samples analyzed. The value increased to 75% in the second collection, reducing to 50% in the fourth collection. Still regarding the presence of thermo tolerant coliforms, it is important to emphasize the poor quality of the water samples coming from School A. This can be explained by the fact that these samples present values of free residual chlorine below the minimum value required by law. This study was conducted on children's schools, whose commitment is to provide, in addition to education, security for the students through an appropriate infrastructure, with proper cleanliness, hygiene and nutrition. 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https://openalex.org/W2509651511	https://discovery.ucl.ac.uk/1530849/1/Rylance%20Household%20air%20pollution%20and%20the%20lung%202016.pdf	English	null	Household air pollution and the lung microbiome of healthy adults in Malawi: a cross-sectional study	BMC Microbiology	2,016	cc-by	6,336	© 2016 The Author(s). 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: jamie.rylance@lstmed.ac.uk 1Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool L3 5QA, UK 3Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Blantyre, Malawi Full list of author information is available at the end of the article Household air pollution and the lung microbiome of healthy adults in Malawi: a cross-sectional study Jamie Rylance1,3* , Anstead Kankwatira3, David E. Nelson4, Evelyn Toh4, Richard B. Day2, Huaiying Lin5, Xiang Gao5, Qunfeng Dong5, Erica Sodergren6, George M. Weinstock6, Robert S Heyderman3, Homer L. Twigg III2 and Stephen B. Gordon1,3 Rylance et al. BMC Microbiology (2016) 16:182 DOI 10.1186/s12866-016-0803-7 Rylance et al. BMC Microbiology (2016) 16:182 DOI 10.1186/s12866-016-0803-7 Abstract Background: Domestic combustion of biomass fuels, such as wood, charcoal, crop residue and dung causes Household Air Pollution (HAP). These inhaled particulates affect more than half of the world’s population, causing respiratory problems such as infection and inflammatory lung disease. We examined whether the presence of black carbon in alveolar macrophages was associated with alterations in the lung microbiome in a Malawi population. Methods: Bronchoalveolar lavage samples from 44 healthy adults were sequenced using 16S rDNA amplification to assess microbial diversity, richness and relative taxa abundance. Individuals were classified as high or low particulate exposure as determined by questionnaire and the percentage of black carbon within their alveolar macrophages. Results: Subjects in the low and high particulate groups did not differ in terms of source of fuels used for cooking or lighting. There was no difference in alpha or beta diversity by particulate group. Neisseria and Streptococcus were significantly more abundant in samples from high particulate exposed individuals, and Tropheryma was found less abundant. Petrobacter abundance was higher in people using biomass fuel for household cooking and lighting, compared with exclusive use of electricity. Conclusions: Healthy adults in Malawi exposed to higher levels of particulates have higher abundances of potentially pathogenic bacteria (Streptococcus, Neisseria) within their lung microbiome. Domestic biomass fuel use was associated with an uncommon environmental bacterium (Petrobacter) associated with oil-rich niches. Keywords: Respiratory microbiome, Household air pollution, Alveolar macrophage, Petrobacter known to drive inflammation in the lung, to alter micro- bial binding to respiratory epithelium, and to act as vehi- cles delivering microbial molecules to the distal airways. Data analysis C i Comparison of alveolar macrophage black carbon con- tent with subject demographics: Two-way contingency tables were created using high/low alveolar macrophage black carbon content as one category and subject demo- graphics as the other category. Forty-four demographics features include sex, cook fuel, cook location, heat, smoking, light fuel, and living conditions were examined. Fisher’s exact test was applied for the analysis [16]. Microbiome Analysis: To account for the uneven se- quencing depth of each sample, all 44 samples were normalized using subsampling without replacement at depth 843 reads, and the subsampling was repeated for 10 times. The averaged read count among the 10 permutations was used in subsequent analysis. Alpha diversity richness was measured using Observed taxa number, Chao 1 and ACE indices, and diversity even- ness was assessed using Shannon, Simpson’s (1-D), and Pielou indices [17]. These were compared between par- ticulate groups using Wilcoxon Rank Sum tests. Differ- ence of alpha diversity between groups was analysed by linear model with and without confounding factors, such as age, gender, cooking location, type of cook fuel and light fuel. Beta-diversity was visualized by non- metric multidimensional scaling using Bray-Curtis dissimilarity [18, 19] by the R ecodist package [20]. The Microbiome analysis DNA was extracted from BAL supernatants using DNAse/RNase free reagents and materials and a DNeasy kit (Qiagen, CA, USA). Ribosomal 16S subunit rDNA sequencing was performed at the Genome Insti- tute (Washington University, MO, USA) as previously described [9]. Briefly, 27 F-534R primers for the hyper- variable regions 1 to 3 (V1V3) were utilized. The 16 s rDNA sequencing was performed on the Roche 454 FLX Titanium platform and processed with the Mothur package v1.29 [14] based on its standard operative pro- cedure (http://www.mothur.org/wiki/454_SOP). Briefly, sequence reads were demultiplexed into individual samples based on perfect match to the barcode se- quences. Primers and barcodes were trimmed from each read and low-quality and chimeric sequences were removed with default Mothur parameters with one minor adjustment: the trump symbol was not included at filter.seqs() step due to our observation that its resulting in over-removal of aligned reads. The remaining high-quality 16S sequences (420 ± 15.9 bp) from each sample were classified using the RDP Classi- fier v2.5 with the default threshold value of 0.8 from phylum to genus level [15]. We hypothesised that, in healthy people, HAP would be associated with alterations in the lung microbiome in terms of diversity, richness and the relative abundance of various microbial taxa. Furthermore, prior compara- tive analysis of geographical differences in microbiota prevalence identified Petrobacter in more than a third of a group sampled in Malawi, but none in a US cohort. Petrobacter is a gram negative, aerobic bacterium identi- fied in 2004 from oil reservoir samples. Given this niche, we hypothesised that the prevalence in a Malawi group would be due to inhalation of smoke from biomass fuel use: we present evidence of this association in this paper [10, 11]. Participants and bronchoscopy Healthy, non-smoking, HIV-negative adults aged 18 to 50 were recruited from peri-urban communities in Blan- tyre, Malawi from May 2009 to December 2012. Ethical approval was granted by the College of Medicine REC, University of Malawi and Liverpool School of Tropical Medicine REC (P.03/10/916 and 09.69 respectively), and written consent was obtained. Bronchoalveolar lavage (BAL) fluid was obtained as previously described [12], filtered through gauze and centrifuged immediately at 400 g for 10 min. The cell pellet was processed as de- scribed below. Acellular supernatants were stored at -80 °C for batch extraction and sequencing. Structured interviews determined participants’ demographics and type of fuel used for heating, cooking and lighting. The stated “main source” of each was used as a classifier in analyses. Background Globally, most inhaled particulate matter derives from the domestic combustion of biomass fuels such as wood, charcoal, crop residue and dung [1, 2]. This Household Air Pollution (HAP) is associated with 4.3 million deaths per year from respiratory disease, including 900,000 childhood deaths from pneumonia [3–6]. Alterations in microbial populations in the lung caused by particle ex- posure could explain increased rates of respiratory infec- tion in subjects exposed to HAP. Inhaled particulates are In healthy lungs, previously thought to be sterile environments, communities of bacteria, together with fungi and viruses form the microbiome. Variations in this microbiome may either reflect or drive mucosal inflammation and immune function [7]. Extensive sequencing of bacterial 16S rDNA from the lungs of healthy individuals has revealed the common presence of phyla such as Proteobacteria, Bacteroidetes, Acti- nobacteria and Firmicutes [8]. * Correspondence: jamie.rylance@lstmed.ac.uk 1Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool L3 5QA, UK 3Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Blantyre, Malawi Full list of author information is available at the end of the article Study of the alterations in lung microbiota resulting from environmental exposures is a nascent field with the potential to explain the pathophysiological mechanisms Rylance et al. BMC Microbiology (2016) 16:182 Page 2 of 7 BAL samples. No further stratification or selection strat- egy was used. of lung disease. Cigarette smoking does not appear to be associated with significant changes in the lung microbiome in a US cohort [9]. However, the effects of exposure to other environmental sources of particu- lates, particularly in low income countries, have not yet been described. Quantification of particulate within cells, and participant selection The BAL cell pellet was re-suspended in RPMI 1640, and cells were counted using a Neubauer chamber. Cytospin preparations of macrophages (Thermo Shan- don, UK) were imaged at 40x by light microscopy. Fifty fields from each experiment were analysed using freely available digital image analysis software (Image SXM, www.ImageSXM.org.uk), as previously described [13]. The samples for this study were drawn from a larger bronchoscopic study of 128 volunteers, all of whom had particulate imaging and quantification. Samples were sequentially identified for microbiome analysis from the highest and lowest particulate of available and adequate Page 3 of 7 Rylance et al. BMC Microbiology (2016) 16:182 Page 3 of 7 PERMANOVA test in the R vegan package was used to test whether high biomass and low biomass cohorts form distinct clusters based on Bray-Curtis dissimilar- ities among the samples [21]. Multivariate dispersion of groups was compared using the betadisper() command in R vegan package to test for homogeneity of variance in high biomass and low biomass cohorts [22]. [SD 4283] vs 6545 [SD 3183] respectively). There was no difference in alpha diversity metrics between low and high particulate groups by any measure at either genus or phylum level (Observed taxa number, Chao 1, ACE indices, Shannon, Simpson’s (1-D), and Pielou all p > 0.05). PERMANOVA test in the R vegan package was used to test whether high biomass and low biomass cohorts form distinct clusters based on Bray-Curtis dissimilar- ities among the samples [21]. Multivariate dispersion of groups was compared using the betadisper() command in R vegan package to test for homogeneity of variance in high biomass and low biomass cohorts [22]. Beta diversity was no different in low and high par- ticulate groups for genus level (PERMANOVA p = 0.209). Analysis on phylum level shows no difference between low and high particulate groups (PERMANOVA p = 0.397). Interestingly, when dispersion of the two commu- nities was analysed, the low particulate population tended to be more spread out compared to high particulate group, though this did not quite reach statistical signifi- cance (average distance to centroid: 0.544 for the low bio- mass group, 0.484 for the high biomass group, p = 0.096) at genus level. Participants Forty-four participants were selected for 16S RNA se- quencing (23 from low particulate and 21 from the high particulate group as determined by alveolar macrophage carbon content from an available set of 128 samples (representative images are shown in Fig. 1). Baseline characteristics are given in Table 1. Participants in the low and high particulate groups did not differ signifi- cantly in terms of sex, BMI, lung function, source of fuels used for cooking or lighting, or bronchoalveolar lavage differential cell counts (see Table 1). High particu- late individuals were older (mean 34.1 years vs. 29.2 years, p = 0.03). All individuals did have a potential domestic source of particulate exposure for either cooking or lighting. There were no significant differences between low and high particulate groups at the phylum level (data not shown). Table 2 shows the relative abundance of the twenty genera represented at a frequency of 1 % or more of the total sequence reads. Neisseria abundance was signifi- cantly associated with the high particulate group, ac- counting for 4.98 % (SD 7.71) of total reads compared with 1.00 % (1.80) in the low particulate group (p = 0.01). This relationship was maintained after adjustment for age, sex, and cooking location (p = 0.046). Tropheryma was identified significantly less frequently in the high compared with low particulate group (0.97 % [2.99] vs 13.37 % [29.5] respectively, p = 0.046 unadjusted and p = 0.01 adjusted). Streptococcus was observed at higher relative abundance in the high particulate group (13.71 % [13.09] vs 6.77 % [7.28]): this was non-significant in the Quantification of particulate within cells, and participant selection Differences in the abundance of specific genera be- tween groups was analysed using negative binomial (NB) models and adjusted for differences in age, gender, cooking location, and smoking status prior to 6 months before the study (all participants were non-smokers for the 6 months immediately prior). To filter extremely low abundant taxa in our analysis, we limited this ana- lysis to bacteria that were present in greater than 1 % abundance in at least one cohort. Alpha and beta diversity The mean total of high quality sequences was 7268, and was similar in low and high particulate groups (7928 Fig. 1 Representative images of macrophage staining and particulate density. Ex vivo alveolar macrophages have undergone cytospin preparation, and staining with Fields B. Panels a and b show representative 40x light microscopy images of macrophages from low and high particulate groups respectively Fig. 1 Representative images of macrophage staining and particulate density. Ex vivo alveolar macrophages have undergone cytospin preparation, and staining with Fields B. Panels a and b show representative 40x light microscopy images of macrophages from low and high particulate groups respectively Rylance et al. BMC Microbiology (2016) 16:182 Page 4 of 7 Table 1 Characteristics of participants classified as low and high macrophage particulate burden Low particulate (n = 23) High particulate (n = 21) p Age, mean years (SD) 29.2 (7.7) 34.1 (6.8) 0.03 Sex, female (%) 9 (39) 12 (57) 0.37 Ethnicity – African, n (%) 23 (100) 21 (100) - BMI, mean (SD) 22.4 (4.2) 22.4 (2.6) 0.97 FEV1 % predicted, mean (SD) 96.2 (15.2) 98.2 (9.7) 0.62 FVC % predicted, mean (SD) 96.3 (15.1) 100.4 (9.5) 0.31 Cooking, n (%) Electricity only 2 (8.7) 0 (0) 0.60 Charcoal stove 14 (60.9) 13 (61.9) . Wood 7 (30.4) 8 (38.1) . Lighting, n (%) Electricity only 6 (26.1) 4 (19.1) 0.11 Candle mostly 10 (43.5) 4 (19.1) . Paraffin mostly 7 (30.4) 13 (61.9) . BAL differential count, % (SD) Macrophage 95.5 (4.6) 95.3 (3.2) 0.85 Lymphocyte 4.0 (4.1) 4.2 (2.6) 0.87 Macrophage carbon, % (SD) 0.1 (0.1) 2.5 (1.8) <0.0001 Data are presented as n (%) or mean ± SD. Alpha and beta diversity Significance testing used Fisher’s exact tests BMI Body Mass Index, SD standard deviation Table 2 Genus level differences between low and high particulate groups Reads, % of total (SD) p value High Low Unadjusted Adjusted Streptococcus 13.71 (13.09) 6.77 (7.28) 0.062 0.045* Prevotella 7.28 (7.95) 7.61 (11.00) 0.938 0.899 Tropheryma 0.97 (2.99) 13.37 (29.50) 0.046* 0.006* Paenibacillus 4.68 (11.37) 3.04 (9.67) 0.702 0.355 Corynebacterium 5.43 (11.84) 2.22 (4.11) 0.127 0.498 Petrobacter 3.78 (8.83) 3.37 (8.08) 0.924 0.560 Acidovorax 3.12 (12.28) 4.02 (8.31) 0.825 0.198 Neisseria 4.98 (7.71) 1.00 (1.80) 0.010* 0.043* Propionibacterium 2.86 (4.72) 2.03 (4.21) 0.592 0.430 Veillonella 2.50 (3.52) 2.28 (3.25) 0.907 0.763 Sphingomonas 1.68 (2.62) 2.37 (3.39) 0.591 0.909 Ralstonia 0.15 (0.40) 3.70 (17.05) 0.027* 0.991 Bacillus 1.67 (3.10) 1.80 (4.97) 0.917 0.619 Akkermansia 1.88 (2.85) 1.43 (1.71) 0.482 0.221 Fusobacterium 1.22 (2.39) 1.70 (2.96) 0.684 0.303 Actinomyces 1.41 (2.41) 1.20 (2.05) 0.825 0.980 Porphyromonas 1.24 (1.99) 1.27 (2.87) 0.984 0.784 Gemella 1.66 (2.05) 0.66 (0.81) 0.055 0.338 Staphylococcus 0.98 (1.98) 1.14 (2.32) 0.838 0.677 Cloacibacterium 0.79 (2.63) 1.21 (4.43) 0.775 0.129 The relative abundance of bacteria in the lung microbiome was compared by using negative binomial test. Raw and adjusted p values are given. Adjustments were made for differences in age, gender and cooking location, between high and low biomass groups denotes significant at p < 0.05. Rows are presented in descending order of relative abundance Table 1 Characteristics of participants classified as low and high macrophage particulate burden Table 2 Genus level differences between low and high particulate groups unadjusted analysis (p = 0.062), but significant (p = 0.045) after adjustment for potential confounding variables. Ralstonia appeared more abundant in high particulate groups, only before adjustment (p = 0.027, after adjust- ment p = 0.991). Discussion Our study found that high particulate exposure as de- fined by alveolar macrophage carbon content was associ- ated with altered relative abundance of bacteria within the lungs of healthy Malawian adults. Specifically, we re- port higher proportions of Neisseria and Streptococcus, Petrobacter in Malawi A comparative analysis of the lung microbiome in Malawian and US samples identified Petrobacter as commonly isolated amongst the Malawi participants (17/44). Given both the original and recent descriptions of this organism in oil reservoirs, we hypothesised that Petrobacter prevalence could be explained by exposure to domestic fuel combustion, in particular to paraffin for lighting [10, 11]. Therefore, we analysed individual factors which might associate with Petrobacter (see Table 3), particularly in relation to domestic biomass fuel exposure. For those participants, who used electri- city exclusively for lighting, have significantly lower bacteria abundance (0.025 ± 0.080) compared to those people who use candle, no-glass paraffin lights, instead (4.61 ± 9.26, p = 0.00044). Cooking predominantly out- side rather than indoors was associated with lower copy numbers (cooking outdoor 0.50 ± 1.20 vs. cooking in- door 5.53 ± 10.20, p = 0.014). Table 3 Associations of Petrobacter abundance in the lung microbiome of healthy Malawians Petrobacter abundance (%) p Yes No Sex: female 6.32 ± 10.37 1.32 ± 5.53 0.40 Cooking fuel: electricity only 0 ± 0 3.74 ± 8.51 <0.001 Cooking location: mostly outdoor 0.51 ± 1.20 5.53 ± 10.20 0.015* Lighting fuel: electricity only 0.025 ± 0.080 4.61 ± 9.27 0.0004* “Ex-smoking” status 4.36 ± 9.06 0 ± 0 0.79 GLM with negative binomial model shows associations of Petrobacter within the lung microbiome of Malawians, focussing on potential exposures to particulate exposures from domestic fuel use. The relative abundance of Petrobacter in each cohort was recorded as mean ± standard deviation significance at p < 0.05 level Table 3 Associations of Petrobacter abundance in the lung microbiome of healthy Malawians GLM with negative binomial model shows associations of Petrobacter within the lung microbiome of Malawians, focussing on potential exposures to particulate exposures from domestic fuel use. The relative abundance of Petrobacter in each cohort was recorded as mean ± standard deviation significance at p < 0.05 level Rylance et al. BMC Microbiology (2016) 16:182 Page 5 of 7 Page 5 of 7 and lower proportions of Tropheryma. However, lung microbiome diversity and the relative abundance at the phyla level were not significantly different based on our current sample size. Petrobacter was more abundant in individuals who used biomass fuels for cooking or light- ing than in those who did not. Petrobacter in Malawi Finally, particulate matter in alveolar macrophages did not directly correlate with biomass exposure as determined by questionnaire, sug- gesting in this small study that other factors contributed to alveolar macrophage particulate ingestion. These likely include ingress of pollution from neighbouring homes, traffic pollution when commuting to work and occupational exposures. comparisons between smokers and non-smokers in the US [9]. Our study is compatible with prior descriptions of high diversity in the lung microbiome, with Streptococcus and Prevotella most frequently represented [9, 24]. Despite the absence of differences in the global lung microbiome between high and low particulate exposed subjects, interesting differences in specific taxa were observed. Streptococcus was more abundant in high particulate exposed participants after adjustment of po- tential confounders. In humans, ambient air pollution increases the risk of pneumonia in adults and children, [29, 30] and is commonly caused by Streptococcus pneumoniae. Analagous changes occur in cigarette smokers [31]. Bronchial epithelium exposed to urban particulates demonstrates increase expression of platelet activating factor and S. pneumoniae binding [32]. Nasopharyngeal carriage of S. pneumoniae is high in Malawi, occurs early, and pathogen specific mucosal T- cell regulation may contribute to prolonged carriage [33]. High levels of Streptococcus have also been demonstrated in the upper airway microbiome in a case-control study of infants in Ecuador [34]. Similarities with the Malawian study are low levels of pneumococcal immunisation and antibiotic use, and significant rural poverty. Taken to- gether, these studies would provide a mechanistic link be- tween our findings and the epidemiological associations of particulate concentration and pneumonia incidence. This study provides a novel analysis of the lung micro- biome from individuals in a low income country with exposure to high biomass fuels. We characterised high particulate exposures according to the particulate bur- den in the cells of the distal airways, which reflects the cumulative exposure to respirable-size particulates. This is likely to be more relevant to changes in the micro- biome than air sampling methods [23]. We did not find any significant differences in the lung microbiome as a whole between high and low particulate groups. However, both richness and abundance of the lower respiratory microbiome may be significantly altered by sampling techniques which vary in their potential for introducing “carryover” contamination from the upper airways [24]. Petrobacter in Malawi Individual lower respiratory tract microbiome demon- strates less similarity to that in other individuals than to the upper respiratory tract of the same person [9, 25, 26]. Nevertheless, samples obtained directly from explanted lungs and those taken indirectly by bronchoscopy demon- strate similar patterns [24]. Our study did not include sampling of the upper airway. Therefore, one cannot de- termine conclusively whether our BAL findings reflect true differences in the lung microbiome or differences in upper airway carriage between subjects with low and high exposure to particulates. However, since micro-aspiration is common in humans [17], it is likely that a true lung microbiome will contain many of the same taxa found in the oral cavity. Our study was performed on stored sam- ples: not all of the potential environmental controls were available. However, since all subjects underwent the same bronchoscopy protocol, findings between high and low biomass fuel groups are highly likely to represent non- artefactual differences. Sterile saline controls from the bronchoscope using the same kit as this study have been reported, and contain a very low number of reads [27]. Neisseria was also more prominent in high particulate group. This organism is not usually considered in the context of the lower airways, although serogroup Y has been associated with pneumonia in the elderly and Army recruits [35]. Nasopharyngeal carriage of N. meningitidis is increased amongst those exposed to cigarette smoke, [36] and ex vivo human epithelial cell models demon- strate bacterial binding is also increased [37]. The role of other ambient particulates is not well defined. Tropheryma showed the inverse relationship, in that it was more frequently represented in the low particulate group. The pathogenic significance of this is uncertain: while T. whipplei is the aetiological agent of Whipple’s disease, it is commonly found in health in other studies of lung microbiome [9]. It occurs in especially high levels in microbiome studies of HIV-infected individuals from the US [38]. However, the lower levels in our par- ticulate exposed healthy volunteers is unexplained. While we hypothesised that particulate exposure could alter the microbiome, it is also possible that difference in the microbiome could affect particulate uptake in mac- rophages through effects on immune activation and clearance responses [39]. Reduced diversity of the lung microbiome has been seen in disease states, such as COPD, [24] and with medical treatments, such as inhaled corticosteroids [28]. Petrobacter in Malawi In our HIV-negative, healthy volunteers, however, we found no significant differences in diversity or richness between high and low particulate groups, mirroring findings of Funding h k 7. Segal LN, Alekseyenko AV, Clemente JC, Kulkarni R, Wu B, Chen H, Berger KI, Goldring RM, Rom WN, Blaser MJ, et al. Enrichment of lung microbiome with supraglottic taxa is associated with increased pulmonary inflammation. Microbiome. 2013;1(1):19. 7. Segal LN, Alekseyenko AV, Clemente JC, Kulkarni R, Wu B, Chen H, Berger KI, Goldring RM, Rom WN, Blaser MJ, et al. Enrichment of lung microbiome with supraglottic taxa is associated with increased pulmonary inflammation. Microbiome. 2013;1(1):19. g This work was funded and supported by: Wellcome Trust grant 086756/B/08/Z (JR); a Wellcome Trust Project Grant 083606/A/07/Z (RSH); a strategic award from the Wellcome Trust for the Malawi-Liverpool-Wellcome Trust Clinical Research Programme 084679/Z/08/Z (RSH). NIH/NHLBI U01 HL098960 (HLT). In addition, this project was supported by the Indiana Clinical and Translational Sciences Institute funded in part by NIH Grant Number UL1 TR001108. 8. Twigg 3rd HL, Morris A, Ghedin E, Curtis JL, Huffnagle GB, Crothers K, Campbell TB, Flores SC, Fontenot AP, Beck JM, et al. Use of bronchoalveolar lavage to assess the respiratory microbiome: signal in the noise. Lancet Respir Med. 2013;1(5):354–6. 8. Twigg 3rd HL, Morris A, Ghedin E, Curtis JL, Huffnagle GB, Crothers K, Campbell TB, Flores SC, Fontenot AP, Beck JM, et al. Use of bronchoalveolar lavage to assess the respiratory microbiome: signal in the noise. Lancet Respir Med. 2013;1(5):354–6. Ethics approval and consent to participate All participants provided informed, written consent. Ethical approval for this study of humans was granted by the University of Malawi College of Medicine Research Ethics Committee (proposal P.03/10/916) and Liverpool School of Tropical Medicine Research Ethics Committee in the UK (proposal 09.69). Authors’ contributions JR: Study design, ethics, laboratory work, data analysis, writing the paper. AK: Volunteer recruitment, sample collection, writing the paper. DEN and ET: Laboratory work (DNA isolation), writing the paper. RBD: Laboratory work and analysis, writing the paper. HL, XG and QD: Data analysis, paper drafting and writing. ES and GMW: Laboratory work (microbiome sequencing and QC), writing the paper. RSH: Study design, writing the paper. HLT and SBG: Study design, data analysis, writing the paper. All authors read and approved the final manuscript. Petrobacter and fuel use The finding of Petrobacter, an unusual organism associ- ated with fossil fuels, in lung lavage led us to speculate Rylance et al. BMC Microbiology (2016) 16:182 Rylance et al. BMC Microbiology (2016) 16:182 Page 6 of 7 Page 6 of 7 that biomass fuel use could be the source of this bac- terium. Our analysis demonstrates the presence of Petrobacter is negatively associated with use of clean fuel (electricity) for cooking and lighting, and with practices that reduce household air pollution exposure (i.e. cooking outside). This, and the tendency to be in- creased in females, who are most highly exposed to HAP, [2] suggests that Petrobacter is associated with burning of biomass fuel in Malawians. Only recognised as a genus in 2004, Petrobacter are non-spore forming gram negative aerobic rods with flagella [10]. First iso- lated from an Australian terrestrial oil reservoir, the organism appears tolerant of high temperatures, and there are no relevant reports of human disease or pathological association. Overall, it is plausible, and supported by our study, that Petrobacter in the lungs derives from biomass fuels. Interestingly, there was no difference in the amount of Petrobacter in BAL from individuals with high and low particulate matter in al- veolar macrophages. This may suggest that other sources of lower airway particulate matter are more important than using oil for cooking (i.e. use of other fossil fuels for cooking and heating, tobacco smoking). Received: 1 March 2016 Accepted: 4 August 2016 There are significant differences in the composition of the lung microbiome of Malawians with differing levels of particulate exposure as determined by macrophage carbon content. These differences might contribute to the excess respiratory infections associated with par- ticulate exposure. We have further demonstrated that the finding of Petrobacter in the lung is associated with household biomass fuel exposure. Interventions to im- prove air quality have the potential to alter the micro- biome of the lower respiratory tract and ultimately improve the lung health of Malawians. Author details 1 1Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool L3 5QA, UK. 2Department of Medicine, Indiana University, Indianapolis, IN, USA. 3Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Blantyre, Malawi. 4Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA. 5Center for Biomedical Informatics, Department of Public Health Sciences, Loyola University Chicago, Maywood, IL 60153, USA. 6The Jackson Laboratory for Genomic Medicine, Farmington, Connecticut 06032, USA. Acknowledgements Th h ld lik 5. Mortimer K, Gordon SB, Jindal SK, Accinelli RA, Balmes J, Martin 2nd WJ. Household air pollution is a major avoidable risk factor for cardiorespiratory disease. Chest. 2012;142(5):1308–15. 6. Lim S, et al. 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Scott JA, Hall AJ, Muyodi C, Lowe B, Ross M, Chohan B, Mandaliya K, Getambu E, Gleeson F, Drobniewski F, et al. Aetiology, outcome, and risk factors for mortality among adults with acute pneumonia in Kenya. Lancet. 2000;355(9211):1225–30. 3. Scott JA, Hall AJ, Muyodi C, Lowe B, Ross M, Chohan B, Mandaliya K, Getambu E, Gleeson F, Drobniewski F, et al. Aetiology, outcome, and risk factors for mortality among adults with acute pneumonia in Kenya. Lancet. 2000;355(9211):1225–30. 4. Pandey MR, Boleij JS, Smith KR, Wafula EM. Indoor air pollution in developing countries and acute respiratory infection in children. Lancet. 1989;1(8635):427–9. Acknowledgements The authors would like to thank the participants at Queen Elizabeth Central Hospital, Blantyre and staff at MLW especially Rose Malamba for their help with volunteer recruitment and bronchoscopy. We also thank Ruichen Rong’s contribution to the initial stage of the data analysis. 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Sequencing data from this study is deposited at NCBI, and openly available (SRA accession number SRP043676), weblink: http://www.ncbi.nlm.nih.gov/ gquery/?term=SRP043676 Sequencing data from this study is deposited at NCBI, and openly available (SRA accession number SRP043676), weblink: http://www.ncbi.nlm.nih.gov/ gquery/?term=SRP043676 10. Salinas MB, Fardeau ML, Cayol JL, Casalot L, Patel BK, Thomas P, Garcia JL, Ollivier B. Petrobacter succinatimandens gen. nov., sp. nov., a moderately 10. Salinas MB, Fardeau ML, Cayol JL, Casalot L, Patel BK, Thomas P, Garcia JL, Ollivier B. Petrobacter succinatimandens gen. nov., sp. nov., a moderately Fully anonymised participant level data is available at http://doi.org/10.6070/ H4513W8T (deposited at LabArchives). Fully anonymised participant level data is available at http://doi.org/10.6070/ H4513W8T (deposited at LabArchives). Page 7 of 7 Page 7 of 7 Rylance et al. 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W4232933041.txt	null	en		Analysis of nucleation events in the European boundary layer using the regional aerosol-climate model REMO-HAM with a solar radiation-driven OH-proxy	null	2,014	cc-by	14,682	Atmos. Chem. Phys., 14, 11711–11729, 2014 www.atmos-chem-phys.net/14/11711/2014/ doi:10.5194/acp-14-11711-2014 © Author(s) 2014. CC Attribution 3.0 License. Analysis of nucleation events in the European boundary layer using the regional aerosol–climate model REMO-HAM with a solar radiation-driven OH-proxy J.-P. Pietikäinen1 , S. Mikkonen2 , A. Hamed2,† , A. I. Hienola1 , W. Birmili3 , M. Kulmala4 , and A. Laaksonen1,2 1 Finnish Meteorological Institute, P. O. Box 503, 00101 Helsinki, Finland of Applied Physics, University of Eastern Finland, P. O. Box 1627, 70211 Kuopio, Finland 3 Leibniz Institute for Tropospheric Research (IfT), Permoserstr. 15, 04318 Leipzig, Germany 4 Department of Physics, University of Helsinki, P. O. Box 44, 00014 Helsinki, Finland † deceased in September 2013 2 Department Correspondence to: J.-P. Pietikäinen (joni-pekka.pietikainen@fmi.fi) Received: 13 January 2014 – Published in Atmos. Chem. Phys. Discuss.: 2 April 2014 Revised: 12 September 2014 – Accepted: 22 September 2014 – Published: 7 November 2014 Abstract. This work describes improvements in the regional aerosol–climate model REMO-HAM in order to simulate more realistically the process of atmospheric new particle formation (NPF). A new scheme was implemented to simulate OH radical concentrations using a proxy approach based on observations and also accounting for the effects of clouds upon OH concentrations. Second, the nucleation rate calculation was modified to directly simulate the formation rates of 3 nm particles, which removes some unnecessary steps in the formation rate calculations used earlier in the model. Using the updated model version, NPF over Europe was simulated for the periods 2003–2004 and 2008–2009. The statistics of the simulated particle formation events were subsequently compared to observations from 13 ground-based measurement sites. The new model shows improved agreement with the observed NPF rates compared to former versions and can simulate the event statistics realistically for most parts of Europe. 1 Introduction Atmospheric aerosols influence our quality of life in many different ways, from affecting human health and diminishing visibility, to changing the climate patterns and the hydrological cycle. An important phenomenon associated with the atmospheric aerosol system is the formation of new aerosol particles through gas-to-particle conversion, a process that seems to occur almost everywhere in the troposphere (Kulmala et al., 2004). The climate relevance of new particle formation has been demonstrated in several studies (e.g. Spracklen et al., 2006; Wang and Penner, 2009; Matsui et al., 2011). NPF strongly influences aerosol number concentrations and makes an important contribution to global and local cloud condensation nuclei (CCN) concentrations (Lihavainen et al., 2003; Kerminen et al., 2005; Laaksonen et al., 2005; Merikanto et al., 2009). As such, nucleation is among the key processes that need to be represented in stateof-the-art regional and global aerosol–climate models. Modelling nucleation and the subsequent growth is a difficult task. Based on the assumption that sulfuric acid (H2 SO4 ) is the main driving force in the process of nucleation, several parametrizations have been proposed to explain NPF: binary water–sulfuric acid nucleation (Vehkamäki et al., 2002), ternary water–sulfuric acid–ammonia nucleation (Napari et al., 2002; Merikanto et al., 2007), ion-induced nucleation involving water and sulfuric acid (Modgil et al., 2005), an ion-mediated nucleation (IMN) mechanism (Yu, 2010) and combined neutral and ion-induced nucleation (Kazil and Lovejoy, 2007), as well as two nucleation parametrizations for the forested boundary layer (BL) – the cluster activation mechanism (Kulmala et al., 2006; Sihto et al., 2006) and the kinetic mechanism (Laakso et al., 2004; Kuang et al., 2008). These parametrizations are designed to estimate the number Published by Copernicus Publications on behalf of the European Geosciences Union. 11712 J.-P. Pietikäinen: Modelling European boundary layer nucleation of nucleated particles as a function of the main controlling parameter (H2 SO4 ) at the expense of compromising the realism of the simulated process. For example, Metzger et al. (2010) showed that using the product of the concentrations of H2 SO4 and organic molecules, the modelled nucleation rates were in better accord with measured values. The ability of global and regional models to predict NPF events has been tested before. Spracklen et al. (2008) used a global chemistry transport model with aerosol microphysics to predict the contribution of boundary layer nucleation to regional and global distributions of CCN. They found that by using the cluster activation scheme, the modelled particle size distributions and total particle number concentrations at three continental sites in Europe were improved. Makkonen et al. (2009) modified a global aerosol– climate model with respect to NPF by including several optional nucleation parametrizations that could be run together with binary homogeneous sulfuric acid–water nucleation. By adding the cluster activation parametrization to the boundary layer, the authors found that the particle number concentration in the lower atmosphere increased more than 10fold, while in the upper atmosphere the increase was even larger. The study also showed that the cloud droplet number concentration depends on the nucleation mechanism used. Kazil et al. (2010) implemented a new scheme for neutral and ion-induced nucleation of sulfuric acid and water in a global aerosol–climate model, considering that such a nucleation mechanism is a good candidate to explain NPF over the oceans and free troposphere. The combination of the new scheme and nucleation via cluster activation seemed to better explain the observations of ultrafine aerosol concentrations over the Pacific Ocean than the cluster activation alone. Many other studies using global aerosol–climate models have demonstrated the importance of atmospheric NPF for regional and global aerosol number concentration and cloud condensation nuclei budgets (Merikanto et al., 2009; Pierce et al., 2007; Pierce and Adams, 2009; Wang and Penner, 2009; Yu and Luo, 2009; Trivitayanurak et al., 2008; Jung et al., 2010; Laakso et al., 2013; Scott et al., 2014), each study assessing which parametrization leads to the best agreement with observations in their model. Global models have a large grid size (usually 200–300 km when aerosols are included), hence the number concentration of newly formed particles and size distribution are prone to large uncertainties. Regional climate models, on the other hand, have resolution varying from kilometres to tens of kilometres and hence resolve much greater variability in emissions and processing, and provide a better framework to calibrate potential nucleation mechanisms against observations. Numerous regional climate models exist, but only a few have been used to analyse NPF. Sotiropoulou et al. (2006) used an air quality model based gas/aerosol model to study the impact of NPF on regional air quality and CCN formation. They concluded that an online coupled regional aerosol–climate model would improve the nucleation analyAtmos. Chem. Phys., 14, 11711–11729, 2014 sis done in their work. Matsui et al. (2011) used a weather research and forecasting model coupled with chemistry to study NPF over the Beijing region in China. The authors showed that the model is able to reproduce the timing of NPF events and non-NPF days. Matsui et al. (2011) reported that reductions in primary aerosol emissions do not necessarily lead to lower CCN concentrations because NPF generates a stronger source of CCN in conditions with lower condensation sink. Fountoukis et al. (2012) used a three dimensional chemical transport model with a microphysical module to simulate NPF on the European scale. They showed that in some regions the total particle number concentrations can be increased by a factor of 3 when nucleation is included. They also found that a semi-empirical ternary sulfuric acid–ammonia–water parametrization shows better agreement with measurements of particles larger than 10 nm than kinetic or activation parametrization. In this study, the predictive capability of the NPF of the regional aerosol–climate model REMO-HAM is investigated. The results are compared with measurements from 13 European sites covering years 2003–2004 and 2008–2009, which allows us to test the nucleation in the model against the observations covering a range of seasons and environments. REMO-HAM is modified in this work to include a new measurement-based OH-proxy. The advantage thereof is that the incoming solar radiation is linked to the OH concentrations, thus taking into account the effects of clouds. The method shown here can be very useful for other types of models where nucleation is important to resolve adequately, but for whom a tropospheric chemistry scheme would be prohibitively expensive. In addition, the particle formation rate of ∼ 1 nm clusters is replaced by the direct formation of 3 nm particles. This study is (to our knowledge) the first to compare nucleation rates from the model to those from observations. In the previous studies, the focus has been in comparing simulated and measured particle concentrations. Comparing the model nucleation rate against that derived from the observations is a stronger constraint than comparing particle concentrations to observed particle concentrations, because the latter has greater possibility for compensating errors (for example via biases in coagulation sink or particle growth rates). The paper is structured as follows: first, the models with their modifications and the methods are described in Sect. 2; Sect. 3 presents a detailed analysis of the results, followed by Sect. 4, where the main conclusions are listed and further steps are discussed. www.atmos-chem-phys.net/14/11711/2014/ J.-P. Pietikäinen: Modelling European boundary layer nucleation 2 Methods 2.1 2.1.1 Model description ECHAM5-HAM global aerosol–climate model In this work, the updated version ECHAM5-HAM2 (Roeckner et al., 2003; Stier et al., 2005; Zhang et al., 2012) is used to provide lateral aerosol boundary data for the regional model simulations. ECHAM-HAM2 is a global aerosol– climate model that includes the updated HAM2 aerosol module (Stier et al., 2005; Zhang et al., 2012) and the microphysical module M7 (Vignati et al., 2004). 2.1.2 REMO-HAM regional aerosol–climate model In this study, the main tool is the regional aerosol–climate model REMO-HAM (Pietikäinen et al., 2012). The core of REMO-HAM is a hydrostatic, three-dimensional atmosphere model developed at the Max Planck Institute for Meteorology in Hamburg, and is based on the Europa Model, the former numerical weather prediction model of the German Weather Service (Jacob and Podzun, 1996; Jacob, 2001). The physical core of REMO is based on the physical packages of the global circulation model ECHAM4 (Roeckner et al., 1996). Many parts of the model, for example the cloud and soil treatments, have been updated (Pfeifer, 2003; Semmler et al., 2004; Hagemann, 2002; Rechid, 2009; Kotlarski, 2007). With respect to the aerosol module, REMO-HAM incorporates many of the updates in physics that are included in the recent ECHAM5-HAM2 version (REMO-HAM has the HAM suffix because it does not have all the HAM2 updates). The main deficiencies of REMO-HAM are the missing SOA module and the online coupling of the HAM module with the radiation scheme (Pietikäinen et al., 2012). 2.2 OH-proxy The chemistry modules of ECHAM-HAM and REMO-HAM are based on a sulfate aerosol chemistry module described by Feichter et al. (1996). In this module, dimethyl sulfide (DMS), sulfur dioxide (SO2 ) and sulfate (SO2− 4 ) are treated as prognostic variables. For oxidation, the module uses three-dimensional monthly mean oxidant fields from hydroxyl (OH), hydrogen peroxide (H2 O2 ), ozone (O3 ) and nitrogen dioxide (NO2 ) (Stier et al., 2005). These fields are calculated/provided by the comprehensive MOZART chemical transport model (Horowitz et al., 2003). Both gas- and aqueous-phase oxidations are included. In the gas phase, SO2 and DMS are oxidized by OH during the day time while DMS reacts with the nitrate radical (NO3 ) during the night. NO3 is assumed to be in steady state with its production and loss terms, which both include reactions with NO2 . The reactions of O3 , SO2 and H2 O2 are considered in the aqueous phase. www.atmos-chem-phys.net/14/11711/2014/ 11713 The formation of sulfuric acid (H2 SO4 ) occurs via the reaction between the hydroxyl radical OH and sulfur dioxide SO2 ; which, in turn, is directly emitted from various anthropogenic and natural sources. SO2 is also produced in a reaction between DMS and OH. The OH concentrations are higher during the day time due to photolysis reactions (source terms) (Seinfeld and Pandis, 1998). As mentioned before, the models use monthly mean fields for OH, which is not a very realistic approach. To overcome this problem, both ECHAM-HAM and REMO-HAM use an artificial diurnal cycle. This is obtained by using the monthly mean values as a baseline and multiplying them with a diurnal coefficient. This coefficient follows a cosine peak between sunrise and sunset and its amplitude is scaled with the day length (thus, the monthly mean values for OH are preserved). Although this approach is more realistic than the original, where the constant values were used, it has some disadvantages: it can overestimate the values for short days, and it is not connected to radiation (for example, below clouds, the concentrations are not affected by the decreased solar radiation). In order to preserve the speed of the chemical module (keep it as usable as possible for long-term simulations), the calculation method for OH concentrations is replaced with an OH-proxy. Rohrer and Berresheim (2006) presented an equation for approximating OH concentration by using a nonlinear function of the photolysis frequency of ozone J (O1 D) as a predictor. The approach is to build the proxy by using variables that are commonly measured in different sites and can be easily accessed with atmospheric models. Thus, the downward short-wave flux (SWF↓) is used as the main predictor instead of J (O1 D). The reasons for this are that the correlation between these two variables is evident, SWF↓ is often measured, and SWF↓ is available in the climate models. The construction of the proxy follows a similar approach to that used by Mikkonen et al. (2011) for H2 SO4 concentration. A nonlinear fitting procedure is applied to the measurement data, where the functional form for the proxy is given by [OH] = a × (SWF ↓)b + c, (1) where the exponent b reflects the combined effects of all photolytic processes that generate OH either directly or via production of and recycling from HO2 . The dependence of OH on reactants such as NOx , hydrocarbons, O3 or H2 O is condensed into the single pre-exponential coefficient, a. The coefficient c includes all processes that are light-independent; for example, OH production at night time. These coefficients were estimated with OH-measurement data recorded in Hyytiälä, Finland (Petäjä et al., 2009). The implemented OH-proxy (OHproxy ) is 3081.0 · (SWF ↓)0.8397 day time OHproxy = (2) 6.033 × 104 night time, where the units are [molec cm−3 ] for OH-proxy and [W m−2 ] for SWF↓. With this approach, the OH concentrations used Atmos. Chem. Phys., 14, 11711–11729, 2014 11714 J.-P. Pietikäinen: Modelling European boundary layer nucleation by the model are more realistic and are linked to the incoming solar radiation in each grid box on every model level. 2.3 Nucleation scheme ECHAM-HAM and REMO-HAM use a binary sulfuric acid– water nucleation scheme by Vehkamäki et al. (2002) and a neutral and charged H2 SO4 /H2 O nucleation scheme by Kazil and Lovejoy (2007) along with two nucleation mechanisms restricted to the forested boundary layer: the cluster activation (Kulmala et al., 2006) and the kinetic nucleation scheme (Laakso et al., 2004). These empirical schemes are usually employed to calculate the formation rates of 1 (or 1.5) nm clusters. However, the empirical formulae are not based on directly measured cluster formation rates, as the 1 nm rates have been obtained by extrapolation from measured 3 nm particle formation rates (Kerminen and Kulmala, 2002). The extrapolation requires, as input, the cluster growth rate, which often has quite large uncertainty. Furthermore, condensable organics (Kulmala et al., 2013), which are known to participate in cluster growth between 1 and 3 nm, are not included in the current model setup. Taken together, the extrapolation from 3 nm to 1 nm and the modelling of the growth from 1 nm back to 3 nm creates an error in the modelled 3 nm particle formation rates. This unnecessary calculation cycle can be bypassed as the 3 nm formation rate can be directly parametrized based on observations. In this work, the formation rate of 3 nm particles J3 nm [cm−3 s−1 ] is calculated using the kinetic nucleation scheme J3 nm = K × [H2 SO4 ]2 , (3) where K = 1.417 × 10−15 [cm3 s−1 ] is the kinetic coefficient and [H2 SO4 ] is the sulfuric acid concentration in molec cm−3 . The value of the kinetic coefficient, K, is based on a comparison of the model results and measurements conducted within this work (not shown). We compared measured H2 SO4 concentrations against different K values from Hyytiälä, Melpitz and San Pietro Capofiume, and derived the best fit. The 3 nm particles are assumed to consist of sulfuric acid only (and thus a corresponding amount of H2 SO4 is removed from the gas phase as the particles are formed). The default approach of nucleation rate is also modified: kinetic nucleation is not restricted to occur only at the forested boundary layer, but is instead calculated in every grid box. As the nucleation mechanism(s) at higher altitudes are unknown, this approach may generate some error. However, our focus is on boundary layer nucleation, and therefore our conclusions are more or less independent of the assumed free tropospheric nucleation mechanism. 2.4 Simulations The ECHAM5-HAM data are used at the lateral boundaries of REMO-HAM (Pietikäinen et al., 2012) for aerosol species Atmos. Chem. Phys., 14, 11711–11729, 2014 2 ] Figure 1. The orography of the REMO domain and the analysed locations. with an update frequency of 6 h. ERA-Interim data are used to nudge ECHAM5-HAM and as a lateral meteorological boundary forcing for REMO-HAM (Dee et al., 2011). The resolution of T63L31 is applied for ECHAM5-HAM (horizontally 210 km, vertically 31 levels), while for REMOHAM a resolution of 0.44◦ (50 km × 50 km) is used with 27 vertical levels. The models have been run for the years 2003–2004 and 2008–2009 with spin-up times of 3 months. The domain for REMO-HAM covers the whole of Europe. To study the nucleation events in more detail, one-hour output resolution for the REMO-HAM simulations is used. For 2003 and 2004, two model versions are used: OH-proxy version including 3 nm nucleation in all grid boxes (henceforth called REMO-OHP), and a normal chemistry version including 3 nm nucleation in all grid boxes (henceforth called REMO-NCH). Figure 1 shows the orography of the model domain and the measurement sites used in this study. Detailed information about the measurement sites is presented in Table 1. 2.5 Measurement sites and data Two different approaches for comparing the simulated nucleation events against measurement data are used. Firstly, observation data from three stations, Hyytiälä, Melpitz and San Pietro Capofiume, are used. Details about measurement data and instruments used can be found in Birmili and Wiedensohler (2000); Jaatinen et al. (2009) and Engler et al. (2007). The aerosol size distributions, from which the event statistics were calculated, were measured using a twin Differential Mobility Particle Sizer (DMPS) at all sites. Secondly, literature-based observation data are used to analyse the www.atmos-chem-phys.net/14/11711/2014/ J.-P. Pietikäinen: Modelling European boundary layer nucleation 11715 Table 1. Measurement sites with long-term observations of the new particle formation events analysed in this work. Observation site Coordinates Altitude (m a.s.l.) Measurement period Reference Hyytiälä, Finland 61◦ 500 N, 24◦ 180 E 181 1 Jan 2003–31 Dec 2004 Mar 2008–Apr 2009 Hari and Kulmala (2005) Manninen et al. (2010) Melpitz, Germany 51◦ 320 N, 12◦ 540 E 87 1 Jul 2003–31 Dec 2004 May 2008–Apr 2009 Birmili and Wiedensohler (2000) Engler et al. (2007) Manninen et al. (2010) San Pietro Capofiume, Italy 44◦ 370 N, 11◦ 400 E 11 2003–Aug 2004 (partly Oct) Mar 2008–Sep 2008 Jaatinen et al. (2009) Manninen et al. (2010) Mace Head, Ireland 53◦ 190 N, 09◦ 530 E 5 Aug 2002–Jul 2004 Jun 2008–Apr 2009 Yoon et al. (2006) Manninen et al. (2010) Hohenpeißenberg, Germany 47◦ 480 N, 11◦ 000 E 985 Apr 1998–Aug 2000 Apr 2008–Apr 2009 Birmili et al. (2003) Manninen et al. (2010) Värriö, Finland 67◦ 460 N, 29◦ 350 E 400 2003–2004 Dal Maso et al. (2007) Pallas, Finland 67◦ 580 N, 24◦ 070 E 560 2003–2004 Apr 2008–Apr 2009 Dal Maso et al. (2007) Manninen et al. (2010) Vavihill, Sweden 56◦ 010 N, 13◦ 090 E 172 Feb 2001–May 2004 Apr 2008–Feb 2009 Kristensson et al. (2011) Manninen et al. (2010) Finokalia, Greece 35◦ 200 N, 25◦ 400 E 250 Apr 2008–Apr 2009 Apr 2008–Apr 2009 Pikridas et al. (2012) Manninen et al. (2010) Cabauw, Netherlands 51◦ 570 N, 04◦ 530 E 0 Apr 2008–Mar 2009 Manninen et al. (2010) K-Puszta, Hungary 46◦ 580 N, 19◦ 350 E 125 Mar 2008–Feb 2009 Manninen et al. (2010) Puy de Dôme, France 45◦ 420 N, 03◦ 130 E 1465 Feb 2007–Jun 2010 Apr 2008–Apr 2009 Boulon et al. (2011) Manninen et al. (2010) Jungfraujoch, Switzerland 46◦ 320 N, 07◦ 570 E 3580 Apr 2008–Apr 2009 Apr 2008–Apr 2009 Boulon et al. (2010) Manninen et al. (2010) model results for all 13 stations. The measurement sites, the measurement periods and references to the data are given in Table 1. 2.6 Event classification The classification of modelled nucleation events is based on two criteria. First, the J3 nm values have to be over 0.01 [cm−3 s−1 ] for two sequential hours. This limit comes from the lower detection limit of the instruments used in Hyytiälä and San Pietro Capofiume. Second, for the same time period, the rate of number concentration change with respect to change in logarithmic diameter for 3 nm particles has to be over 2000 dN/d log10 Dp [cm−3 ]. This value is derived directly from the aerosol size distributions by comparing them and the J3 nm values. According to our tests, this approach classifies the event days realistically, but some error is introduced in specific cases; for example, when a nucleation event is terminated prematurely due to rain, etc. Nevertheless, these cases are not very common in the model and the criteria work very well for the modelled data. www.atmos-chem-phys.net/14/11711/2014/ The event classification used for measurements (Hyytiälä, Melpitz and San Pietro Capofiume) was conducted by Jaatinen et al. (2009) with the method based on Dal Maso et al. (2005). A day is considered an event day when the formation of new aerosol particles starts at the lowest measurable particle size (diameter 3 nm) and subsequent growth of the newly formed particles is observed for several hours. The nucleation event classification is based on event clarity – i.e. the number concentrations of the freshly formed particles, and their formation and growth rates. For more details on the classification method, see Hamed et al. (2007). 3 Comparison with measurements 3.1 J3 nm values The measured and modelled J3 nm values are compared in Fig. 2. Since the measurement data are only for the nucleation event days, the same approach is made to model data using the event classification method described in Sect. 2.6. Atmos. Chem. Phys., 14, 11711–11729, 2014 11716 J.-P. Pietikäinen: Modelling European boundary layer nucleation HYYTIÄLÄ J3nm [cm−3 s−1] 10 1 0.1 0.01 0.001 Jan FebMarAprMayJun Jul Aug Sep Oct NovDec Jan FebMarAprMayJun Jul Aug Sep Oct NovDec REMO-OHP J3nm [cm−3 s−1] 100 REMO-NCH Measurements MELPITZ 10 1 0.1 0.01 0.001 Jan FebMarAprMayJun Jul Aug Sep Oct NovDec Jan FebMarAprMayJun Jul Aug Sep Oct NovDec J3nm [cm−3 s−1] 100 SAN PIETRO CAPOFIUME 10 1 0.1 0.01 393 %. REMO-OHP underestimates the rates during the autumn peaks in Melpitz, whereas in San Pietro Capofiume the autumn rates are in good agreement. During the summer, REMO-OHP underestimates the values in Melpitz and San Pietro Capofiume, especially in the latter. Although not perfect, REMO-OHP produces quite realistic J3 nm values and performs clearly better in this respect than REMO-NCH. The underestimation in REMO-OHP may come from the chemistry part, but also from the nucleation parametrization. For example, better representation of organics and their influence to the nucleation rates could lead to more realistic J3 nm values (in both model versions). Currently, the influence of organics comes indirectly from the kinetic coefficient K in Eq. (3), which is based on measurement and includes the effect of organics (if any). We chose this approach as the model does not have an SOA module. Besides the nucleation rates, the length of the events is also an important factor for the total number of nucleated particles. This is analysed in the next section. 0.001 Jan FebMarAprMayJun Jul Aug Sep Oct NovDec Jan FebMarAprMayJun Jul Aug Sep Oct NovDec Years 2003-2004 Figure 2. Measured and modelled daily mean J3 nm rates for event days at Hyytiälä, Melpitz and San Pietro Capofiume. 3.2 Start and end time/duration of events The measurement data for Hyytiälä, Melpitz and San Pietro Capofiume also includes the nucleation event start time, end time and (calculated) length. For these variables, monthly statistics for the measurements and modelled results are derived. Figure 3 shows that, at Hyytiälä, REMO-OHP can reproduce the event length realistically for most of the modelled period, excluding some overestimation periods during summer/autumn of 2004. In REMO-NCH, the overestimation of event length can be seen throughout the year, excluding spring, where the model reproduces measured values fairly well. For the event start times, REMO-OHP results are in good agreement with the measurements, although it has delayed start times during the spring and summer of 2003. On the other hand, the REMO-NCH events start 1–3 h too early and the difference is biggest during the summer months, especially during 2004. The end times of the events show more fluctuations, but overall the agreement between the measurements and REMO-OHP is good. However, during the summer/autumn of 2004, REMO-OHP shows a strong delay in event end times (up to 5 h). Similar behaviour can be seen with REMO-NCH, which tends to delay the event ends for almost the whole modelled period. At Melpitz, REMO-OHP overestimates the event lengths. Seasonally, the model shows 4 h overestimations in the spring, 0–4 h during the summer and 2–4 h in the autumn. REMO-NCH has similar trend, but the overestimations are worse; 8–10 h in the spring, 6–8 h in the summer and 6 h in the autumn. REMO-OHP captures event start times very well for 2003, but during 2004, the model gives too early start times for the first half of the year. For the second half, the start times are delayed, but the difference stays within a couple of hours. In REMO-NCH, the events start a few hours too Fig. 2. Measured and modelled daily mean J3nm rates for event days at Hyytiälä, Melpitz and San Pietro Capofiume. Overall, the measurements show that Hyytiälä and Melpitz have the highest nucleation rates in the spring and autumn, whereas in San Pietro Capofiume the values are quite high during all seasons except winter. Both of the model versions show similar features, although REMO-OHP cannot reproduce the high rates in Hyytiälä during the autumn. REMONCH shows overall much higher values at all locations and the values have a maximum during the summer. The mean J3 nm values show that REMO-OHP is able to reproduce measured NPF rates at Hyytiälä, although overall some underestimation can be seen; the relative difference of 2-year mean 1r , calculated by first subtracting the measured mean from the model mean, then dividing this by the measured mean and finally multiplying this by 100 %, is 1r = −71 %. The highest measured rates are not captured during the spring by REMO-OHP, whereas the summer values are in good agreement. For REMO-NCH, the values are also quite realistic, but overestimated (1r = 66 %). During summer, the REMO-NCH values are over 10 times too high, but in the spring, REMO-NCH reproduces the measured rates more realistically than REMO-OHP, which underestimates the values by a factor of 5–10. This shows that, seasonally, the new model version still has deficiencies. Similar behaviour as in Hyytiälä can also be seen at Melpitz and San Pietro Capofiume. At these locations, the overestimation of REMO-NCH is larger, especially at San Pietro Capofiume. The 1r -values for REMO-OHP are −35 % and −60 % at Melpitz and San Pietro Capofiume, respectively. For REMO-NCH, the corresponding values are 590 % and Atmos. Chem. Phys., 14, 11711–11729, 2014 www.atmos-chem-phys.net/14/11711/2014/ J.-P. Pietikäinen: Modelling European boundary layer nucleation J.-P. Pietikäinen: Modelling European boundary layer nucleation 11717 08:00 06:00 REMO-NCH REMO-NCH Measurements 16 14 12 10 08 06 04 02 n b r r y n l g p ct v c n b r r y n l g p ct v c Ja Fe Ma Ap Ma Ju Ju Au Se O No De Ja Fe Ma Ap Ma Ju Ju Au Se O No De n b r r y n l g p ct v c n b r r y n l g p ct v c Ja Fe Ma Ap Ma Ju Ju Au Se O No De Ja Fe Ma Ap Ma Ju Ju Au Se O No De Years 2003-2004 Years 2003-2004 SAN PIETRO CAPOFIUME REMO-OHP Length [h] REMO-OHP Length [h] Length [h] Start time End time 14 12 10 08 06 04 22:00 20:00 18:00 16:00 14:00 22:00 20:00 18:00 16:00 14:00 12:00 Measurements 24:00 22:00 20:00 18:00 16:00 14:00 16:00 14:00 12:00 10:00 08:00 06:00 Start time 10:00 REMO-OHP End time MELPITZ 12:00 10:00 08:00 06:00 04:00 End time Start time HYYTIÄLÄ 12:00 REMO-NCH Measurements 18 16 14 12 10 08 06 04 n b r r y n l g p ct v c n b r r y n l g p ct v c Ja Fe Ma Ap Ma Ju Ju Au Se O No De Ja Fe Ma Ap Ma Ju Ju Au Se O No De Years 2003-2004 Figure 3. Monthlymean mean event event start endend timetime and length at Hyytiälä, Melpitz and San Pietro Months without data or events Fig. 3. Monthly starttime, time, and length at Hyytiälä, Melpitz andCapofiume. San Pietro Capofiume. Months without data or have been dismissed. have been dismissed. Fraction of event days the other hand, higher pre-existing condensation sink (which would be expected if SOA was included in the model) would lead to lower H2 SO4 concentrations and decrease the J3 nm values. This effect, however, would not be very strong, because the nucleation events usually start when the air is clean (measurements show low condensation sink) and during this time H2 SO4 concentrations would stayMelpitz almost as high as 2003-2004 REMO-OHP without SOA in the1.2 model. This eventuallyMeasurements leads back to the REMO-NCH point that nucleation events would be shorter with SOA in 1.0 the model due to increased condensation sink and faster de0.8 pletion of H2 SO4 , 0.6 as the events progress. The results from0.4San Pietro Capofiume show that REMOOHP overestimates0.2 the event lengths by 2 h, throughout the 0.0 year, whereas REMO-NCH r y h y n l g p ct them v c n by b r 2–10 n b r roverestimates n l g p ct v c Ja Fe Ma Ap Ma Ju Ju Au Se O No De Ja Fe Ma Ap Ma Ju Ju Au Se O No De (maximum being in the summer). The event start times in REMO-OHP are almost identical with measurements in 2003, but during the beginning of 2004, the model has a tendency to initiate nucleation slightly too early. This bias, however, decreases during the summer. REMO-NCH has a systematical bias to start the events too early and seasonally, the difference is smallest during the winter and highest during the summer. The same mechanism applies here as for Fraction of event days Fraction of event days early and the difference is highest during the summer and almost disappears during the late autumn and early spring (no data for winter, unfortunately). The end time of the events at Melpitz is not very well captured by either of the models; both show much later end times than the measurements. In particular, REMO-NCH has a tendency to delay the ending of the nucleation events substantially. Hyytiälä 2003-2004 REMO-OHP The distributionsMeasurements were also compared with the 1.0 aerosol REMO-NCH measurements analysed by Hamed et al. (2010) (not shown). 0.8 This 0.6comparison showed that the model results underestimate the number concentration of particles > 100 nm by 0.4 a factor of 2 (similar behaviour can be also seen for the 0.2 aerosol distributions in an earlier study by Pietikäinen et al., 0.0 2012). One l g p ct for r r y n reason r y missing v c this n b possible n b isr the n l g p growth ct v c Ja Fe Ma Ap Ma Ju Ju Au Se O No De Ja Fe Ma Ap Ma Ju Ju Au Se O No De caused by condensable which2003-2004 would lead to higher Sanorganics, Pietro Capofiume REMO-OHP Measurements concentrations of particles > 100 nm. Either way, the lower 1.2 REMO-NCH particle number concentration leads to smaller surface area 1.0 0.8 and condensation sink. This might be the key factor in un0.6 derstanding why the model overestimates the event lengths 0.4 at Melpitz: if the condensation of H2 SO4 is too slow dur0.2 ing the nucleation event, H2 SO4 will continue to cause nu0.0 l g p ct v On r rbeen c r y n lperiod y n removed. v c nit bhas g p ct until b ar longer cleationJanfor Fe Ma Ap Ma Ju Ju Au Se O No De Ja Fe Ma Ap Ma Ju Ju Au Se O No De Fig. 4. The fraction of days with NPF events, 2003–2004, at 3 European observation sites on a monthly basis. The graph compares www.atmos-chem-phys.net/14/11711/2014/ Atmos. Chem. Phys., 14, 11711–11729, 2014 simulations with observational evidence. 1.0 REMO-OHP REMO-NCH Hyytiälä 2003-2004 Measurements 0.8 0.6 0.4 0.2 0.0 Fraction of event days 1.2 REMO-OHP REMO-NCH Melpitz 2003-2004 Measurements 1.0 0.8 0.6 0.4 0.2 0.0 n b r r y n l g p ct v c n b r r y n l g p ct v c Ja Fe Ma Ap Ma Ju Ju Au Se O No De Ja Fe Ma Ap Ma Ju Ju Au Se O No De 1.2 Fraction of event days J.-P. Pietikäinen: Modelling European boundary layer nucleation Fraction of event days 11718 n b r r y n l g p ct v c n b r r y n l g p ct v c Ja Fe Ma Ap Ma Ju Ju Au Se O No De Ja Fe Ma Ap Ma Ju Ju Au Se O No De San Pietro Capofiume 2003-2004 REMO-OHP Measurements REMO-NCH 1.0 0.8 0.6 0.4 0.2 0.0 n b r r y n l g p ct v c n b r r y n l g p ct v c Ja Fe Ma Ap Ma Ju Ju Au Se O No De Ja Fe Ma Ap Ma Ju Ju Au Se O No De Figure 4. The fraction of days with NPF events, 2003–2004, at 3 European observation sites on a monthly basis. The graph compares model simulations with observational evidence. Melpitz: the low condensation sink of H2 SO4 in the models is the most probable reason for the delays in the nucleation end times. The simplified sulfate chemistry module could also be one reason for the continuation of the Melpitz and San Pietro Capofiume events. The OH-proxy is based on measurements from Hyytiälä, which means that the influences of other relevant chemical species to OH concentrations are based on Hyytiälä conditions. For example, nitrogen oxides (NOx) and volatile organic compounds (VOCs) are species competing for the reaction with OH (Seinfeld and Pandis, 1998). The VOC / NOx ratio dictates which species is predominant in the reaction. As the information about the average VOC / NOx ratio is now implicitly included in the proxy through measurements from Hyytiälä, error may be caused in environments where the typical VOC / NOx ratios differ from those in Hyytiälä. This will impact the H2 SO4 concentrations and could partially explain why the J3 nm values have different biases and why the length of events is not captured similarly in different locations. 3.3 Fraction of event days The fraction of event days per month is analysed from all measurement stations. This section is divided into two parts, which are based on the simulation periods. 3.3.1 Years 2003 and 2004 The measured and modelled monthly fractions of nucleation days for Hyytiälä, Melpitz and San Pietro Capofiume are shown in Fig. 4. The measurement data have some gaps, because measurements were not available for the entire twoyear period (details in Table 1). Atmos. Chem. Phys., 14, 11711–11729, 2014 REMO-OHP underestimates the fraction of nucleation days per month in spring and overestimates it in early summer at Hyytiälä. For autumn, the model underestimates the fraction in 2003 (reproducing only half of the nucleation days), but captures the events in 2004. REMO-NCH overestimates the fraction almost throughout the modelling period, going up to five times higher event frequency. Late autumn in 2003 and spring 2004 are the only times when REMONCH underestimates or comes even close to the measurements. Overall, the values from the model simulations are not a perfect match, but REMO-OHP shows much better agreement. For Melpitz, Fig. 4 shows that REMO-OHP slightly overestimates the nucleation events for the year 2003 (0–15 %). For 2004, REMO-OHP overestimates the values for the first half of the year (up to a factor of 5) and underestimates them for the second; for example, getting less than half of the events during September. With REMO-NCH, the fraction of monthly nucleation days is overestimated in every month. The low fraction in measurements for summer 2004 can be partly explained by the high number of undefined days (up to 14 days per month) (Jaatinen et al., 2009). At San Pietro Capofiume, REMO-OHP tends to predict nucleation events too frequently by 30–50 % for both years, especially during wintertime (Fig. 4). If January and February are disregarded, the pattern of the first year is well captured by REMO-OHP. REMO-NCH shows high overestimations, especially during summertime. For many months, REMO-NCH show nucleation fractions of 1.0. Even during the winter, more than 60 % of the days show nucleation events. Pietikäinen et al. (2012) showed that the model has a positive SO2 bias, which can lead to elevated H2 SO4 values. The bias is relatively high in polluted areas, and location such as San Pietro Capofiume falls into this category (Laaksonen et al., 2005, and references therein). Despite the www.atmos-chem-phys.net/14/11711/2014/ J.-P. Pietikäinen: Modelling European boundary layer nucleation improved OH chemistry presented in this work, the results for San Pietro Capofiume are affected by the positive SO2 bias. In addition to the measurement-based analysis conducted for Hyytiälä, Melpitz and San Pietro Capofiume, an analysis based on observation data from literature was performed. Figure 5 shows the fraction of nucleation days per month for these locations (more details in Table 1). For Mace Head, data from Yoon et al. (2006) are used. Two types of nucleation events are observed in Mace Head: the coastal events, driven by iodine species emitted by algae during low tides, and the continental type of events; i.e. sulfuric acid-driven events similar to those observed at the other stations. The former type of nucleation is not included in REMO-HAM, making the comparison between simulations and observations somewhat complicated. However, Yoon et al. (2006) provided two kinds of nucleation event statistics: the total number of events, and the number of events for cases in which clean marine air masses advected over tidal areas to the measurement station. While some of the latter events may be of the continental type, it is clear that most of them are coastal (see also O’Dowd et al., 2002). Similarly, it is likely that the majority of the rest of the events (polluted cases, i.e. total events minus clean events) are of the continental type. Figure 5 shows the total number of nucleation events and the difference between the total and clean air mass cases (shown as 1Yoon et al., 2006). The model results for Mace Head show that, if compared to all event cases, REMO-OHP underestimates the nucleation days for the whole simulation period. On the other hand, REMO-NCH gives reasonably realistic results. In addition, the overestimation seen before in REMO-NCH is not present. However, if the 1Yoon et al. (2006) results are compared, results from REMO-OHP show better agreement. The model still underestimates the event numbers during both winter and spring 2003, but the absolute difference is much smaller. During spring 2004, and both summers and autumns, REMO-OHP is able to capture the measured statistics that have even slight overestimations in some cases. REMO-NCH overestimates the values for all months. At Hohenpeißenberg, REMO-OHP reproduces the measured values with good accuracy. Also, the yearly cycle is in reasonable agreement with measurements. There are some months, for example during spring, when the model overestimates the number of event days. On the other hand, underestimation occurs in autumn and winter, but the absolute difference is quite small. REMO-NCH shows realistic results only during the winter time. During other periods, the model overestimates the event day fraction 3–5 times. The results from Värriö show that REMO-OHP underestimates the measured nucleation event frequencies by roughly a factor of two. The biggest difference is that the model fails to reproduce the observed autumnal nucleation events. This is more realistically captured with REMO-NCH, which overestimates the values for the first half of the year, but is in www.atmos-chem-phys.net/14/11711/2014/ 11719 good agreement with measurements otherwise. Similarly, the missing autumn nucleation in REMO-OHP can be seen at Pallas. There, REMO-OHP does not underestimate the values as much as at Värriö. Besides autumn, only the spring of 2003 is underestimated; otherwise, values are close to measurements. REMO-NCH has similar behaviour at Pallas as at Värriö, although the overestimation is slightly more frequent. Autumn nucleation events also seem to be a problem for REMO-OHP at Vavihill. In addition, the winter nucleation is underestimated or missing, but otherwise the model is able to reproduce the event fractions realistically. REMO-NCH is able to get the late-winter events, but overestimates the summer values. Moreover, the autumn is better captured with REMO-NCH than REMO-OHP. It is not clear why the autumn nucleation is missing from the simulated climate. In order to rule out problems in the nucleation classification method, the banana plots showing the evolution of aerosol size distribution during the day were studied (details not shown here). The banana plots did not show any clear nucleation events during autumn, which means that the classification does work. There are few candidates to explain why the autumn time nucleation is not captured by the model. It is possible that the sulfuric acid concentrations are too low. This is supported by the earlier study on black carbon concentration over Finland by Hienola et al. (2013), who reported deficiencies in the used emission database. Although the analysis in their study was done for black carbon, the database could also have similar problems for other species, such as SO2 . Higher-resolution data (spatial and temporal) could help to improve the sulfuric acid concentrations, especially at remote places like Värriö and Pallas, where small concentration changes could have a big impact on nucleation. On the other hand, the kinetic nucleation scheme employed in the model may well be too simple. Taking into account condensable organics could improve the results (Andreae, 2013). Also, the kinetic coefficient used should ideally not be treated as a constant, as the nucleation rates probably vary with meteorological parameters and some chemical species. However, the current level of understanding of the nucleation process does not permit accounting for these factors. 3.3.2 Years 2008 and 2009 For 2008 and 2009 the simulations are conducted only with REMO-OHP. As the previous sections have shown, REMONCH produces too-high nucleation rates and event frequencies. For this reason, in Fig. 6, only the REMO-OHP model run is shown. At Hyytiälä, REMO-OHP shows that the predicted nucleation events in springtime are underestimated, during summer some overestimation can be seen and in autumn the nucleation seems to be missing. Nevertheless, the yearly cycle is captured (autumn excluded) and the values are reasonably close to the measurements. In Melpitz, the model Atmos. Chem. Phys., 14, 11711–11729, 2014 1.0 REMO-OHP REMO-NCH Mace Head 2003-2004 Yoon et al. (2006) ∆ Yoon et al. (2006) 0.8 0.6 0.4 0.2 Fraction of event days J.-P. Pietikäinen: Modelling European boundary layer nucleation Fraction of event days 11720 0.0 1.2 0.8 0.6 0.4 0.2 0.0 0.3 0.2 0.1 0.0 0.5 REMO-OHP REMO-NCH Pallas 2003-2004 Dal Maso et al. (2007) 0.4 0.3 0.2 0.1 0.0 n b r r y n l g p ct v c n b r r y n l g p ct v c Ja Fe Ma Ap Ma Ju Ju Au Se O No De Ja Fe Ma Ap Ma Ju Ju Au Se O No De Fraction of event days n b r r y n l g p ct v c n b r r y n l g p ct v c Ja Fe Ma Ap Ma Ju Ju Au Se O No De Ja Fe Ma Ap Ma Ju Ju Au Se O No De Fraction of event days Fraction of event days REMO-OHP REMO-NCH Värriö 2003-2004 Dal Maso et al. (2007) 0.4 1.2 Hohenpeißenberg 2003-2004 Birmili et al. (2003) 1.0 n b r r y n l g p ct v c n b r r y n l g p ct v c Ja Fe Ma Ap Ma Ju Ju Au Se O No De Ja Fe Ma Ap Ma Ju Ju Au Se O No De 0.5 REMO-OHP REMO-NCH REMO-OHP REMO-NCH n b r r y n l g p ct v c n b r r y n l g p ct v c Ja Fe Ma Ap Ma Ju Ju Au Se O No De Ja Fe Ma Ap Ma Ju Ju Au Se O No De Vavihill 2003-2004 Kristensson et al. (2011) 1.0 0.8 0.6 0.4 0.2 0.0 n b r r y n l g p ct v c n b r r y n l g p ct v c Ja Fe Ma Ap Ma Ju Ju Au Se O No De Ja Fe Ma Ap Ma Ju Ju Au Se O No De Figure 5. The fraction of days with NPF events, 2003–2004, at five European observation sites on a monthly basis. underestimates the fraction of events, while the analysis for 2003 and 2004 showed overestimations (Fig. 4). The underestimation is fairly strong for both years. The yearly cycle is captured, although the winter events are missing. The emission database used is for the year 2000 (Dentener et al., 2006), and it is surprising that the model is underestimating the 2008 and 2009 result, because the SO2 emissions are known to have decreased over the last 2–3 decades (Hamed et al., 2010, and references therein). On the other hand, this could imply the same possible cause described in the previous section: the nucleation scheme used needs to have more input parameters in terms of other compounds. For San Pietro Capofiume, data coverage from the literature is quite limited. Still, the same features as for 2003 and 2004 can be seen: the model overestimates the number of nucleation events. At Mace Head, the results show similar underestimation as in 2003 and 2004. The results from REMOOHP at Hohenpeißenberg for 2003 and 2004 were very close to measurements. For 2008 and 2009, the model does not capture all the events. Again, taking into account the emission reductions for sulfuric species, this result is surprising. It appears that, although sulfuric acid can be considered the main driver for nucleation, the simplistic approach using it as the only participating species should be improved. The same applies to Pallas, where similar underestimation can be seen. At Vavihill, the model can reproduce the measured values better, although it has a slightly underestimating bias. The Finokalia results show large overestimations in spring, summer and autumn. In winter, the model tends to underesAtmos. Chem. Phys., 14, 11711–11729, 2014 timate the results when compared to both literature sources. The reason for the overestimation could stem from too-high solar radiation levels in the model. The model cloudiness was, therefore, compared against ERA-Interim data, but no clear bias was found. Another possible reason could be the DMS and OH concentrations. As mentioned in Sect. 2.2, DMS is oxidized by OH during the day time. The location of Finokalia provides enough sunlight for OH; so, if these two are overestimated, the nucleation will show patterns similar to Fig. 6. The influence of other sulfuric acid sources cannot be excluded; but, taking the Finokalia location into account, the combination of overestimated DMS and OH appears to be the most credible explanation. Also, the proxy is quite simple and the results from Finokalia show that more input parameters should be employed in order to get a better representation of the regional characteristics. At Cabauw, the model predicts a yearly nucleation maximum during the spring, whereas, in measurements, it is in the summer. The modelled values are slightly lower than the measured, and the autumn peaks are missing. At K-Puszta, the values are closer to the measurements. For summer, the nucleation event frequency is even overestimated. The measurements show that the yearly maximum should be during springtime, whereas in the model, the peak occurs in summertime. The measurements show that it should be during the springtime. Overall, the values are quite realistic and of the same magnitude as the measurements. www.atmos-chem-phys.net/14/11711/2014/ Fraction of event days 1.0 REMO-OHP Hyytiälä 2008-2009 Manninen et al. (2010) 0.8 0.6 0.4 0.2 Fraction of event days J.-P. Pietikäinen: Modelling European boundary layer nucleation 0.0 REMO-OHP 0.8 0.6 0.4 0.2 0.0 n b r r y n l g p ct v c n b r r y n l g p ct v c Ja Fe Ma Ap Ma Ju Ju Au Se O No De Ja Fe Ma Ap Ma Ju Ju Au Se O No De 1.0 0.8 0.6 0.4 0.2 Fraction of event days Fraction of event days San Pietro Capofiume 2008-2009 Manninen et al. (2010) REMO-OHP 0.0 REMO-OHP 0.8 0.6 0.4 0.2 0.0 0.6 0.4 0.2 n b r r y n l g p ct v c n b r r y n l g p ct v c Ja Fe Ma Ap Ma Ju Ju Au Se O No De Ja Fe Ma Ap Ma Ju Ju Au Se O No De Fraction of event days Fraction of event days Hohenpeißenberg 2008-2009 Manninen et al. (2010) REMO-OHP 0.8 0.0 REMO-OHP 0.4 0.3 0.2 0.1 0.0 n b r r y n l g p ct v c n b r r y n l g p ct v c Ja Fe Ma Ap Ma Ju Ju Au Se O No De Ja Fe Ma Ap Ma Ju Ju Au Se O No De 1.0 0.8 0.6 0.4 0.2 Fraction of event days Fraction of event days REMO-OHP Vavihill 2008-2009 Manninen et al. (2010) 0.0 1.0 0.6 0.4 0.2 0.0 REMO-OHP Cabauw 2008-2009 Manninen et al. (2010) 1.0 0.8 0.6 0.4 0.2 n b r r y n l g p ct v c n b r r y n l g p ct v c Ja Fe Ma Ap Ma Ju Ju Au Se O No De Ja Fe Ma Ap Ma Ju Ju Au Se O No De Fraction of event days Fraction of event days Finokalia 2008-2009 Pikridas et al. (2012) REMO-OHP Manninen et al. (2010) 0.8 n b r r y n l g p ct v c n b r r y n l g p ct v c Ja Fe Ma Ap Ma Ju Ju Au Se O No De Ja Fe Ma Ap Ma Ju Ju Au Se O No De 0.0 REMO-OHP K-Puszta 2008-2009 Manninen et al. (2010) 1.0 0.8 0.6 0.4 0.2 0.0 n b r r y n l g p ct v c n b r r y n l g p ct v c Ja Fe Ma Ap Ma Ju Ju Au Se O No De Ja Fe Ma Ap Ma Ju Ju Au Se O No De Puy de Dôme 2008-2009 Boulon et al. (2011) REMO-OHP Manninen et al. (2010) 0.6 0.4 0.2 0.0 n b r r y n l g p ct v c n b r r y n l g p ct v c Ja Fe Ma Ap Ma Ju Ju Au Se O No De Ja Fe Ma Ap Ma Ju Ju Au Se O No De 0.8 Fraction of event days Fraction of event days Pallas 2008-2009 Manninen et al. (2010) 0.5 n b r r y n l g p ct v c n b r r y n l g p ct v c Ja Fe Ma Ap Ma Ju Ju Au Se O No De Ja Fe Ma Ap Ma Ju Ju Au Se O No De 0.8 Mace Head 2008-2009 Manninen et al. (2010) 1.0 n b r r y n l g p ct v c n b r r y n l g p ct v c Ja Fe Ma Ap Ma Ju Ju Au Se O No De Ja Fe Ma Ap Ma Ju Ju Au Se O No De 1.0 Melpitz 2008-2009 Manninen et al. (2010) 1.0 n b r r y n l g p ct v c n b r r y n l g p ct v c Ja Fe Ma Ap Ma Ju Ju Au Se O No De Ja Fe Ma Ap Ma Ju Ju Au Se O No De 1.0 11721 0.7 0.6 Jungfraujoch 2008-2009 Boulon et al. (2010) REMO-OHP Manninen et al. (2010) 0.5 0.4 0.3 0.2 0.1 0.0 n b r r y n l g p ct v c n b r r y n l g p ct v c Ja Fe Ma Ap Ma Ju Ju Au Se O No De Ja Fe Ma Ap Ma Ju Ju Au Se O No De n b r r y n l g p ct v c n b r r y n l g p ct v c Ja Fe Ma Ap Ma Ju Ju Au Se O No De Ja Fe Ma Ap Ma Ju Ju Au Se O No De Figure 6. The fraction of days with NPF events, 2008–2009, at 12 European observation sites on a monthly resolution. Puy de Dôme is a location where the model gives very realistic results. The overall tendency is slightly underestimated, but the yearly cycle is well captured. This also holds true for Jungfraujoch, although there the model produces some overestimation. Overall, these results are very good considering the mountainous location, which are known to be difficult for the model dynamics (Pietikäinen et al., 2012). www.atmos-chem-phys.net/14/11711/2014/ 3.4 Vertical extent of nucleation Figure 7 shows example periods of modelled nucleation at Hyytiälä, Melpitz and San Pietro Capofiume. The nucleation events are strong at Hyytiälä, but the growth seems to be missing. There are at least two possible explanations for this: the model lacks condensable organics, and the representation of the aerosol population with seven log-normal modes leads to problems, as is shown by Korhola et al. (2013). In the latter case, the particles grow due to the condensation Atmos. Chem. Phys., 14, 11711–11729, 2014 J.-P. Pietikäinen: Modelling European boundary layer nucleation 10 4000 3500 3000 2500 2000 1500 1000 500 0 06/07 08/07 10/07 12/07 14/07 06/07 08/07 10/07 12/07 14/07 100 Date h [m.a.s.l.] 12/02 28/05 30/05 01/06 03/05 100 10000 1000 26/05 28/05 30/05 01/06 03/05 100 14/02 16/02 D [nm] dn/dlog10 (D) [cm−3 ] 10 5 10/02 26/05 100 10000 1000 08/02 10/02 12/02 14/02 16/02 Ntot [cm−3 ] D [nm] 1000 08/02 4000 3500 3000 2500 2000 1500 1000 500 0 10000 100 4000 3500 3000 2500 2000 1500 1000 500 0 1000 Date SAN PIETRO CAPOFIUME 2004 1000 100 3 10000 1000 10000 10 100 h [m.a.s.l.] h [m.a.s.l.] 3 dn/dlog10 (D) [cm−3 ] 1000 Ntot [cm−3 ] D [nm] 100 MELPITZ 2003 1000 10000 dn/dlog10 (D) [cm−3 ] HYYTIÄLÄ 2004 1000 Ntot [cm−3 ] 11722 100 Date Figure 7. Nucleation events and total number concentration from Hyytiälä (5 to 15 July 2004), Melpitz (25 May to 4 June 2003) and San Fig. 7. Nucleation events and total number concentration from Hyytiälä (5 to 15 July 2004), Melpitz (25 May to 4 June 2003) and San Pietro Pietro Capofiume (7 to 17 February 2004). The black line shows the height of the boundary layer. Capofiume (7 to 17 February 2004). The black line shows the height of the boundary layer. of sulfuric acid and coagulation, but the mode structure is unable to show this as a continuous phenomenon. Instead, Fig. 7 shows how the particles have “moved” directly to Aitken/accumulation mode sizes. The vertical evolution of events reveals that, at Hyytiälä, nucleation takes place mostly inside the boundary layer. In some cases, the concentrations above the boundary layer are also very high. This is a known phenomena in ECHAM5HAM (Kazil et al., 2010) and has also been shown to exist REMO-HAM (Pietikäinen et al., 2012). In addition, the OH proxy is a function of radiation and is based on surface measurements. This might cause some error at higher altitudes. At Melpitz, the nucleation bursts are much stronger than at Hyytiälä (Fig. 7). It is noticeable that, during the night time, the accumulation mode number concentration increases. This happens when the particles in Aitken mode coagulate with the accumulation mode particles. As mentioned before, the model does not have an online SOA module, which means that the only condensable species is sulfuric acid. During the night, the H2 SO4 concentrations are low, so only coagulation is active. As the accumulation and coarse modes do not co- Atmos. Chem. Phys., 14, 11711–11729, 2014 agulate in M7, the number concentration starts to increase. Like at Hyytiälä (although shown much more clearly), the Aitken/accumulation mode is flushed away during the morning. This can be also seen from measurements (not shown here). The reason for this is the boundary layer mixing during the morning, which is caused by solar heating. At the same time, nucleation bursts can be seen. Vertically the situation is similar to that at Hyytiälä: in some cases, nucleation bursts exceed the boundary layer. There are also some high number concentrations well above the boundary layer height. This can be explained by convective clouds: the vertical transport moves SO2 and H2 SO4 to the mid and upper troposphere. There, the gases have the potential to trigger nucleation and, eventually, the particles will come down (Kazil et al., 2006). In the model, all the gas-phase sulfate is assumed to condense to cloud droplets in stratiform clouds, but this is missing for convective clouds, because the grid box cloud fraction is not defined in this case. The wet deposition is calculated in and below convective clouds, but during the vertical transport no gas-phase sulfate is assumed to condense to cloud droplets. www.atmos-chem-phys.net/14/11711/2014/ J.-P. Pietikäinen: Modelling European boundary layer nucleation 11723 Figure 8. The yearly mean nucleation rates. Means are calculated only for data that met the event classification criteria presented in Sect. 3.3. Fig. 8. The yearly mean nucleation rates. Means are calculated only for data that meets the event classification criteria presented in This means that the convective clouds act in the model as an emissions sources (industrial areas, cities, etc.). The ship 3.3. species. effective elevator forSect. the aerosol tracks are also visible from the averaged nucleation values. Laaksonen et al. (2005) reported that, at San Pietro Capofiume, the nucleated particles grow to 100 nm size in 10 h (on average, measurements from 24 March 2002 to 24 August 2004). This fits quite reasonably to our results (Fig. 7). Laaksonen et al. (2005) also showed that the largest particles reach sizes larger than 200 nm by midnight. The model also seems to be able to reproduce this behaviour. During 12–13 February 2004, the influence of precipitation can be seen: almost all of the particles are flushed from the boundary layer. 3.5 Mean nucleation rates in Europe One interesting aspect of climate models is that the spatial extent of nucleation events can be studied. The approach used here is to apply the classification method explained in Sect. 3.3 for all grid boxes in every output step (1 h) and average only these cases. Figure 8 shows the simulated average nucleation rates J3 nm (for periods when event classification criteria is met). On average, nucleation occurs in the model throughout Europe, with “hot spots” of strong nucleation near the peak www.atmos-chem-phys.net/14/11711/2014/ More locally, for example, at Melpitz, the high nucleation rates seem to be linked to big industrial-point SO2 sources (power generation) in the easternmost parts of Germany and neighbouring countries (Czech Republic, Poland). In order to calculate the strongest nucleation events in Europe, J3 nm is averaged for all output steps. Figure 9 shows the seasonal mean values for 2003 and 2004 (results are almost identical for 2008 and 2009 and are, thus, not shown). Nucleation is strongest during the spring and summer, as expected. Again, strong emission sources, as well as ship tracks, can be clearly seen from the maps. During autumn, nucleation rates are low in Fennoscandia, as was also seen in the nucleation event frequency statistics in Sect. 3.3. This may be due to model biases in meteorological conditions (especially cloud cover), emissions, and/or process parametrizations (kinetic nucleation, OH-proxy). 3.6 Spatial extent of events Nucleation events are naturally influenced by meteorological variables. This leads to very different nucleation events on Atmos. Chem. Phys., 14, 11711–11729, 2014 11724 J.-P. Pietikäinen: Modelling European boundary layer nucleation Figure 9. The seasonal mean nucleation rates for 2003 and 2004. Figure 10. Snapshot examples of 6 different European nucleation events. a spatial scale. Figure 10 shows six nucleation event snapshots taken from the years 2008 and 2009. The top left panel (3 March 2008 12:00 UTC) shows how most parts of Europe are without substantial NPF, whereas northern Africa has quite strong events. The top centre panel (16 June 2008 11:00 UTC) shows nucleation happening mostly near the Atmos. Chem. Phys., 14, 11711–11729, 2014 eastern part of Mediterranean Sea. The top right (24 December 2008 10:00 UTC) is an example of weak nucleation. The lower left (1 February 2009 10:00 UTC) shows strong nucleation events over Ukraine and western Russia, whereas western Europe is without events. Almost the opposite is seen in the lower centre (21 April 2009 12:00 UTC) panel, where www.atmos-chem-phys.net/14/11711/2014/ J.-P. Pietikäinen: Modelling European boundary layer nucleation 11725 Table 2. Annual production of nucleated 3 nm particles in the boundary layer. Year San Pietro Capofiume [m−2 ] Europe (land points) [m−2 ] 2003 2004 2008 2009 2.4 × 1015 2.1 × 1015 2.3 × 1015 2.3 × 1015 2.0 × 1015 1.7 × 1015 1.9 × 1015 1.9 × 1015 Figure 11. Monthly nucleated 3 nm particle burden calculated only for the boundary layer. eastern Europe is without nucleation, but western and central Europe are experiencing a strong nucleation event. The last panel on the lower right (16 September 2009 12:00 UTC) shows a situation where central Europe is without nucleation, but western and eastern Europe are having events. These plots show that the nucleation events in the model can go from very local scales to hundreds of kilometres, which is in good agreement with previous studies of the spatial extent of nucleation (for example, over North America by Crippa and Pryor, 2013). 3.7 Boundary layer analysis Using the information of the mean nucleation event length, the number of nucleation days per year and the mean formation rate from Hamed et al. (2007), combined with the height information of a well-mixed boundary layer from Laaksonen et al. (2005), a rough estimate of the yearly number of nucleated 3 nm particles in the boundary layer over San Pietro Capofiume can be calculated: 3.6×1015 m−2 . The equivalent value can be calculated from the model output for the grid box where San Pietro Capofiume is located without any estimations. The results are in Table 2, where the values for San Pietro Capofiume and Europe (only land points) are shown. The values for San Pietro Capofiume are lower than the literature estimate. However, the difference is less than a factor of 2. Both the model and the literature estimates, especially the latter, have a number of possible (unquantified) error sources; therefore, such a difference appears quite reasonable. The monthly production of 3 nm particles in the European boundary layer is shown in Fig. 11. The production has a minimum during the winter and a maximum during the summer. This shows that, overall, the simulated annual cycle of nucleation in the European boundary layer is more similar to that observed in San Pietro Capofiume (summer maximum, winter minimum Hamed et al., 2007) than the cycle in Hyytiälä (spring and autumn maxima Kulmala et al., 2004). www.atmos-chem-phys.net/14/11711/2014/ 4 Conclusions A measurement-based OH proxy was implemented in the regional aerosol–climate model REMO-HAM. This supersedes a former version that used monthly mean fields for OH with an artificial diurnal cycle. The newly implemented proxy is a function of radiation, thus linking the cloudiness of the model to the OH concentrations. In addition, the nucleation rate expression was changed to directly calculate the 3 nm particles (in diameter). Despite some underestimation in different regions, the new model version gives more realistic nucleation rates for 3 nm particles compared to the original model version, which overestimated the observed nucleation rates. Overall, the agreement with observations has been considerably improved. Nucleation event statistics were analysed at 13 different European sites. The results show good agreement at some sites, but for some the yearly cycle was not captured. Also, for many (northern) sites, the OH-proxy model fails to predict nucleation events during autumn, whereas they are frequently observed. A more detailed analysis was done for three measurement sites (Hyytiälä, Melpitz and San Pietro Capofiume). The results show that the monthly means for start time, end time and length of nucleation events are quite well captured. The main problem is that the nucleation in the model tends to continue longer than in observations. The main reason for this can be the missing organic growth of particles, which leads to lower number concentration of particles > 100 nm. This decreases the condensation sink of sulfuric acid and the remaining sulfuric acid will keep the nucleation active for longer period of time. The vertical extension of nucleation events was also analysed. As expected, the events mainly happen inside the boundary layer. Because of the simple form of the proxy, the model simulates nucleation also in the upper troposphere. On the other hand, this feature has been reported also in earlier versions and in the global model ECHAM-HAM (Kazil et al., 2010; Pietikäinen et al., 2012). The distribution plots show that nucleation bursts are realistically captured, but the growth to larger particles is not as continuous as in measurements due to the missing organic condensation and the structure of the modal aerosol model (Korhola et al., 2013). Atmos. Chem. Phys., 14, 11711–11729, 2014 11726 J.-P. Pietikäinen: Modelling European boundary layer nucleation The spatial distribution of nucleation events showed that strongest events occur close to the major sources of sulfur dioxide. It is worth noting that large point sources of SO2 , such as in adjacent East European countries, seem to contribute to the strong nucleation events happening at Melpitz. Seasonally, the trend over Europe is to have strong nucleation during the summer and less during the winter. The same was shown when the total nucleation was calculated in the European boundary layer. Small changes in the simple chemistry module can lead to big improvements in results, as is shown in this study. In addition, using a proxy does not increase the computational burden of the model at all. This makes the approach very useful in aerosol–climate models. To improve the system, more work should be targeted to connect the coefficients used in the proxy with regional features. This could mean, for example, two-dimensional maps for the coefficients. Also, taking into account the seasonal effects, the proxy could provide even more realistic results; this will be studied in a subsequent analysis. The same applies also for the nucleation coefficient (activation/kinetic). The regional meteorological and chemical features play an important role in shaping the nucleation events. Acknowledgements. This work was supported by the Academy of Finland (Research Programme for Climate Change FICCA, project 140748, and the Center of Excellence Programme, project 1118615), by the strategic funding of the University of Eastern Finland, and by the EU FP7 IP PEGASOS (FP7-ENV-2010/265148). The authors would like to thank T. Nieminen for providing the Hyytiälä event classification data and Declan O’Donnell for proofreading and correcting the English language. Edited by: E. Nemitz References Andreae, M. O.: The aerosol nucleation puzzle, Science, 339, 6122, 911–912, doi:10.1126/science.1233798, 2013. Birmili, W. and Wiedensohler, A.: New particle formation in the continental boundary layer: meteorological and gas phase parameter influence, Geophys. Res. Lett., 27, 3325–3328, 2000. 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https://openalex.org/W2893065453	https://bmcwomenshealth.biomedcentral.com/track/pdf/10.1186/s12905-018-0645-6	English	null	Complications related to induced abortion: a combined retrospective and longitudinal follow-up study	BMC women's health	2,018	cc-by	7,147	Open Access Abstract Background: Induced abortion is one of the most common gynecological procedures in Sweden, but there is still little knowledge about the adverse effects. The aims of this study are to provide an overview of complications of medical and surgical abortions and to evaluate the impact of bacterial screening to prevent postabortal infections. Methods: All women who underwent induced abortion at Skaraborg Hospital between 2008 and 2015 are included in the study. Bacterial screening for chlamydia, gonorrhea, mycoplasma, and bacterial vaginosis was performed prior to the abortions. Abortion complications, categorized as bleeding, infection, or incomplete abortion were assessed in women who came in contact with the gynecological clinic within 30 days after the procedure. Results: A total of 4945 induced abortions were performed during the study period. Nearly all, 4945 (99.7%) were eligible for inclusion in the study. Medical abortions < 12 weeks were the most common procedure (74.7%), followed by surgical abortions (17.5%), and medical abortion > 12 weeks (7.8%). Complications were registered in 333 (6.7%) of all abortions. Among medical abortions < 12 weeks, the complication frequency increased significantly, from 4.2% in 2008 to 8.2% in 2015 (RR 1.49, 95% 1.04–2.15). An incomplete abortion was the most common complication related to medical abortions < 12 weeks. Of all women who tested positive for one or several bacteria at the screening and therefore received antibiotics, 1.4% developed a postabortal infection. Among those who tested negative at the screening, 1.7% developed infectious complications. Conclusions: The share of complications related to medical abortions < 12 weeks increased significantly during 2008–2015 without any evident cause. Women who tested positive for one or several bacteria upon screening and received antibiotics experienced almost an equal proportion of postabortal infections compared to women who tested negative upon screening. The screening process seems to fulfill its purpose of reducing the risk of infectious complications. Keywords: Induced abortion, Pregnancy, Endometritis, Vacuum curettage, Sexually transmitted diseases method was a medical abortion, which was used in 88% of all cases. [1] Early medical abortions (< 9 weeks of gesta- tion) were introduced in Sweden 1992 when Mifepristone became available [2]. Before that, surgical abortions were the only legal option < 12 gestational weeks. © 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. Complications related to induced abortion: a combined retrospective and longitudinal follow-up study Isabelle Carlsson1* , Karin Breding2 and P.-G. Larsson2,3 Background An induced abortion is one of the most common gynecological procedures in Sweden. There were 36,600 induced abortions reported to The National Board of Health and Welfare (Socialstyrelsen) during 2014. The majority (93%) of which, were performed before the 12th week of pregnancy. The rate of abortions was 20 per 1000 women by the age of 15–44. The most common Despite well-developed abortion methods, there are known risks and adverse effects that must be considered. Potential complications related to abortions include pain, bleeding, an incomplete abortion, or an infection in the upper genital tract that causes endometritis, oophoritis, parametritis, and salpingitis. In Finland, in the upper genital tract that causes endomet oophoritis, parametritis, and salpingitis. In Finl * Correspondence: isabelle.carlsson@vgregion.se 1Skaraborg Hospital Skövde, Lövängsvägen, Skövde, Sweden 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. * Correspondence: isabelle.carlsson@vgregion.se 1Skaraborg Hospital Skövde, Lövängsvägen, Skövde, Sweden Full list of author information is available at the end of the article * Correspondence: isabelle.carlsson@vgregion.se 1Skaraborg Hospital Skövde, Lövängsvägen, Skövde, Sweden Full list of author information is available at the end of the article Carlsson et al. BMC Women's Health (2018) 18:158 https://doi.org/10.1186/s12905-018-0645-6 Carlsson et al. BMC Women's Health (2018) 18:158 https://doi.org/10.1186/s12905-018-0645-6 Methods This is a combined retrospective and prospective follow-up study of women who had an induced abor- tion at Skaraborg Hospital between 2008 and 2015. From 2011 all complications are prospectively regis- tered. Skaraborg Hospital consists of the hospitals in Skövde, Lidköping, Mariestad, and Falköping. Skara- borg Hospital system is the only provider of abortions in the area. The abortions were managed by the gynecological clinics at all four hospitals. Lidköping hospital was however not included until 2013 when it became part of Skaraborg Hospital. Before that it was a separate hospital that managed both medical and surgical abortions. Lidköping Hospital is located a 1 hour drive from Skövde Hospital. The reported incidence of postabortal infections varies between studies, likely depending on local differences in diagnostics, the study population, laws and regulations, and the prevalence of sexually transmitted diseases. In Skaraborg, where the present study takes place, the number of postabortal infections was evaluated by Char- onis and Larsson in 2006 [5]. They reported a frequency of 2.4% for infectious complications from medical abor- tions and 4.9% from surgical abortions. Another study conducted in Sweden and Norway investigated the infec- tion rate after surgical abortions. They reported infec- tions among 4.8% of the patients [6]. These patients had not received any prophylactic antibiotics. Several studies have shown that pre-abortion treatment for BV [6, 7] is effective in reducing the rate of postabortal infections. Women considering an induced abortion were seen by a gynecologist at the gynecological clinic. The length of the pregnancy was established through a vaginal ultra- sound by measuring the crown rump length (CRL). The gestational age was reported as weeks + days (7 + 3 etc.). The doctor also performed a gynecological examination, and samples were taken for bacterial screening. The bac- terial screening program at Skaraborg Hospital included C. trachomatis, N. gonorrhoeae, M. genitalium, and bac- terial vaginosis. Women who did not proceed with an abortion are not included in the study. For most women, medical abortion at home was the recommended pro- cedure. Medical abortion completed in the hospital was recommended for women who were young, nulliparous, or more than 12 weeks gestation. Surgical abortion, using vacuum curettage, was performed if the women demanded this or had difficulties understanding the in- formation about medical abortion. Carlsson et al. BMC Women's Health (2018) 18:158 Carlsson et al. BMC Women's Health (2018) 18:158 Page 2 of 9 In that way, it has a positive impact on contamination and reinfection from sexual partners. Niinimäki et al. investigated the rate of complications re- lated to medical abortions. Of the 24,006 adult partici- pants who underwent a medical abortion, 15.4% were later diagnosed with bleeding, 2.0% experienced an in- fection, 10.2% experienced an incomplete abortion, and 13.0% had to proceed with a vacuum curettage [3]. The main purpose of this study was to provide an overview of complications related to induced abortion and to investigate the difference in complications be- tween medical and surgical abortions. The second pur- pose was to evaluate the impact of bacterial screening on the rate of postabortal infections. Infections related to abortions are often caused by an ascending bacterial infection such as chlamydia, gonor- rhea, mycoplasma and bacterial vaginosis (BV) that pro- ceeds from the lower genitals and moves through the cervix to the uterus [4]. The infection, if untreated, can spread to the fallopian tubes and may lead to infertility. Page 2 of 9 Outcome definitions An infection in this context is referred to as postabortal endometritis, which is an inflammation in the uterus caused by a bacterial infection. To be categorized as an infection, a patient must have had symptoms of infection and received antibiotics from a doctor. The symptoms may be bleeding or discharge, lower abdominal pains, a fever, and/or an elevated CRP (C-reactive protein). An infection can sometimes be caused by an incomplete abortion. If an infection and an incomplete abortion occurred at the same time, it was categorized as an incomplete abortion. p Women who had medical abortions < 12 weeks were informed that a woman should expect heavy bleeding 4–12 h after taking of the misoprostol and then bleeding similar to a heavy period for the next 2–5 days. Women who had a surgical abortion were informed to expect a bleeding for 1 week. All women were scheduled for a follow-up visit with a midwife approximately 1 month after the abortion. The main purpose of the follow-up visit was to ensure that the pregnancy had been terminated properly by analyzing the levels of human chorionic gonadotropin hormone in the urine and to discuss contraceptive methods. Complications in the other hand were always handled by a doctor. Women were encouraged to contact one of the gynecological clinics at Skaraborg Hospital for gynecological evaluation with a doctor if they experi- enced significant bleeding after 7 days, abdominal pains, fever or malodorous discharge/bleeding after the abortion. Bleeding is often a part of the normal course of an abortion. Only heavy bleeding lasting longer than 12 h or longstanding bleeding (> 21 days) was categorized as a bleeding complication. The diagnosis of bleeding complications was set by the doctor when the patients came to the gynecological department. Furthermore, to categorize the complication as bleeding, the patient could not have had a simultaneous infection or incom- plete abortion. If the vaginal ultrasound revealed uterine contents of 15 mm or more, resembling placental tissue, treatment with mifepristone was performed. If the bleeding stopped with this treatment, it was classified as a bleed- ing complication. However, if any significant evidence of retained products of conception were detected on ultra- sound scan, vacuum curettage was scheduled and the complication was defined as an incomplete abortion. Women with an incomplete abortion and simultaneous signs of infection were counted as incomplete abortions since incomplete abortions are a natural cause of infections. Statistical analysis The data were described using frequencies and propor- tions. SPSS version 22 was used for calculating a relative risk with a 95% confidence interval. Carlsson et al. BMC Women's Health (2018) 18:158 Carlsson et al. BMC Women's Health (2018) 18:158 Page 3 of 9 Page 3 of 9 vaginally and then 2 orally every 3rd hour until abortion. Medical abortions > 12 weeks are often completed by a vacuum curettage as part of the normal course. A vacuum curettage is in these cases not counted as treat- ment for a complication. Outcome definitions During 2008–2010, abortion records were collected retrospectively. Data collected included the number of previous induced and spontaneous abortions, parity, length of current pregnancy, results of bacterial screen- ing, prescribed antibiotic treatment, and the chosen abortion method. Women were followed through the pa- tient records system to determine the rate of complica- tions. All patients who had a visit with a doctor at the gynaecological clinic within 30 days after the abortion were included. The cause of the visit was determined through review of patient records and entered into the spreadsheet as an infection, bleeding, incomplete abortion, or other (e.g contraceptive consulting (not a complication)). In abortions, after 12th week of gestation, the remaining placental tissue can be treated with extra mifepristone or vacuum curettage. However, this is not considered a complication if this is done during the hos- pital visit. If the patients return after discharge from the hospital with retained placental tissue, this is regarded as an incomplete abortion complication. From 2011 to 2015, data from the abortion complications were collected prospectively by an admin- istrator at the gynecological clinic. If a patient visited the gynecological clinic within two calendar months of the abortion, paper copies of the patient records were made and then given to one of the gynecologists. The gynecologist evaluated the time and the cause of the visit (infection, bleeding, incomplete abortion, or other) by reading the patient records and reported back so that complications within 30 days from the abortion could be recorded. Methods A surgical abortion, also called vacuum curettage, is allowed up to 12 + 0 gestational weeks but is not recommended before the 7th week because of the risk of an incomplete abortion. The World Health organization (WHO) recommends antibiotic prophylaxis to prevent infectious complica- tions associated with abortions. However, the WHO points out that there is only evidence for prophylactic antibiotics performing a surgical abortion when the risk of infections is more evident [8]. Additionally, antibiotic prophylaxis for all women undergoing abortion procedures likely results in overuse of antibiotics, which is associated with antibiotic resistance [9]. To address the problem of antibiotic overuse, it has been sug- gested that a screen and treat method may be more appropriate [10]. When using the screen and treat method, all patients undergoing abortion procedures will be tested for a number of selected bacteria. Antibiotic treatment is given if/when a bacterial infection is identified, which could be before, during, or after the abortion. Prior re- search indicated that the timing of antibiotic administra- tion relative to the timing of the abortion does not affect the rate of postabortal infection [5–7]. The screen and treat strategy has several benefits. It enables targeted antibiotic treatment, which helps in reducing the use of antibiotics. This counteracts the negative aspects of anti- biotic treatment, such as drug reactions and the develop- ment of antibiotic resistance among bacterial strains. The screening process also identifies patients with sexu- ally transmitted diseases, which enables contact tracing. A medical abortion < 12 weeks is initiated by giving the patient mifepristone (Mifegyne ®) 200 mg × 1 orally day one to stop the development of the pregnancy. The treatment continues on the third day with misoprostol (Cytotec ®) 0.2 mg × 4 vaginally, which will help the uterus contract and expel the pregnancy. A medical abortion that is performed after 12 gestational weeks (12 + 1) takes place at the hospital. The protocol is the same with mifepristone and the misoprostol first 4 Carlsson et al. BMC Women's Health (2018) 18:158 Carlsson et al. BMC Women's Health (2018) 18:158 Page 4 of 9 Page 4 of 9 The number of induced abortions gradually increased throughout the study, from 533 to 805 abortions per year (Fig. 1; Table 1). Medical abortions < 12 weeks were the most frequently used method. The share of medical abortions < 12 weeks increased from 58.2 to 86.6% between 2008 and 2015 while the share of surgical abor- tions (31.1% vs 6.8%) and medical abortions > 12 weeks (10.7% vs 6.6%) has decreased. difference between patients undergoing abortion in 2008–2010 and 2015. The distribution of medical abor- tions by gestational weeks was examined. There was an increase in the proportion of medical abortions < 7 + 0 gestational weeks from 51.2% in 2008–2010 to 57.1% in 2015. The number of medical abortions at 9–12 gestational weeks also increased from 0.2% in 2008– 2010 to 7.9% in 2015. Over the course of the study, complications occurred in 6.7% (n = 333) of all induced abortions. Table 2 pre- sents data on postabortal complications by method. Complications occurred in 7.3% of the medical abortions < 12 weeks. Over time, the rate of complications for medical abortions < 12 weeks increased from 4.2% in 2008 to 8.2% in 2015 (Fig. 2). This difference was signifi- cant (RR 1.49, 95% 1.04–2.15). The most common com- plication (57%) related to medical abortions < 12 weeks was incomplete abortions, which occurred 153 times, comprising 4.1% of all medical abortions. The frequency of infections related to medical abortions < 12 weeks was only 1.2%. Table 3 compares the proportion of complications re- lated to medical abortions during different gestational weeks. The rate of complications rose in abortions per- formed before the 7th week (RR 1.21 (95% CI 0.71–2.06) and between the 7th and 9th weeks (RR 1.62 (95% CI 0.94–2.78), but no one of these increases are significant separately. The share of medical abortions performed at home was also calculated. During 2008–2010, 74.6% of all medical abortions before the 9th gestational week were carried out at home compared with 85.2% during 2015. This is an increase of just over 10%. All medical abortions < 9 weeks during 2015 were sep- arated by the location of the abortion (Table 4). Carlsson et al. BMC Women's Health (2018) 18:158 Medical abortions at the hospital between 9 and 12 gestational weeks were not included in this calculation because home abortions are not an option after 9th week. The purpose was to investigate whether there was any differ- ence in the number of complications between medical abortions performed at home or at the hospital. The complication frequency was significantly higher among women < 7 gestational weeks who had their abortions at home (RR 2.99 (95% CI 0.42–21.4)). There were 864 surgical abortions and 45 cases of complications, which gives a total complication fre- quency of 5.2% (Table 2; Fig. 3). There was an increase in the share of complications during 2011–2013 with a maximum of 8.0% during 2012. Infections were the most common complications related to surgical abortions, representing more than half of the postoperative compli- cations. Bleeding was a rare complication; only 4 cases of abnormal bleeding following surgical abortions were observed. Only 385 abortions were performed after the 12th week during 2008–2015. In 18 of them, there were complications afterwards, which represent a complica- tion frequency of 4.7%. Nine were incomplete abortions, six were infections, and three were bleeding. The prevalence of chlamydia, mycoplasma, and bacter- ial vaginosis is illustrated in Fig. 4. The bacterial screen- ing detected 116 chlamydial infections during 2008– 2015. The prevalence of chlamydia among women undergoing abortions varied between 1.0–3.0% per year. M. genitalium was found in 132 cases and had a total prevalence of 2.7%. Bacterial vaginosis was the most common infection, with 775 cases. The prevalence of Since there was a significant increase in complications related to medical abortions < 12 weeks, a comparison was made to determine whether there is any evident Fig. 1 Number of induced abortions per year from 2008 to 2015 at Skaraborg Hospital. The total number of abortions per year is illustrated together with the three different abortion methods: medical abortion > 12 weeks, medical abortion < 12 weeks and surgical abortion. Data from 2008 to 2012 include the clinics in Skövde, Falköping, and Mariestad. Lidköping’s hospital was added to the statistics in 2013 Fig. 1 Number of induced abortions per year from 2008 to 2015 at Skaraborg Hospital. The total number of abortions per year is illustrated together with the three different abortion methods: medical abortion > 12 weeks, medical abortion < 12 weeks and surgical abortion. Results A total of 4945 induced abortions were performed dur- ing the study period. Sixteen patients were excluded, resulting in 4945 induced abortions included in the study. The mean age of the women receiving abortions was 26 years and half were nulliparous (53.9%). The majority of women (63.5%) had not had a prior induced abortion (range 0–6 induced abortions). Skaraborgs hospital is located in a rural area. As such, there are very few alternatives to seek for medical treatment except going back to the hospital. The hospital has three outpatient clinics using the same computer medical record. Carlsson et al. BMC Women's Health (2018) 18:158 Data from 2008 to 2012 include the clinics in Skövde, Falköping, and Mariestad. Lidköping’s hospital was added to the statistics in 2013 Fig. 1 Number of induced abortions per year from 2008 to 2015 at Skaraborg Hospital. The total number of abortions per year is illustrated together with the three different abortion methods: medical abortion > 12 weeks, medical abortion < 12 weeks and surgical abortion. Data from 2008 to 2012 include the clinics in Skövde, Falköping, and Mariestad. Lidköping’s hospital was added to the statistics in 2013 Carlsson et al. BMC Women's Health (2018) 18:158 Page 5 of 9 Table 1 All induced abortions at Skaraborg Hospital in 2008–2015. This table shows the number and share of induced abortions performed by the three abortion methods 2008 2009 2010 2011 2012 2013 2014 2015 Total n (%) Medical Abortions < 12 weeks 310 (58.2) 311 (61.8) 311 (65.3) 390 (72.4) 432 (75.1) 628 (82.4) 617 (82.0) 697 (86.6) 3696 (74.7) Surgical Abortions 166 (31.1) 144 (28.6) 133 (27.9) 99 (18.4) 88 (15.3) 82 (10.8) 97 (12.9) 55 (6.8) 864 (17.5) Medical Abortions > 12 weeks 57 (10.7) 48 (9.5) 32 (6.7) 50 (9.3) 55 (9.6) 52 (6.8) 38 (5.1) 53 (6.6) 385 (7.8) Total Abortions 533 (10.8) 503 (10.2) 476 (9.6) 539 (10.9) 575 (11.6) 762 (15.4) 752 (15.2) 805 (16.3) 4945 bacterial vaginosis varied between 12.4–19.5% per year. Only one patient was diagnosed with gonorrhea during the study and therefore is not included in the figure. including only surgical abortions, 1.1% had an infec- tion after testing positive at the screening. Among those who did not have a bacterial infection upon screening, 3.2% developed a postabortal infection. This difference was not statistically significant (OR 0.330, 95% 0.042–2.592). The presence of coinfections was investigated in data collected from 2008 to 2010. A correlation was found between bacterial vaginosis and chlamydia. Among pa- tients infected with chlamydia, 24.3% also had bacterial vaginosis (RR 1.56 (95% CI 1.12–2.17)). Among those who tested negative for chlamydia, 16.5% had bacterial vaginosis. An even stronger correlation was found be- tween bacterial vaginosis and mycoplasma. Of all pa- tients who tested positive for mycoplasma, 37.5% had bacterial vaginosis compared with 16.0% among women without mycoplasma (RR 2.46 (95% CI 1.95–3.10)). No correlation was found between chlamydia and myco- plasma. They affected the same age group but different individuals. Excluded patients Of all 4961 induced abortions, 16 were excluded from the study. Six were failed medical abortions in which the pregnancy had proceeded. Four patients were excluded since they underwent diagnostic laparoscopy for sus- pected ectopic pregnancy at the same time as a vacuum curettage. One patient was excluded because she did not contact the gynecological clinic until the 38th day after her abortion. She was later diagnosed with an incom- plete abortion and had a vacuum curettage. One patient terminated her pregnancy by a hysterectomy and was therefore excluded. This patient had been treated earlier with endometrial ablation for menorrhagia. She became pregnant after several years of amenorrhea, and the pregnancy was not discovered until the 18th week. Of all women who tested positive for one or several bacteria at the screening and therefore received anti- biotics, 1.4% developed a postabortal infection. Among those who tested negative at the screening 1.7% developed infectious complications. When Table 2 The relative number and share of complications in each abortion method between 2008 and 2015. The complications are categorized as incomplete abortion, bleeding or infection 2008 2009 2010 2011 2012 2013 2014 2015 Total n (%) Complications following Medical Abortions < 12 weeks 13 (4.2) 13 (4.2) 25 (8.0) 24 (6.2) 36 (8.3) 55 (8.8) 47 (7.6) 57 (8.2) 270 (7.3) Incomplete Abortions 8 6 17 11 17 32 27 35 153 Bleeding 4 5 3 7 13 14 12 14 72 Infections 1 2 5 6 6 9 8 8 45 Complications following Surgical Abortions 11 (6.6%) 6 (4.2%) 4 (3.0%) 7 7.1%) 7 (8.0%) 6 (7.3%) 2 (2.1%) 2 (3.6%) 45 (5.2%) Incomplete Abortions 5 3 1 0 2 2 1 1 15 Bleeding 0 0 0 2 1 1 0 0 4 Infections 6 3 3 5 4 3 1 1 26 Complications following Medical Abortions > 12 weeks 2 (3.5%) 4 (8.3%) 2 (6.3%) 3 (6.0%) 2 (3.6%) 1 (1.9%) 3 (7.9%) 1 (1.9%) 18 (4.7%) Incomplete Abortions 0 0 1 2 2 0 3 1 9 Bleeding 0 1 1 1 0 0 0 0 3 Infections 2 3 0 0 0 1 0 0 6 Table 2 The relative number and share of complications in each abortion method between 2008 and 2015. categorized as incomplete abortion, bleeding or infection Carlsson et al. BMC Women's Health (2018) 18:158 Page 6 of 9 Fig. Excluded patients 2 Complication frequency among medical abortions < 12 weeks in Skaraborg i 2008–2015. The diagram shows separate graphs for incomplete abortions, bleeding, infections, and the total complication frequency One patient was excluded because the surgical abor- tion had to be discontinued. The reason was that a false passage to the uterus had been created when dilating the cervix. Subsequently, the pregnancy was terminated successfully by a surgical abortion. One patient was ex- cluded because of an interstitial pregnancy close to the fallopian tube. The patient had an incomplete medical abortion with a subsequent vacuum curettage. Despite this, she had abdominal pains and bleeding. When the interstitial pregnancy was finally discovered, the patient was treated with methotrexate and later, a subtotal hysterectomy. One patient was excluded because her personal code number could not be found in the patient records system. Another patient was excluded since there no information could be found on the length of her pregnancy, her number of previous abortions, and parities. of complications after medical abortions < 12 weeks that was statistically significant. Incomplete abortions were found to be the most common complication after med- ical abortions < 12 weeks. Concerning medical abortions > 12 weeks and surgical abortions, it has been difficult to discern whether there are any trends since the numbers of medical abortions > 12 weeks and surgical abortions are low and the cases of complications are few. The sec- ond aim was to evaluate the impact of bacterial screen- ing on postabortal infections. The frequency of infections after induced abortions appeared to be equal or even lower among patients who had a bacterial infec- tion upon screening and therefore did receive antibiotics. Despite bacterial screening, there were still patients who suffered from infectious complications. When looking at the number of abortions each year (Fig. 1, Table 1), we can see an increase from 2008 when 533 abortions were performed, to 2015 when there were 805 abortions. This increase can primarily be explained by the addition of abortions being performed at Lidköp- ings Hospital from 2013 and forward. But this may not be the only reason. An increase in the number of Discussion When performing a vacuum curettage, there is always a risk that bacteria from the lower genitals will be brought up to the uterus, causing endometritis. Figure 3 shows a peak in the com- plication frequency during 2011–2013. However, accord- ing to Table 2, this increase only consists of a few more cases, so it could be just a coincidence. The total complication frequency after surgical abortions was 5.2%, which is consistent with previous research [5]. There was a significant increase in the share of com- plications related to medical abortions < 12 weeks (RR 1.49, 95% 1.04–2.15). One potential reason is that the proportion of induced abortions performed at home has risen. It is likely that women who have medical abortions at home will visit our outpatient clinic in a greater ex- tent since they do not have the direct help and support from a midwife. Later, they may have been diagnosed with a postabortal complication even though their symp- toms are mild and could have resolved without any treatment. Table 4 shows that abortions at home are as- sociated with a greater proportion of complications, but this is not statistically significant. This is likely due to of the low number of abortions performed at the hospital. The prevalence of chlamydia among women undergo- ing induced abortions was consistent with previous research. One of the latest studies on the subject from Sweden by Bjartling et al. (2010) reported a prevalence of chlamydia of 2.8% compared to our average of 2.3% [4]. Mycoplasma was present in 2.7% of all the patients, which was nearly the same as Bjartling et al. (2010) (2.5%). The prevalence of gonorrhea was very low, as expected. Only one patient had gonorrhea, which is 0.02% of all patients. Bacterial vaginosis seems to be less prevalent in this study compared with others. The preva- lence of bacterial vaginosis was 15.7%, compared with approximately 20% in two other Swedish studies [5, 6]. The number of infections after medical abortions < 12 weeks is 1.2%, which is low compared with Charonis et al.’s (2006) reported frequency of 2.4% [5]. The actual number of infections after medical abortions < 12 weeks in our study is, however, low: only 45 patients in 8 years, which makes the results difficult to validate. Discussion The first aim was to investigate the number of complica- tions associated with induced abortions. The main find- ing after compiling the results was an increasing number Fig. 3 Complication frequency among surgical abortions in Skaraborg in 2008–2015. The diagram shows separate graphs for incomplete abortions, bleeding, infections, and the total complication frequency Fig. 3 Complication frequency among surgical abortions in Skaraborg in 2008–2015. The diagram shows separate graphs for incomplete abortions, bleeding, infections, and the total complication frequency Carlsson et al. BMC Women's Health (2018) 18:158 Page 7 of 9 Page 7 of 9 Table 3 Complication frequency related to medical abortions < 12 weeks separated by different gestational weeks. Comparison between 2008 and 2010 and 2015 Gestational weeks 2008–2010 2015 Total 50/931 (5.4%) 57/697 (8.2%) < 7 + 0 25/477 (5.2%) 27/398 (6.8%) 7 + 1–9 + 0 25/452 (5.5%) 24/244 (9.8%) 9 + 1–12 + 0 0/2 (0.0%) 6/55 (10.9%) Table 3 Complication frequency related to medical abortions < 12 weeks separated by different gestational weeks. Comparison between 2008 and 2010 and 2015 4.1% of the medical abortions < 12 weeks had to be sup- plemented by a vacuum curettage. There were also six failed medical abortions that were excluded from the statistics (see excluded cases). A failed abortion must be interpreted as the worst kind of in- complete abortion. When reading the patient records, it was evident that many patients did not attend the follow-up visits. It is of utmost importance to explain to the patients why this appointment is so essential and to motivate them to come. abortions could be expected at this time because of the high number of children that were born in the early 1990’s [11]. Girls born during that decade should have reached an age of 20–24 years by now, and this is the age where the proportion of induced abortions is the highest. According to a statistical report from The National Board of Health and Welfare [1], the propor- tion of medical abortions has increased the last 15 years, while the proportion of surgical abortions has decreased. This matches our findings in Fig. 1 and Table 1. There were few surgical abortions compared to medical abortions, and the relative number of complica- tions related to surgical abortions was low; most were infections, which we expected. Discussion As many as A comparison was made between all patients who tested positive for one or several bacteria upon screening and received antibiotics and those whose tests were negative. We found that the share of infectious compli- cations was almost the same among patients who had tested positive at the screening. The screen and treat policy, where patients have to wait for the screening re- sults before treatment does not appear to increase the risk and might be a better choice than antibiotic prophy- laxis for all patients. Table 4 Complications related to medical abortions < 12 weeks during 2015 separated by the different gestational weeks and the location of the abortion. The relative number of abortions and complications are presented in each group together with the complication frequency. The complication frequency was higher among women who had their abortions at home, but the difference was not significant (RR 1.30, 95% 0.57–2.97) Gestational weeks At home At the hospital Total 45/547 (8.2%) 12/150 (8.0%) < 7 26/357 (7.3%) 1/41 (2.4%) 7 + 1–9 + 0 19/190 (10.0%) 5/54 (9.3%) 9 + 1–12 + 0 – 6/55 (10.9%) Table 4 Complications related to medical abortions < 12 weeks during 2015 separated by the different gestational weeks and the location of the abortion. The relative number of abortions and complications are presented in each group together with the complication frequency. The complication frequency was higher among women who had their abortions at home, but the difference was not significant (RR 1.30, 95% 0.57–2.97) When including only surgical abortions, there was a greater difference between the two groups. There were fewer infectious complications among patients treated with antibiotics for chlamydia, gonorrhea, mycoplasma, or bacterial vaginosis. This indicates that antibiotics may have a positive impact, beyond treating the screened in- fections. There was, however, no significant difference between these groups, probably because of the low number of surgical abortions. g , Gestational weeks At home At the hospital Total 45/547 (8.2%) 12/150 (8.0%) < 7 26/357 (7.3%) 1/41 (2.4%) 7 + 1–9 + 0 19/190 (10.0%) 5/54 (9.3%) 9 + 1–12 + 0 – 6/55 (10.9%) Carlsson et al. BMC Women's Health (2018) 18:158 Page 8 of 9 Fig. 4 Prevalence of chlamydia, mycoplasma, and bacterial vaginosis upon bacterial screening It is, however, clear that bacterial screening cannot prevent all cases of postabortal infections. Funding g The first part of this project was partly financed by the unit of research and development (FoU) at Skaraborg Hospital. The cowriter Larsson has also received compensation from Astellas Company for holding a one-hour lecture on female incontinence. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Acknowledgements Another limitation is that antibiotic treatment for the screened infections could not always be given prior to the abortion since the analysis for chlamydia, gonorrhea, and mycoplasma takes 2 days. This means that antibi- otics were sometimes given one or 2 days after the day of the abortions. This dilemma primarily affects medical abortions, which are often initiated the same day as the appointment at the gynecologist. Surgical abortions are usually performed after a week or so, which facilitates the timing of the treatment. According to a study by Larsson et al. [7], the time from abortion to infection is usually four to 5 days. We would like to thank all participants at the outpatient ward in Skaraborg Hospital. Abbreviations BV B l BV: Bacterial vaginosis; CI: Confidence interval; CRL: Crown rump length; EPN: Regional ethical review board; PCR: Poly chain reaction; RR: Relative risk; WHO: World Health Organization Conclusions The rate of complications associated with medical abortions < 12 weeks has increased from 4.2% in 2008 to 8.2% in 2015. The cause of this is unknown but it may be associated with a shift from hospital to home medical abortions. Women who tested positive for one or several bacteria upon screening and received antibiotics had almost an equal proportion of postabortal infections as women who tested negative upon screening. The screen- ing process seems to fulfill its purpose of reducing the risk of infectious complications. However, the use of bacterial screening did not eliminate all cases of posta- bortal infections. There are likely other bacteria involved that we do not know about yet. This study has several limitations. The timeline of 30 days regarding the follow-up of the patients proved to have disadvantages. Some patients did not contact the gynecological clinic for their postabortal problems until after 30 days, which leads to an underestimation in the amount of complications. One limitation is that some patients may be missing in our statistics since they sought medical help somewhere other than at Skaraborg Hospital. However, there are few private gynecological clinics in the area, so it can be assumed that the majority of the complications are represented in this material. Discussion There must be other bacteria involved, beyond those that are screened for. In a study from 2009, the presence of pathogens in cervical samples among 114 women with upper genital tract infections was investigated [12]. It showed that bacteria from the upper respira- tory tract were detected more times than sexually transmitted bacteria. Haemophilus influenza, Group A streptococci, and Streptococcus pneumoniae were de- tected in nine cases compared with C. trachomatis and N. gonorrhoeae, which were identified in seven of the patients. Perhaps bacteria from the upper respiratory tract are involved in developing postabor- tal infections or increasing bacterial resistance could play a role. appeared to be similar to each other might have been categorized differently. One of the strengths in this study is the size of the study group. A total of 4945 induced abortions were in- cluded, which gave a fairly accurate depiction of the inci- dence of bacterial infections in the screening process and of the overall complication frequency. Competing interests Competing interests The authors declare that they have no competing interests. 9. Brian G Bell FS, Stobberingh E, Goossens H, Pringle M. A systematic review and meta-analysis of the effects of antibiotic consumption on antibiotic resistance. BMC Infectious Diseases. 2014;14:13. https://doi.org/10.1186/ 1471-2334-14-13. 10. Penney GC TM, Norman J, McKenzie H, Vale L, Smith R, Imrie M. A randomised comparison of strategies for reducing infective complications of induced abortion. Br J Obstet Gynaecol. 1998. 11. Socialstyrelsen. Statistics on Pregnancies, Deliveries and Newborn Infants 2016 2018. Available from: https://www.socialstyrelsen.se/publikationer2018/ 2018-1-7. Accessed 26 Mar 2016. 12. Charonis G, Larsson PG. Prolonged use of intrauterine contraceptive device as a risk factor for tubo-ovarian abscess. Acta Obstet Gynecol Scand. 2009; 88(6):680–4. 9. Brian G Bell FS, Stobberingh E, Goossens H, Pringle M. A systematic review and meta-analysis of the effects of antibiotic consumption on antibiotic resistance. BMC Infectious Diseases. 2014;14:13. https://doi.org/10.1186/ 1471-2334-14-13. Carlsson et al. BMC Women's Health (2018) 18:158 Carlsson et al. BMC Women's Health (2018) 18:158 Carlsson et al. BMC Women's Health (2018) 18:158 participated sufficiently in the work to take public responsibility for appropriate portions of the content. All authors (IC, KB, PL) agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. participated sufficiently in the work to take public responsibility for appropriate portions of the content. All authors (IC, KB, PL) agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Author details 1 1Skaraborg Hospital Skövde, Lövängsvägen, Skövde, Sweden. 2Department of Obstetrics and Gynecology, Skaraborg Hospital Skövde, Skövde, Sweden. 3Department of Clinical and Experimental Medicine, University of Linköping, Linköping, Sweden. 3Department of Clinical and Experimental Medicine, University of Linköping, Linköping, Sweden. Received: 10 October 2017 Accepted: 12 September 2018 Received: 10 October 2017 Accepted: 12 September 2018 Ethics approval and consent to participate Ethical approval by the regional ethical review board (EPN) in Gothenburg was given in 2010, number 658–09, for an extra screening of Mycoplasma genitalium in order to lower the amount of post abortion infections. All therapeutic abortions in Sweden are reported to the National Board of Health and Welfare. No consent was therefore needed to collect information about gestational age, the number of previous abortions, abortion method or the age of patients. At our hospital, we register all given antibiotics and the reason for treatment as a quality control and for statistical purposes. Therefore, there is no need for consent to collect data from the bacterial screening process. No consent was needed for the prospective part. Follow up appointments 1 month after the abortions were standard procedures even before we started collecting data for this study. 12. Charonis G, Larsson PG. Prolonged use of intrauterine contraceptive device as a risk factor for tubo-ovarian abscess. Acta Obstet Gynecol Scand. 2009; 88(6):680–4. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 10. Penney GC TM, Norman J, McKenzie H, Vale L, Smith R, Imrie M. A randomised comparison of strategies for reducing infective complications of induced abortion. Br J Obstet Gynaecol. 1998. Consent for publication p Consent for publication was not applicable. Competing interests The authors declare that they have no competing interests. 11. Socialstyrelsen. Statistics on Pregnancies, Deliveries and Newborn Infants 2016 2018. Available from: https://www.socialstyrelsen.se/publikationer2018/ 2018-1-7. Accessed 26 Mar 2016. Authors’ contributions All authors (IC, KB, PL) have made substantial contributions to the conception and design, or the acquisition of data, or the analysis and interpretation of data. IC and PL were involved in drafting the manuscript or revising it critically for important intellectual content. All authors (IC, KB, PL) have given final approval of the version to be published. Each author Another limitation is that various individuals were in- volved in categorizing the complications. This may have resulted in misdiagnosis. In other words, patients who Page 9 of 9 Page 9 of 9 Page 9 of 9 12. Charonis G, Larsson PG. Prolonged use of intrauterine contraceptive device as a risk factor for tubo-ovarian abscess. Acta Obstet Gynecol Scand. 2009; 88(6):680–4. References 1. Socialstyrelsen. Abortstatistik 2014 – Statistics on induced abortions 2014 2015. [Available from: https://www.socialstyrelsen.se/publikationer2015/2015-9-4. Accessed 25 Jan 2016. 1. Socialstyrelsen. Abortstatistik 2014 – Statistics on induced abortions 2014 2015. [Available from: https://www.socialstyrelsen.se/publikationer2015/2015-9-4. Accessed 25 Jan 2016. 2. Skånberg T. Motion 1992/93:So435 Abortpillret Mifegyne 1993. Available from: http://www.riksdagen.se/sv/Dokument-Lagar/Forslag/Motioner/ Abortpillret-Mifegyne_GG02So435/?text=true. Accessed 9 Feb 2016. 3. Niinimaki M, Suhonen S, Mentula M, Hemminki E, Heikinheimo O, Gissler M. Comparison of rates of adverse events in adolescent and adult women undergoing medical abortion: population register based study. BMJ (Clinical research ed). 2011;d2111:342. 4. Bjartling C, Osser S, Persson K. The association between mycoplasma genitalium and pelvic inflammatory disease after termination of pregnancy. BJOG. 2010;117(3):361–4. 5. Charonis G, Larsson PG. Use of pH/whiff test or QuickVue advanced pH and amines test for the diagnosis of bacterial vaginosis and prevention of postabortion pelvic inflammatory disease. Acta Obstet Gynecol Scand. 2006; 85(7):837–43. 6. Larsson PG, Platz-Christensen JJ, Dalaker K, Eriksson K, Fahraeus L, Irminger K, et al. Treatment with 2% clindamycin vaginal cream prior to first trimester surgical abortion to reduce signs of postoperative infection: a prospective, double-blinded, placebo-controlled, multicenter study. Acta Obstet Gynecol Scand. 2000;79(5):390–6. 7. Larsson PG, Platz-Christensen JJ, Thejls H, Forsum U, Pahlson C. Incidence of pelvic inflammatory disease after first-trimester legal abortion in women with bacterial vaginosis after treatment with metronidazole: a double-blind, randomized study. Am J Obstet Gynecol. 1992;166(1 Pt 1):100–3. 8. World Health Organization DoRHaR. Safe abortion: technical and policy guidance for health systems. Second edition 2012. [Available from: http:// www.who.int/reproductivehealth/publications/unsafe_abortion/ 9789241548434/en/. Accessed 30 Mar 2016.
https://openalex.org/W4293227853	https://vestnik.tsuab.ru/jour/article/download/1149/797	Russian	null	Kontseptual'nye podkhody k formirovaniyu arkhitektury ekoposelenii [Conceptual approaches to ecovillage architecture]	Vestnik Tomskogo gosudarstvennogo arhitekturno-stroitelʹnogo universiteta	2,022	cc-by	3,343	И.В. СЛИМАК, Алтайский государственный институт культуры, Алтайский государственный политехнический университет им. И.И. Ползунова И.В. СЛИМАК, Алтайский государственный институт культуры, Алтайский государственный политехнический университет им. И.И. Ползунова КОНЦЕПТУАЛЬНЫЕ ПОДХОДЫ К ФОРМИРОВАНИЮ АРХИТЕКТУРЫ ЭКОПОСЕЛЕНИЙ Статья посвящена анализу способов гармонизации урбанистического строительства с природными условиями и естественным ландшафтом. Актуальность данной статьи обусловлена усилением негативного воздействия гло- бальных технологических процессов на природу и истощение ее ресурсов, напрямую оказывающих влияние на среду обитания человека, его образ жизни, физическое и пси- хологическое здоровье. В этой связи представляет научный интерес авторское видение и индивидуальный язык архитектора как особый творческий метод в создании архитек- турного произведения, предназначенного для создания экопозитивной среды жизнедея- тельности. Основной целью статьи является анализ зарубежных и отечественных архитектурных концепций интеграции градостроительного проектирования с окружающей средой и на этой основе определение архитектурно-планировочных принципов и факторов форми- рования архитектурной структуры экопоселений как наиболее востребованных органи- ческих способов обеспечения здорового образа жизни и качественной жилой среды. В рамках исследования определено, что экологические принципы в организации сре- ды обитания человека формировались параллельно с развитием архитектуры жилища и концептуально видоизменялись в зависимости от исторических условий, потребностей социума, уровня развития науки и техники, а также личностных взглядов архитектора- творца, его миропонимания и мировосприятия, предлагавшего идеи архитектурно- художественных и строительных решений интеграции природы и жилища. Выявлены основные тенденции повышения качества среды обитания человека с уче- том его экологической безопасности и рационального природопользования. Сделан вы- вод, что в настоящее время формирование экопоселений выступает новой парадигмой обитания, требующей комплексных исследований по поиску рациональных архитектур- но-строительных решений их формирования. Ключевые слова: экопоселение; экологическая архитектура; архитектурные концепции; энергоэффективность; альтернативные источники энергии. Для цитирования: Слимак И.В. Концептуальные подходы к формированию архитектуры экопоселений // Вестник Томского государственного архитектур- но-строительного университета. 2022. Т. 24. № 1. С. 44–53. DOI: 10.31675/1607-1859-2022-24-1-44-53 I.V. SLIMAK, Altai State Institute of Culture, Polzunov Altai State Technical University I.V. SLIMAK, Altai State Institute of Culture, Polzunov Altai State Technical University Вестник ТГАСУ Т. 24, № 1, 2022 Вестник ТГАСУ Т. 24, № 1, 2022 44 УДК 728.1 DOI: 10.31675/1607-1859-2022-24-1-44-53 DOI: 10.31675/1607-1859-2022-24-1-44-53 CONCEPTUAL APPROACHES TO ECOVILLAGE ARCHITECTURE Purpose: The analysis of Russian and foreign conceptual approaches to ecovillage archi- tecture and identification of ecovillage architecture and planning principles as the most popular © Слимак И.В., 2022 45 Концептуальные подходы к формированию архитектуры экопоселений ways to provide a healthy lifestyle and a high-quality living environment. Approach: The analysis of urban construction harmonization with natural conditions and landscape. Research findings: The ecological principles of the environment are developed together with the build- ing architecture, that are conceptually modified depending on historical conditions, social needs, science and technology, author’s worldview. The main tendencies of the quality im- provement are identified for the living environment with respect to its safety and nature man- agement. Originality/value: Due to the increased negative impact of global technological pro- cesses on the nature and the resource depletion affecting the human environment, lifestyle, physical and psychological health, the author’s view on this problem is interesting for the crea- tion of eco-friendly living environment. Practical implications: Ecovillages is a new para- digm of habitation, which require comprehensive research into an efficient architectural and construction solutions. Keywords: ecovillage; ecological architecture; conceptual approach; energy effi- ciency; alternative energy sources. For citation: Slimak I.V. Kontseptual'nye podkhody k formirovaniyu arkhitektury ekoposelenii [Conceptual approaches to ecovillage architecture]. Vestnik Tomskogo gosudarstvennogo arkhitekturno-stroitel'nogo universiteta – Journal of Construction and Architecture. 2022. V. 24. No. 1. Pp. 44–53. DOI: 10 31675/1607 1859 2022 24 1 44 53 Введение Стремительное развитие науки и техники оказывает прямое влияние на архитектурное творчество, активизируя архитекторов-новаторов к поискам новых концептуальных идей и идейно-образных архитектурных объектов, со- ответствующих своему назначению и способных преодолевать пространство и время. При этом в настоящее время в связи с усугубляющим негативным влиянием технологических факторов на среду обитания человека актуализи- руются проблемы интеграции градостроительных аспектов с природными условиями. Поиск решения проблемы гармонизации урбанистического строи- тельства с природными условиями и естественным ландшафтом обусловлива- ет актуальность данного исследования, в рамках которого наибольший инте- рес представляет авторское видение архитекторами способов и принципов создания экологической среды обитания человека. Поэтому основной целью настоящей статья является анализ зарубежных и отечественных архитектур- ных концепций интеграции градостроительного проектирования с окружаю- щей средой и на этой основе определение архитектурно-планировочных принципов и факторов формирования архитектурной структуры экопоселе- ний, как наиболее востребованных органических способов обеспечения здо- рового образа жизни и качественной жилой среды. К проблеме создания экожилища и формирования экопоселений в насто- ящее время обращаются многие ученые-архитекторы, такие как Д.А. Куликова, А.Н. Тетиора, А.Л. Гельфонд, анализирующие опыт проектных разработок в области урбоэкологии и формирования отдельных поселений в отдаленных регионах. Однако ученые отмечают недостаточность научных исследований в этой области и, соответственно, нерешенность проблемы гармонизации и ин- теграции архитектуры и природы. В то же время многие ученые-исследователи отмечают отсутствие единства в становлении современной архитектуры, проявляющееся в совер- 46 И.В. Слимак шенно разном стилевом выражении. Вместе с тем в научных трудах подчер- кивается преобладание в проектировании архитектурных объектов авторского видения и индивидуального языка архитектора как особого творческого мето- да в создании архитектурного произведения. Следовательно, авторская кон- цепция является основой зарождения архитектурных образов и форм, опреде- ляющая целесообразность дальнейшей работы и выступающая важным эта- пом проектирования. В словаре архитектора концепция определяется как «совокупность ин- формационных материалов и оформленных требований, которые позволяют произвести оценку направления движения проекта и сформировать четкие требования к дальнейшему проектированию» [1, с. 54]. Т.Ю. Быстрова в своих научных трудах характеризует концепцию как «систему основополагающих взглядов, идей и принципов, общий замысел, т. е. комплекс методологических положений, определяющих подход к работе и ор- ганизации ее проведения, способствующих разрешению проблем» [2, с. 31]. Ученый поясняет, что в структурное содержание концепции органично входит характеристика объекта проектирования, цель и задачи, основные принципы и основные направления деятельности, а также механизм ее реализации. Т.Ю. Быстрова в своих научных трудах характеризует концепцию как «систему основополагающих взглядов, идей и принципов, общий замысел, т. е. комплекс методологических положений, определяющих подход к работе и ор- ганизации ее проведения, способствующих разрешению проблем» [2, с. Введение 31]. Ученый поясняет, что в структурное содержание концепции органично входит характеристика объекта проектирования, цель и задачи, основные принципы и основные направления деятельности, а также механизм ее реализации. Поиски и выражение концептуального решения в любом виде искус- ства, в том числе и архитектуре, часто проявляются в авторском видении ар- хитектурного творчества, обусловленного сформированным мировоззрением и мироощущением архитектора, его профессиональными знаниями, конкрет- ной школой и многими другими факторами. «У каждого автора свой язык – своя “морфология”, своя “грамматика” и свой “синтаксис”» [3, с. 54]. При этом важно подчеркнуть, что социальная востребованность архи- тектурного творчества является одним из определяющих факторов его реали- зации и ориентирована на конкретного или потенциального потребителя. «Концепция реализуется в городском пространстве по различным взаимосвя- занным направлениям: средовому, функциональному и образно-символиче- скому» [4, с. 148]. Следовательно, необходим сравнительный ретроспективный анализ концептуальных архитектурных решений, позволяющий определить общий характер образов и форм объектов архитектуры. В рамках настоящего иссле- дования для нас наибольший интерес представляют архитектурные концепции экопоселений, как наиболее востребованных органических способов обеспе- чения здорового образа жизни и качественной жилой среды. Основная часть Первые примеры концепций планировки поселений, связанных с гармо- низацией строительных объектов с окружающей средой, уходят в глубь веков. Так, еще в трудах Витрувия Поллиона «Десять книг по архитектуре» (конец I в. до н. э.) даны советы по выбору места для строительства, обоснованы архитек- турные принципы относительно сторон света и освещения многие другие важ- ные факторы. Помимо этого, и сегодня в архитектуре и дизайне актуальны тре- бования, им сформулированные, – это польза, красота и прочность (рис. 1). Концептуальные подходы к формированию архитектуры экопоселений 47 Рис. 1. Архитектурная концепция Витрувия Рис. 1. Архитектурная концепция Витрувия Важно подчеркнуть, что Витрувий обращал предельное внимание и на тесное взаимодействие строительного объекта с природными условиями, сре- ди которых он выделял грамотный выбор места, наличие рядом природных водоемов и других экологических аспектов, способствующих успешному эко- номическому функционированию будущего поселения и удовлетворению по- требностей человека. Эта архитектурная концепция и сегодня является акту- альной и основополагающей в архитектурном творчестве. К архитектурной концепции экопоселений ученые относят и идеи со- здания садов внутри системы города, предложенные Д. Рёскиным и У. Мор- рисом. По замыслу этих идеалистов-реформаторов, город должен быть окру- жен цветущими полями, при этом приносящими пользу как в качестве про- дуктов питания, так и эстетического наслаждения окружающей красотой. Эти идеи легли в основу архитектурной концепции города-сада, которую впослед- ствии разработал и реализовал Э. Говард (рис. 2). Рис. 2. Взаимодействие концепций Э. Говарда и Д. Рёскина Рис. 2. Взаимодействие концепций Э. Говарда и Д. Рёскина Потребность в улучшении экологического равновесия в этот историче- ский период (XIX в.) была крайне необходимой и обусловлена стремительным И.В. Слимак 48 ростом и индустриализацией городов, вызвавших ухудшение условий жизни человека. Поэтому Э. Говард сконцентрировал внимание на идее архитектур- ной планировки поселения, объединяющей в себе элементы крупного города и деревни, в центре которой местом притяжения всех жителей выступает сад с наличествующими зонами для отдыха (рис. 3). Вокруг сада Э. Говард задумы- вал расположить общественные здания для проведения досуга и повышения культурного уровня жизни горожан и уже только следующим кольцевым стро- ительством возвести промышленные постройки. Он считал, что в таком форма- те поселения «смогут сочетаться преимущества самой энергичной и активной городской жизни со всеми красотами и радостями деревни» [5, с. 29]. Рис. 3. Э. Говард. Архитектурная концепция города-сада будущего Рис. 3. Э. Говард. Архитектурная концепция города-сада будущего К следующей архитектурной концепции, отчасти отражающей элемен- ты экопоселений, относится архитектура устойчивого развития, обоснованная Г.В. Есауловым. Основная часть В ее основе лежит гармонизация строительства с природно- климатическими условиями при обязательной законодательной охране окру- жающей среды. При этом важным фактором выступает экономическая целе- сообразность использования природных ресурсов и, соответственно, умень- шение себестоимости в обеспечении жизнедеятельности и стабилизации уровня жизни людей. Понятие «устойчивая архитектура» легло в основу нового экологиче- ского направления в архитектурном творчестве. Архитектор Фрэнк Ллойд Райт считал, что «…зодчий должен черпать вдохновение в природе, а чело- век – жить по ее законам» [6, с. 71]. Став основателем концепции «Зеленая архитектура», он продвигал идею тесного взаимодействия утилитарно- функциональных решений с их чувственно-психологическим восприятием. «Зеленая» архитектура, по убеждению архитектора, гармоничная и наиболее подходит для комфортного нахождения в ее среде человека, удовлетворяя все его потребности. Такая архитектура в отличие от искусственной среды есте- ственна и не вредит психологическому равновесию (рис. 4). Концептуальные подходы к формированию архитектуры экопоселений 49 Рис. 4. Фрэнк Ллойд Райт. Концепция «зеленой архитектуры» Рис. 4. Фрэнк Ллойд Райт. Концепция «зеленой архитектуры» Основным требованием к строительству архитектурных объектов архитек- тор выдвигал не проектирование отдельных зданий, а функциональное «достраи- вание» фрагментов природного ландшафта как его естественное продолжение, сохраняющее внешний облик и естественные характеристики. При функциони- ровании такого комплекса предлагалось использовать наличествующие природ- ные ресурсы, такие как автономное энергоснабжение и утилизация. Архитектурная концепция Фрэнка Ллойда Райта получила свое про- должение уже в XX в. в творчестве Лье Корбюзье, Райта, Гропиуса и др. Аме- риканский архитектор Стенли Харт Уайтом продолжил веяния «зеленой архи- тектуры» в своих проектах – Urbana-Champaign. Он по-новому осмыслил свой подход к концепции и получил патент на вертикальную фитостену на зданиях, названный новым подходом к ландшафтному дизайну. Следует отметить, что идеи гармонизации крупных городов с природ- ным ландшафтом получили свои развитие в Советском Союзе. Так, одной из наиболее известных архитектурных концепций является идея «зеленого горо- да», предложенная дезурбанистами М.О. Барщом и М.Я. Гинзбургом (рис. 5). Архитекторы предлагали сделать перепланировку одного из пригородных районов Москвы, расположенного вдоль Ярославского шоссе, в виде приго- рода-сада. По их мнению, такой подход позволил бы решить проблему урба- низма и методом «рассасывания» большого города на окраины создать «такой способ расселения человечества, который бы разрешил проблему труда, от- дыха и культуры как единый непрерывный процесс социалистического бы- тия» [7, с. 228]. 50 И.В. Слимак Рис. 5. М. Барщ, М. Гинзбург. Конкурсный проект «Зеленый город». Генеральный план. 1930 г. Рис. 5. М. Барщ, М. Гинзбург. Конкурсный проект «Зеленый город». Генеральный план. 1930 г. Основная часть Таким образом, в течение длительного исторического периода неравно- душных людей, занимающихся градостроительством, всегда волновала про- блема взаимодействия воздвигаемых архитектурных объектов и природного окружения. При этом практически во всех архитектурных концепциях при- родные комплексы и ресурсы выступали как дополнительные аспекты, прино- сящие пользу человеку с точки зрения сохранения здоровья и экономного расходования средств. Но даже при минимальном расходовании природных ресурсов когда-то наступает их истощение, отягощенное регулярным воздей- ствием на них потенциально вредных технологий. Эти факторы требовали нового научного осмысления в строительстве и архитектуре. В русле этих идей на Западе в 1962 г. возникли первые концепции эко- поселений. Здесь следует подчеркнуть, что ведущей целью экопоселений яв- ляются вопросы самоидентификации и самореализации в соответствии с ви- дением, контекстом, культурой и интересами конкретного круга людей. В их идеологических воззрениях часто прослеживаются корни философии опроще- ния, хиппи, дауншифтинга и других контркультур. Так, одно из известных экопоселений этого периода организовала община Фонда Финдхорн, пропа- гандировавшая движение «хиппи». Модель этого поселения, по сути, пред- ставляет экологически рационально обустроенную деревню, жители которой в строительстве использовали интеграцию ландшафта и архитектурной ин- фраструктуры. Помимо этого, в функционировании поселения активно при- менялись возобновляемые источники энергии – солнечные водонагревающие панели, которые обслуживали все общественные и частные здания. Эта общи- на в дальнейшем была переоформлена в Финдхорнский Фонд с огромным ко- личеством участников и сегодня является «центральной образовательной и организационной площадкой сообщества, объединенного позитивным виде- нием духовного совершенствования и объединения человечества» [8, с. 63]. Проект «Экодеревни» очень быстро стал популярным и получил разви- тие во многих странах мира. Одним из продолжателей этого движения являет- ся «Ауровиль», также известный как «Город Зари». Это своеобразный экспе- римент, начавшийся при поддержке индийского правительства и ассамблеи Концептуальные подходы к формированию архитектуры экопоселений 51 ЮНЕСКО. Вся инфраструктура этого города функционирует за счет рацио- нального использования природных ресурсов и альтернативной технологиче- ской переработки используемых продуктов и сырья. В качестве материалов для строительства применяется спрессованная земля, песок и цемент. Не меньший интерес в рамках анализа архитектурных концепций пред- ставляют поселения, сформированные на идее ведения жизнедеятельности по принципу пермакультуры, т. е. без использования в обработке земли различ- ных пестицидов и химических удобрений. Основоположниками этой идеи яв- ляются австралийцы Б. Моллисон и Д. Холмгрен, которые считали необходи- мым выращивать растения на всевозможных поверхностях, включая верти- кальные, горизонтальные и наклонные. В начале 2000 г. особую популярность получил новый вид экопоселения, связанного с образом родового поместья. Основной идеей этой архитектурной концепции выступает комплексное решение социальных и экономических про- блем проживания современного человека. Основная часть Собственно, это близкая по инфра- структуре к городской среде благоустроенная система проживания человека с наличием всех составляющих для проведения культурного досуга, но в то же время находящаяся в тесной гармонии с окружающей природной средой, ис- пользование которой крайне рационально, экологично и возобновляемо. Результаты На основании вышеизложенного можно определить следующие важные для нашего исследования положения. Так к наиболее значимым аспектам в архитектуре и строительстве на всем историческом этапе их развития отно- сится формирование жилой среды обитания человека с учетом его потребно- стей в здоровом образе жизни. Экологические принципы в организации среды обитания концептуально видоизменялись в зависимости от исторических условий, потребностей социума, уровня развития науки и техники, а также личностных взглядов архитектора-творца, его миропонимания и мировоспри- ятия, предлагавшего идеи архитектурно-художественных и строительных ре- шений интеграции природы и жилища. Стремление к экологичности в архитектуре выявляет новые формы жи- лищ, возможности их интеграции с природой. Е.В. Купцова и Т.Н. Колесни- кова конкретизируют современные требования к проектированию экодома, который должен быть «комфортным, гармонично вписанным в природное окружение, с высокими архитектурными качествами, с рациональной объем- но-планировочной структурой, здоровым микроклиматом, безопасным, вы- полненным из экологичных и малоэнергоемких строительных материалов, ресурсосберегающим, с системами регенерации отходов» [9, с. 38]. Эти пока- затели и выступают основными принципами формирования архитектурно- планировочных решений жилых домов в системе экопоселения, среди кото- рых мы выделяем: 1) сохранность территориального природного ландшафта и рациональ- ное использование ресурсов; 2) использование экологических материалов и альтернативных источ- ников энергии; 52 И.В. Слимак 3) эстетичность архитектурного решения; 3) эстетичность архитектурного решения; 4) инфраструктурное удобство; 5) социальный микроклимат. 5) социальный микроклимат. Относительно последнего пункта следует акцентировать внимание на специфической особенности формирования экопоселений, связанных с жела- нием объединения определенных социальных групп в единую систему жиз- ненного территориального обитания. М.И. Тукмакова и И.А. Фахрутдинова такие группы характеризуют как «креативные пространства, объединяющие представителей разных профессий и способствующие сближению единомыш- ленников, обмену идеями и творческими инициативами» [10, с. 118]. Архи- тектурно-пространственная планировка таких сред обитания, включающая «природные формы и другие ассоциативные маркеры, распределенные терри- ториально по всему комплексу, составляют художественно-образную ткань поселения» [11, с. 55]. Заключение Таким образом, сравнительный анализ архитектурных концепций фор- мирования экопоселения дает возможность сделать следующие выводы. Формирование экопоселений явилось результатом раздумий и поисков человеком наиболее гармоничного решения собственного проживания, в ко- тором основополагающими факторами выступали забота о собственном фи- зическом, душевном и психологическом здоровье. Это явилось определенного рода спусковым механизмом возникновения идеи интеграции урбанизации и природно-климатических условий и природного ландшафта, их рациональ- ного использования с учетом возобновляемости ресурсов. Архитектурные концепции планировочных структур экопоселений весьма разнообразны и напрямую зависят от идеологических взглядов автора- архитектора, его мировоззрения и миропонимания, уровня развития науки и техники, а также модных тенденций в мире архитектуры. В настоящее время формирование экопоселений выступает новой пара- дигмой обитания, требующей комплексных исследований по поиску их раци- ональных архитектурно-строительных решений. БИБЛИОГРАФИЧЕСКИЙ СПИСОК 1. Партина А.С. Архитектурные термины : иллюстрированный словарь. Изд. стереотип- ное. Москва : Стройиздат, 2001. 208 с. 2. Быстрова Т.Ю. Философия дизайна. 2-е изд., перераб. Екатеринбург : Изд-во Урал. ун-та, 2015. 128 с. 3. Дуцев М.В. Современные авторские концепции архитектурно-художественного синте- за // Известия Казанского государственного архитектурно-строительного университета. 2012. № 1 (19). С. 7–16. 4. Гельфонд А.Л., Дуцев М.В. Архитектурно-художественный синтез как средство диало- га // Приволжский научный журнал. 2010. № 4. С. 147–152. 5. Мелвин Д. Архитектура: путеводитель по стилям : пер. с англ. Москва : Кладезь-Букс, 2007. 158 с. 6. Архитектура и социальный мир / отв. ред. И.А. Добрицына. Москва : Прогресс- Традиция, 2012. 312 с. Концептуальные подходы к формированию архитектуры экопоселений 53 7. Шульц А.С. Экологические подходы к проектированию устойчивой городской среды // Архитектура и современные информационные технологии. 2021. № 1 (54). С. 227–235. р ур р ф р ( ) 8. Тетиор А.Н. Городская экология. 3-е изд. Москва : Издательский центр «Академия», 2008. 338 с. 9. Купцова Е.В., Колесникова Т.Н. Основные принципы и приемы архитектурной органи- зации экологически позитивного жилища // Промышленное и гражданское строитель- ство. 2009. № 8. С. 37–39. 10. Тукмакова М.И., Фахрутдинова И.А. Архитектурные принципы формирования креатив- ных пространств // Известия КГАСУ. 2018. № 4 (46). С. 116–122. 11. Михайленко Д.В., Резницкая Л.М. Проектная концепция эколого-археологического ком- плекса «Донская Троя» // Вестник Томского государственного архитектурно-строитель- ного университета. 2021. № 23 (2). С. 46–55. REFERENCES 1. Partina A.S. Arkhitekturnye terminy [Architecture terms. Dictionary]. Moscow: Stroiizdat. 2001. 208 p. (rus) T.Yu. Filosofiya dizaina [Philosophy of design], 2nd ed., Ekate . Bystrova T.Yu. Filosofiya dizaina [Philosophy of design], 2n 3. Dutsev M.V. Sovremennye avtorskie kontseptsii arkhitekturno-khudozhestvennogo sinteza [Contemporary author's concepts of architectural and artistic synthesis]. Izvestiya Kazanskogo gosudarstvennogo arkhitekturno-stroitel'nogo universiteta. 2012. No. 1 (19). Pp. 7–16. (rus) 4. Gel'fond A.L., Dutsev M.V. Arkhitekturno-khudozhestvennyi sintez kak sredstvo dialoga [Ar- chitectural and artistic synthesis as a means of dialogue]. Privolzhskii nauchnyi zhurnal. 2010. No. 4. Pp. 147–152. (rus) 5. Melvin J. Arkhitektura: putevoditel' po stilyam [Understanding architectural styles]. Moscow: Kladez'-Buks, 2007. 158 p. (transl. from Engl.) 6. Dobritsyna I.A. (Ed.) Arkhitektura i sotsial'nyi mir [Architecture and social world]. Moscow: Progress-Traditsiya, 2012. 312 p. (rus) 7. Shul'ts A.S. Ekologicheskie podkhody k proektirovaniyu ustoichivoi gorodskoi sredy [Ecologi- cal approaches to urban environment design]. Arkhitektura i sovremennye informatsionnye tekhnologii. 2021. No. 1 (54). Pp. 227–235. (rus) 8. Tetior A.N. Gorodskaya ekologiya [Urban ecology], 3rd ed., Moscow: Akademiya, 2008. 338 p. (rus) 9. Kuptsova E.V., Kolesnikova T.N. Osnovnye printsipy i priemy arkhitekturnoi organizatsii ekologicheski pozitivnogo zhilishcha [Basic principles and techniques for eco-friendly housing architecture]. Promyshlennoe i grazhdanskoe stroitel'stvo. 2009. No. 8. Pp. 37–39. (rus) 10. Tukmakova M.I., Fakhrutdinova I.A. Arkhitekturnye printsipy formirovaniya kreativnykh prostranstv [Architectural principles of creative space formation]. Izvestiya KGASU. 2018. No. 4 (46). Pp. 116–122. (rus) 11. Mikhailenko D.V., Reznitskaya L.M. Proektnaya kontseptsiya ekologo-arkheologicheskogo kompleksa “Donskaya Troya” [The concept of ecological and archeological site "Donskaya Troya"]. Vestnik Tomskogo gosudarstvennogo arkhitekturno-stroitel'nogo universiteta – Jour- nal of Construction and Architecture. 2021. V. 23. No. 2. Pp. 46–55. (rus) Сведения об авторе Слимак Иван Владимирович, ст. преподаватель, Алтайский государственный инсти- тут культуры, 656055, г. Барнаул, ул. Юрина, 277; аспирант, Алтайский государствен- ный политехнический университет им. И.И. Ползунова, ivan-slimak@mail.ru Слимак Иван Владимирович, ст. преподаватель, Алтайский государственный инсти- тут культуры, 656055, г. Барнаул, ул. Юрина, 277; аспирант, Алтайский государствен- ный политехнический университет им. И.И. Ползунова, ivan-slimak@mail.ru Authors Details Ivan V. Slimak, Senior Lecturer, Altai State Institute of Culture, 277, Yurin Str., Barnaul, Russia; Polzunov Altai State Technical University, 46, Lenin Ave., 656038, Barnaul, Russia, ivan-slimak@mail.ru
https://openalex.org/W2752391462	https://www.nature.com/articles/s41598-017-10870-5.pdf	English	null	Peptide-mediated delivery of donor mitochondria improves mitochondrial function and cell viability in human cybrid cells with the MELAS A3243G mutation	Scientific reports	2,017	cc-by	12,690	Peptide-mediated delivery of donor mitochondria improves mitochondrial function and cell viability in human cybrid cells with the MELAS A3243G mutation Received: 27 January 2017 Accepted: 16 August 2017 Published: xx xx xxxx Received: 27 January 2017 Accepted: 16 August 2017 Published: xx xx xxxx Jui-Chih Chang1, Fredrik Hoel 2, Ko-Hung Liu1, Yau-Huei Wei3,4, Fu-Chou Cheng5, Shou-Je Kuo6, Karl Johan Tronstad2 & Chin-San Liu1,7 The cell penetrating peptide, Pep-1, has been shown to facilitate cellular uptake of foreign mitochondria but further research is required to evaluate the use of Pep-1-mediated mitochondrial delivery (PMD) in treating mitochondrial defects. Presently, we sought to determine whether mitochondrial transplantation rescue mitochondrial function in a cybrid cell model of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) disease. Following PMD, recipient cells had internalized donor mitochondria after 1 h, and expressed higher levels of normal mitochondrial DNA, particularly at the end of the treatment and 11 days later. After 4 days, mitochondrial respiratory function had recovered and biogenesis was evident in the Pep-1 and PMD groups, compared to the untreated MELAS group. However, only PMD was able to reverse the fusion-to-fission ratio of mitochondrial morphology, and mitochondria shaping proteins resembled the normal pattern seen in the control group. Cell survival following hydrogen peroxide-induced oxidative stress was also improved in the PMD group. Finally, we observed that PMD partially normalized cytokine expression, including that of interleukin (IL)-7, granulocyte macrophage–colony-stimulating factor (GM-CSF), and vascular endothelial growth factor (VEGF), in the MELAS cells. Presently, our data further confirm the protective effects of PMD as well in MELAS disease. Mitochondria are organelles responsible for a large part of the cellular ATP production. These dynamic organelles have their own DNA, and are constantly adapting their function in accordance with the context-dependent needs of the cell1. Mitochondrial dysfunction is associated with many diseases, and typically leads to metabolic imbal- ance, cellular energy deficiency and ROS production1. Mitochondrial, myopathy, encephalopathy, lactic acidosis and stroke-like episodes syndrome (MELAS) is a genetic mitochondrial disease commonly caused by inherited point mutations in tRNA genes encoded by mitochondrial DNA (mtDNA). This results in defective synthesis of mitochondrial respiratory chain subunits and subsequent impairment of mitochondrial function2. The defects in mitochondrial function gives rise to a complex pathology that has severe consequences for patients. With the exception of mitochondrial replacement therapy, which only can be done on a newly fertilized oocyte, there is no curative treatment for MELAS or similar diseases. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Peptide-mediated delivery of donor mitochondria improves mitochondrial function and cell viability in human cybrid cells with the MELAS A3243G mutation In the present study, we investigated if mitochondrial transplantation enabled by the cell-penetrating peptide Pep-1 rescue mitochondrial function in a cybrid MELAS model. 1Vascular and Genomic Center, Changhua Christian Hospital, Changhua, Taiwan. 2Department of Biomedicine, University of Bergen, Bergen, Norway. 3Department of Biochemistry and Molecular Biology, School of Life Sciences, National Yang-Ming University, Taipei, Taiwan. 4Department of Medicine, Mackay Medical College, Taipei, Taiwan. 5Stem Cell Center, Department of Medical Research, Taichung Veterans General Hospital, Changhua, Taiwan. 6Department of Surgery, Changhua Christian Hospital, Changhua, Taiwan. 7Department of Neurology, Changhua Christian Hospital, Changhua, Taiwan. Jui-Chih Chang, Fredrik Hoel and Ko-Hung Liu contributed equally to this work. Correspondence and requests for materials should be addressed to C.-S.L. (email: 26602@cch.org.tw) SCieNTiFiC REPOrTS \| 7: 10710 \| DOI:10.1038/s41598-017-10870-5 1 www.nature.com/scientificreports/ The mitochondria are double membrane organelles containing two enclosed compartments, the matrix (inner compartment) and the intermembrane space. The inner mitochondrial membrane is the site of the electron trans- port chain (ETC). Here electrons obtained from NADH and FADH2 are transported through four respiratory enzymes (CI-IV) via a series of redox reactions ending with the reduction of oxygen. This electron-transport drives the translocation protons from the matrix-side across the inner membrane, generating an electrochemical gradient (i.e. membrane potential). Reflux of protons through the ATP synthase complex (CV) releases energy used to phosphorylate ADP to ATP. Together, these processes are termed oxidative phosphorylation (OXPHOS)1. Mitochondrial bioenergetics are normally adapting to the physiological requirements of the cells, through regu- lation of oxidative pathways, mitochondrial biogenesis and mitochondrial dynamics3. Mitochondrial biogenesis serves to increase the oxidative capacity under conditions of insufficient ATP production4. Organelle fission and fusion processes are important in mitochondrial quality control, and involves fusion proteins such as OPA15, MFN1 and MFN26 and the fission proteins DRP17 and Fis18. Morphologic changes are seen in response to con- ditions of cellular stress. Mild energy deficiency, which may be due to increased ATP consumption in exercising skeletal muscle9 or sub-lethal inhibition of OXPHOS in cultured cells10 is associated with increased fusion and network complexity of filamentous mitochondria. Severe stress, which may be caused by pathology or toxin expo- sure, is associated with fragmented mitochondria, accompanied by aberrant ROS production and mitochondrial dysfunction11, 12. Specific degradation of dysfunctional mitochondria (mitophagy) has a crucial role in mitochon- drial quality control, serving to sustain cellular energy homeostasis and prevent pathologic ROS production. Peptide-mediated delivery of donor mitochondria improves mitochondrial function and cell viability in human cybrid cells with the MELAS A3243G mutation Deficiencies in mitochondrial quality control are associated with neurodegenerative disorders such as Parkinson’s Disease13, and genetic mitochondrial diseases such as PolG mutations14 and in MELAS15. g Transfer of mitochondria between separate cells has been observed both in vivo and in vitro, a phenomenon that may support cell survival and protect against external stress16–20. The mechanisms involved in such inter- cellular organelle transfers are not fully understood, but nanoscale membrane containing tubes that form inter- cellular connections, tunneling nanotubes, have been reported to traffic mitochondria from mesenchymal stem cells and rescue damaged cells in culture17, 19–21. Another mechanism involving endocytosis and micro-vesicles containing mitochondria has been reported to transfer mitochondria between bone marrow derived mesenchy- mal cells and damaged alveolar epithelium in vivo19. Such observations have given rise to studies addressing the possibility to rescue mitochondrial function by transplanting healthy mitochondria into cells with mitochondrial dysfunction. In an animal study, mitochondria injected directly into the heart after an induced ischemic insult protected against ischemia-reperfusion injury, reduced the infarction size and improved recovery22. In this study it was estimated that only 3–7% of the mitochondria were taken up by the cells, however, it was suggested that extracellular mitochondria also conferred some cardioprotection. Cell culture experiments have indicated that the ability to import exogenous mitochondria may vary between cell types. Whereas mtDNA depleted HeLa cells were found to take up exogenously added mitochondria23, the same was not seen mtDNA depleted A549 cells16. One study reported that mesenchymal stem cells were able to transfer mitochondria to mtDNA depleted cells, but not cybrid cells harboring pathogenic mtDNA with pathogenic mtDNA mutations, including the MELAS A3243G mutation24. Cell-penetrating peptides (CPPs) have been employed to facilitate cellular uptake of substances such as nano- particles, DNA and proteins25, 26. Such proteins have similar functions in nature, such as the HIV-1 protein, TAT, which is a CPP necessary for the entry of HIV-1 virus particles into human cells. Covalent coupling to TAT has been found to mediate import of functionally capable mitochondrial enzymes such as complex I subunits, which promoted recovery of vital cell functions27. Pep-1, another CPP, is able to translocate cargoes via non-covalent self-assembly mechanisms28, 29. Inside the cells, the cargo is released via an endocytosis-independent pathway28, 29. This ability of Pep-1 to act as a chaperone facilitating cellular uptake and intracellular release of cargo, may enable delivery of small organelles into the cell. Peptide-mediated delivery of donor mitochondria improves mitochondrial function and cell viability in human cybrid cells with the MELAS A3243G mutation Recently, we found that mitochondrial transplantation was facilitated when the isolated donor mitochondria were coated with Pep-130, 31. Further, the studies showed that Pep-1 medi- ated mitochondrial delivery (PMD) improved mitochondrial functions in two cell models of the mitochondrial disease Myoclonic Epilepsy and Ragged Red Fibers (MERRF)30, 31.i y p p y gg ( ) Based on these findings, we here investigated if PMD is able to rescue mitochondrial properties in a MELAS cybrid cell model with MELAS A3243G mutation in mtDNA. Our aim was to clarify the effects of PMD on mitochondrial function and morphology to gain better understanding of the processes involved in restoring cell function. We also wanted to determine if Pep1-mediated mitochondrial transplantation influenced oxidative stress responses in the recipient cells because functional restoration could confer an increase of stress tolerance to the MELAS cybrid cells. Results D Donor mitochondria are internalized into MELAS cybrid cells after PMD. To examine the direct effects of Pep-1 on the ultrastructure of isolated mitochondria, rat liver mitochondria were treated with Pep-1 (5, 25 or 50 µM), then analyzed by transmission electron microscopy (Fig. 1). A gap between the mitochondrial outer membranes was observed in mitochondria alone, as shown in Fig. 1A. The shape and membrane structure appeared unchanged in the presence of different concentrations of Pep-1, suggesting that Pep-1 does not affect mitochondrial integrity. However, close contact between the outer membranes was observed after Pep-1 labeling (indicated arrows in Fig. 1A). Membrane fusion was 34 ± 4.3%, 62 ± 13.3% and 77 ± 10.3 after treatment with 5, 25 and 50 µM Pep-1, relative to non-treated controls (11 ± 3.2) (Fig. 1B). To perform Pep-1-mediated mitochondrial delivery (PMD) on MELAS cybrid cells, donor mitochondria were isolated from 143B osteosacroma cybrid cells and labeled with MitoTracker Red immediately before delivery to enable organelle tracing. After an initial incubation of 4 h, red fluorescent mitochondria were observed both inside the MELAS cybrid cells and in mitochondrial aggregates in the medium, as shown using confocal micros- copy and three-dimensional (3D) reconstructions (Fig. 2A). Within the cells, the red fluorescence overlapped SCieNTiFiC REPOrTS \| 7: 10710 \| DOI:10.1038/s41598-017-10870-5 2 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ significantly with green fluorescence from the mitochondria of the host cells (MELAS cybrid cells), which were labeled with MitoTracker Green prior to delivery This co-localization of the two dyes could be due to fusion of Figure 1. Transmission electron microscopy (TEM) analysis of mitochondria coated with various Pep-1 doses (5, 25 or 50 μM). (A) Mitochondria alone (Con); mitochondria coated with 5 μM Pep-1 (Pep-1(5)-Mito); mitochondria coated with 25 μM Pep-1 (Pep-1(25)-Mito); mitochondria coated with 50 μM Pep-1 (Pep- 1(50)-Mito). Gap between outer membranes in the mitochondria alone group and contact between the outer membranes in the Pep-1(5)-Mito group is shown by block box and arrows, respectively. (B) Proportion of membrane-fused mitochondria in total mitochondria calculated from a group of images. p < 0.05 compared to control group; +p < 0.05, compared to Pep-1(5)-Mito group. Figure 1. Transmission electron microscopy (TEM) analysis of mitochondria coated with various Pep-1 doses (5, 25 or 50 μM). (A) Mitochondria alone (Con); mitochondria coated with 5 μM Pep-1 (Pep-1(5)-Mito); mitochondria coated with 25 μM Pep-1 (Pep-1(25)-Mito); mitochondria coated with 50 μM Pep-1 (Pep- 1(50)-Mito). Results D Gap between outer membranes in the mitochondria alone group and contact between the outer membranes in the Pep-1(5)-Mito group is shown by block box and arrows, respectively. (B) Proportion of membrane-fused mitochondria in total mitochondria calculated from a group of images. p < 0.05 compared to control group; +p < 0.05, compared to Pep-1(5)-Mito group. significantly with green fluorescence from the mitochondria of the host cells (MELAS cybrid cells), which were labeled with MitoTracker Green prior to delivery. This co-localization of the two dyes could be due to fusion of host and recipient mitochondria, or to diffusion of the dyes between organelles. To address this, the experiment significantly with green fluorescence from the mitochondria of the host cells (MELAS cybrid cells), which were labeled with MitoTracker Green prior to delivery. This co-localization of the two dyes could be due to fusion of host and recipient mitochondria, or to diffusion of the dyes between organelles. To address this, the experiment SCieNTiFiC REPOrTS \| 7: 10710 \| DOI:10.1038/s41598-017-10870-5 3 www.nature.com/scientificreports/ Figure 2. Translocation of Pep-1-labeled mitochondria (Pep-1-Mito) into MELAS cybrid cells. ( showing mitochondrial translocation from medium into cells. Red (Mitotracker Red) and green Green) fluorescence represent donor Pep-1-Mito and innate mitochondria, respectively. Asterisk aggregates of Pep-1-Mito complexes suspended in medium. Reconstructed three-dimensional (3 images showing co-localization of donor and innate mitochondria inside MELAS cybrid cells, in by arrows (yellow fluorescence). (B) Mitochondria labeled with fluorescent proteins. Donor mito tagged with GFP (MitoGFP, green fluorescence) and innate mitochondria tagged with HcRed1 (M red fluorescence). Nuclei were stained with DAPI (blue fluorescence). Donor Pep-1-labeled Mito 1-MitoGFP) inside recipient MELAS cybrid cells after 2 days; possible sites of fusion between dono innate mitochondria (yellow fluorescence) are indicated by arrows. (C) 3D visualization of Pep-1 internalization and innate MitoRFP in MELAS cybrid cells tracked over time using confocal micro Figure 2. Translocation of Pep-1-labeled mitochondria (Pep-1-Mito) into MELAS cybrid cells. (A) Illustration showing mitochondrial translocation from medium into cells. Red (Mitotracker Red) and green (Mitotracker Green) fluorescence represent donor Pep-1-Mito and innate mitochondria, respectively. Asterisks indicate aggregates of Pep-1-Mito complexes suspended in medium. Reconstructed three-dimensional (3D) confocal images showing co-localization of donor and innate mitochondria inside MELAS cybrid cells, indicated by arrows (yellow fluorescence). (B) Mitochondria labeled with fluorescent proteins. Donor mitochondria tagged with GFP (MitoGFP, green fluorescence) and innate mitochondria tagged with HcRed1 (MitoRFP, red fluorescence). Nuclei were stained with DAPI (blue fluorescence). Results D Pep-1-labeled MitoGFP (Pep-1-MitoGFP) mitochondria were attached to the cell membrane after an initial incubation of 0.5 h, but then had translocated into the cytoplasm after 1 h (Fig. 2C). More obvious co-localization of GFP and RFP was observed after 2 and 4 h of culture (blank arrows in Fig. 2C). After 8 h, extracellular Pep-1-MitoGFP fluores- cence suspended in the medium was significantly reduced (stars in Fig. 2C). Furthermore, the number of foreign MitoGFP and innate MitoRFP, revealed by mean fluorescence area (pixels) per cell in same cell thickness, became enhanced with time (Fig. 2C, insert frame in z-stack image). Moreover, innate MitoRFP in the rescued cells dis- played a concomitant change in mitochondrial morphology, from a dot-like to a rod-like appearance.i p y g p gy pp To confirm the presence of donor mitochondria inside the recipient MELAS cybrid cells, the mtDNA was sequenced. Donor wild-type mtDNA was detected in MELAS cybrid cells after PMD, as validated by DNA sequence analysis of the mitochondrial tRNALeu(UUR) gene at the A3243G mutation locus. This analysis was per- formed 48 h after incubation with Pep-1 carrier alone (Pep-1), naked mitochondria (Mito) or Pep-1-labeled mitochondria (Pep-1-Mito), and was compared to untreated MELAS cybrid cells. The presence of a heterodu- plex mtDNA species (heteroplasmy) at position 3243 was found only in the Pep-1-Mito treated group (Fig. 3A). Moreover, PCR-RFLP analysis showed a significant increase in wild-type mtDNA content: 4.6 ± 2.1% in the untreated MELAS cybrid cells versus 34.6 ± 3.2% in the Pep-1-Mito treated cells at the end of the 48 h incubation period (day 0). Although wild-type mtDNA content was decreased in the subsequent stability period of 1–7 days, it was increased dramatically from approximately 9.0 ± 2.8% to 14.5 ± 2.9% after 11 days of treatment (Fig. 3B). Recovery of respiratory function in MELAS cybrid cells after PMD. The effect of PMD on mito- chondrial function after 4 days of treatment was investigated by monitoring oxygen consumption rate (OCR) in control cybrid cells, MELAS cybrid cells (MELAS), MELAS cybrid cells treated with Pep-1, Mito, Pep-1-Mito or Pep-1-labeled mitochondria with A3243G mutant mtDNA isolated from MELAS cybrid cells (Pep-1-Mito3243). OCR was measured in confluent cells after sequential treatment with specific inhibitors (oligomycin to inhibit ATP synthase, or the uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), rotenone to inhibit complex I) (Fig. 4A). Results D Donor Pep-1-labeled MitoGFP (Pep- 1-MitoGFP) inside recipient MELAS cybrid cells after 2 days; possible sites of fusion between donor and innate mitochondria (yellow fluorescence) are indicated by arrows. (C) 3D visualization of Pep-1-MitoGFP internalization and innate MitoRFP in MELAS cybrid cells tracked over time using confocal microscopy combined with differential interference contrast (DIC). Z-axis yields a longitudinal view showing Pep-1- Figure 2. Translocation of Pep-1-labeled mitochondria (Pep-1-Mito) into MELAS cybrid cells. (A) Illustration showing mitochondrial translocation from medium into cells. Red (Mitotracker Red) and green (Mitotracker Green) fluorescence represent donor Pep-1-Mito and innate mitochondria, respectively. Asterisks indicate aggregates of Pep-1-Mito complexes suspended in medium. Reconstructed three-dimensional (3D) confocal images showing co-localization of donor and innate mitochondria inside MELAS cybrid cells, indicated by arrows (yellow fluorescence). (B) Mitochondria labeled with fluorescent proteins. Donor mitochondria tagged with GFP (MitoGFP, green fluorescence) and innate mitochondria tagged with HcRed1 (MitoRFP, red fluorescence). Nuclei were stained with DAPI (blue fluorescence). Donor Pep-1-labeled MitoGFP (Pep- 1-MitoGFP) inside recipient MELAS cybrid cells after 2 days; possible sites of fusion between donor and innate mitochondria (yellow fluorescence) are indicated by arrows. (C) 3D visualization of Pep-1-MitoGFP internalization and innate MitoRFP in MELAS cybrid cells tracked over time using confocal microscopy combined with differential interference contrast (DIC). Z-axis yields a longitudinal view showing Pep-1- SCieNTiFiC REPOrTS \| 7: 10710 \| DOI:10.1038/s41598-017-10870-5 4 www.nature.com/scientificreports/ MitoGFP stuck to the cell membrane after 0.5 h and penetration into the cell after 1 h, as indicated by an arrow. Blank arrow indicates co-localization of Pep-1-MitoGFP and MitoRFP after 2 h and 4 h. Star symbol indicates decayed fluorescence of Pep-1-MitoGFP suspended in medium after 8 h culture. Insert frame in z-stack image shows quantified Pep-1-MitoGFP and MitoRFP expression at each time point, represented by mean area of red fluorescence (pixel) per cell in same section thickness (μm). was repeated using GFP-tagged donor mitochondria (MitoGFP) and MELAS cybrid cells with HcRed1-tagged mitochondria. Confocal microscopy confirmed that donor mitochondria were internalized into the recipient cells, but did not appear to fuse with the host mitochondria during the course of the experiment; only a few exam- ples of co-localized fluorescence could be identified (arrows in Fig. 2B).hlf pli g Three-dimensional fluorescent imaging of live cells coupled with differential interference contrast (DIC) imaging was used to determine a time course for the entry of foreign mitochondria into host cells. Results D Basal OCR, ATP-linked OCR, and maximum capacity OCR were calculated by sub- tracting the non-respiratory background (post-rotenone rate) (Fig. 4B). Significantly lower rates of basal respira- tion, ATP-linked respiration, and maximal respiratory capacity were found in the untreated MELAS cybrid cells compared to control cells. PMD caused increases in basal respiration (1.7-fold increase), ATP-linked respiration (2.2-fold increase) and maximal respiratory capacity (1.8-fold increase), relative to the untreated MELAS group (Fig. 4B). It is contrary to MELAS cybrid cells treating with Pep-1-Mito3243 and its performance of respiratory function was even lower than the untreated. The result reflects the dependence of mitochondrial function on the manipulation of delivered mitochondria. No significant effects were in MELAS cybrid cells that received uncoated mitochondria. Treatment with Pep-1 alone also increased mitochondrial respiration, but the induction of ATP-linked and maximal respiration was significantly lower than after treatment with Pep-1-Mito. PMD alters expression of mitochondria shaping proteins and mitochondrial biogenesis in PMD alters expression of mitochondria shaping proteins and mitochondrial biogenesis in MELAS cybrid cells. To examine the mitochondrial network in rescued MELAS cybrid cells, cells were treated with Pep-1-Mito and the morphology of mitochondria stained with MitoTracker Green was observed using confocal microscopy after 4 days, and compared with untreated cells (Fig. 5A and B). An abundant mitochondrial network and elongated morphology was observed in both the Pep-1 and Pep-1-Mito groups, compared with the MELAS group that exhibited dot- or sphere-like clusters, indicating mitochondrial frag- mentation (Fig. 5A). However, Pep-1 treatment did not significantly reduce the fragmented proportion of mitochondria despite an increase in the proportion of tubular mitochondria from 24 ± 5.6% to 42 ± 5.1% (Fig. 5B). In contrast, PMD not only increased the proportion of tubular mitochondria from 24% to 61% but also decreased the proportion of spherical fragments from 73 ± 10.7% to 31 ± 3.2%. These mitochondria had a similar morphological pattern of tubular and fragmented mitochondria to those in the control group (Fig. 5B). To determine whether PMD affects the expression of mitochondrial-shaping proteins, we performed western blot analysis (Fig. 5C and D). Results revealed a consistent and significant decrease in mitochondrial fusion proteins OPA1 and MFN2, in MELAS cybrid cells compared to controls, and increased expression of the fission protein DRP1. These effects were associated with a significant reduction in expression of the mitochon- drial marker proteins Tom20 and Tim23. These effects in MELAS cybrid cells were reversed after PMD, causing an increase in expression of OPA1, MFN2, Tom20, and Tim23, and a decrease in DRP1 expression, compared to untreated cells. Pep-1 treatment did not affect mitochondrial shaping proteins but did increase the expres- sion of Tom20 and Tim23 protein in MELAS cybrid cells. Moreover, mitochondrial mass, revealed by NAO staining (Fig. 5E), and expression of mitochondrial biogenetic genes PGC-1α, NRF-1 and Tfam (Fig. 5F) were SCieNTiFiC REPOrTS \| 7: 10710 \| DOI:10.1038/s41598-017-10870-5 5 www.nature.com/scientificreports/ www.nature.com/scientificreports/ m/scientificreports/ Figure 3. Sequence and genotyping analysis of Pep-1-Mito-treated MELAS cybrid cells. (A) Sequence electropherograms of mitochondrial MT-TL1 (tRNALeu) showing an identical sequence change (A3243G) in each sample compared to control. Alterations in nucleotide sequence are indicated by arrows in each of three biological repeats (R). Presence of heteroplasmic mitochondrial DNA was found only in Pep-1-Mito samples. PMD alters expression of mitochondria shaping proteins and mitochondrial biogenesis in Control cybrid cells (Con); MELAS cybrid cells (MELAS); MELAS cybrid cells treated with Pep-1- coated mitochondria (Pep-1-Mito), carrier alone (Pep-1) or naked mitochondria (Mito). (B) Tracking mutate mtDNA in Pep-1-Mito-treated MELAS cybrid cells over time using restriction fragment length polymorphism (RFLP). RFLP from PCR-amplified fragments of mitochondrial tRNALeu indicating the mutation with two digested bands at 233 and 213 bp in MELAS cybrid cells, compared to a single 446 bp band in wild-type mtDNA-containing control cells. Percentage of wild-type mtDNA was shown as mean ± SD in control cells (Control) Untreated MELAS c brid cells (MELAS) and follo ing da s of treatment End of treatment ith P Figure 3. Sequence and genotyping analysis of Pep-1-Mito-treated MELAS cybrid cells. (A) Sequence electropherograms of mitochondrial MT-TL1 (tRNALeu) showing an identical sequence change (A3243G) in each sample compared to control. Alterations in nucleotide sequence are indicated by arrows in each of three biological repeats (R). Presence of heteroplasmic mitochondrial DNA was found only in Pep-1-Mito samples. Control cybrid cells (Con); MELAS cybrid cells (MELAS); MELAS cybrid cells treated with Pep-1- coated mitochondria (Pep-1-Mito), carrier alone (Pep-1) or naked mitochondria (Mito). (B) Tracking mutated mtDNA in Pep-1-Mito-treated MELAS cybrid cells over time using restriction fragment length polymorphism (RFLP). RFLP from PCR-amplified fragments of mitochondrial tRNALeu indicating the mutation with two digested bands at 233 and 213 bp in MELAS cybrid cells, compared to a single 446 bp band in wild-type mtDNA-containing control cells. Percentage of wild-type mtDNA was shown as mean ± SD in control cells (Control), Untreated MELAS cybrid cells (MELAS) and following days of treatment; End of treatment with Pep- 1-Mito complexes (Pep-1-Mito, day 0); Non template control (NTC). p < 0.05, compared to MELAS group; p < 0.05, compared to 7 days post treatment. Figure 3. Sequence and genotyping analysis of Pep-1-Mito-treated MELAS cybrid cells. (A) Sequence electropherograms of mitochondrial MT-TL1 (tRNALeu) showing an identical sequence change (A3243G) in each sample compared to control. Alterations in nucleotide sequence are indicated by arrows in each of three biological repeats (R). Presence of heteroplasmic mitochondrial DNA was found only in Pep-1-Mito samples. Control cybrid cells (Con); MELAS cybrid cells (MELAS); MELAS cybrid cells treated with Pep-1- coated mitochondria (Pep-1-Mito), carrier alone (Pep-1) or naked mitochondria (Mito). (B) Tracking mutated mtDNA in Pep-1-Mito-treated MELAS cybrid cells over time using restriction fragment length polymorphism (RFLP). PMD alters expression of mitochondria shaping proteins and mitochondrial biogenesis in RFLP from PCR-amplified fragments of mitochondrial tRNALeu indicating the mutation with two digested bands at 233 and 213 bp in MELAS cybrid cells, compared to a single 446 bp band in wild-type mtDNA-containing control cells. Percentage of wild-type mtDNA was shown as mean ± SD in control cells (Control), Untreated MELAS cybrid cells (MELAS) and following days of treatment; End of treatment with Pep- 1-Mito complexes (Pep-1-Mito, day 0); Non template control (NTC). p < 0.05, compared to MELAS group; *p < 0.05, compared to 7 days post treatment. 6 SCieNTiFiC REPOrTS \| 7: 10710 \| DOI:10.1038/s41598-017-10870-5 www.nature.com/scientificreports/ Figure 4. Seahorse X-24 analysis of oxygen consumption rate (OCR). (A) OCR measured after 4 days of treatment under normal conditions; after injection of oligomycin (1 μM); after injection of uncoupler FCCP (0.5 μM); after injection of electron transport inhibitor rotenone (2 μM) (n = 5). (B) Basal respiration (Basal), ATP-linked and maximal respiratory capacity (Max) quantified by normalization of OCR to total protein OD value. All data were deducted from the non-respiration background for each group (OCR level post-rotenone treatment). Cell respiration measured in control cybrid cells (Con), MELAS cybrid cells (MELAS); MELAS cybrid cells treated with Pep-1 alone (Pep-1), naked mitochondria (Mito), Pep-1-labeled mitochondria (Pep-1- Mito) or Pep-1-labeled mitochondria carrying the mtDNA3243 mutation (Pep-1-Mito3423). p < 0.05, compared to control group; +p < 0.05, compared to MELAS group; #p < 0.05, compared to Pep-1 group; &p < 0.05, compared to Pep-1-Mito group. Figure 4. Seahorse X-24 analysis of oxygen consumption rate (OCR). (A) OCR measured after 4 days of treatment under normal conditions; after injection of oligomycin (1 μM); after injection of uncoupler FCCP (0.5 μM); after injection of electron transport inhibitor rotenone (2 μM) (n = 5). (B) Basal respiration (Basal), ATP-linked and maximal respiratory capacity (Max) quantified by normalization of OCR to total protein OD value. All data were deducted from the non-respiration background for each group (OCR level post-rotenone treatment). Cell respiration measured in control cybrid cells (Con), MELAS cybrid cells (MELAS); MELAS cybrid cells treated with Pep-1 alone (Pep-1), naked mitochondria (Mito), Pep-1-labeled mitochondria (Pep-1- Mito) or Pep-1-labeled mitochondria carrying the mtDNA3243 mutation (Pep-1-Mito3423). p < 0.05, compared to control group; +p < 0.05, compared to MELAS group; #p < 0.05, compared to Pep-1 group; &p < 0.05, compared to Pep-1-Mito group. significantly restored after PMD, compared to untreated MELAS cybrid cells. PMD alters expression of mitochondria shaping proteins and mitochondrial biogenesis in Pep-1 treatment had the same effect, but the induction of mitochondrial biogenesis was not as good as with PMD treatment, particularly in terms of NRF-1 gene expression (Fig. 5E and F). PMD rescues MELAS cybrid cells from oxidative stress injury. To determine whether PMD improves oxidative stress tolerance in MELAS cybrid cells, cells were cultured in regular medium with 200 μM hydrogen peroxide. After 2 h, cell viability was assessed using Calcein-AM and PI staining. Results revealed a significantly higher number of dead cells (PI positive) in the MELAS cybrid cultures, compared to controls (Fig. 6A and B). PMD partially rescued this effect, but Pep-1 did not. Moreover, the morphology of mitochondrial network in cells that survived oxidative stress still showed an extensive tubular structure and fewer spherical fragments, in both control and Pep-1-Mito groups, compared to the MELAS group (Fig. 5C and D). There was no statistical difference in morphological changes between the Pep-1 and MELAS groups (Fig. 5D). Alterations in cellular cytokine production. To further evaluate the mechanisms of cellular stress after PMD, cytokine production was measured using a Bio-Plex Pro Human Cytokine 27-plex panel. Cytokines that showed a statistical difference between untreated MELAS cells and those that received Pep-1-coated or uncoated donor mitochondria, are shown in Table 1 (p < 0.05). PMD moderately influenced the cytokine profile in MELAS SCieNTiFiC REPOrTS \| 7: 10710 \| DOI:10.1038/s41598-017-10870-5 7 entificreports/ Figure 5. Mitochondrial morphology, mitochondria-shaping proteins and mitochondrial biogenesis in rescued cells after 4 days of treatment. (A) Morphology of mitochondria stained with MitoTracker Green observed by confocal microscopy. (B) Quantification of tubular and fragmented mitochondria was further analyzed using a semi-automatic system. Images from three independent areas containing approximately 200–300 mitochondria from about 6–8 cells each were analyzed from each group (n = 3). (C) Western blot analysis of mitochondrial fusion proteins OPA1 (including long and short OPA1 isoforms) and MFN2, fission protein DRP1 and mitochondrial marker proteins Tom20 and Tim23. (D) Protein expression was quantified by densitometry and normalized to GAPDH (n = 3). (E) Mitochondrial amount analyzed by NAO staining and flow cytometry. (F) Expression of mitochondrial biogenetic genes PGC-1α, NRF-1 and Tfam analyzed by RT-PCR. Relative expression levels were determined relative to β-actin. PMD alters expression of mitochondria shaping proteins and mitochondrial biogenesis in p < 0.05, compared to control group; +p < 0.05, compared to MELAS group; #p < 0.05, compared to Pep-1 group; control cybrid cells (Con); MELAS cybrid cells (MELAS); MELAS cybrid cells treated with Pep-1 alone (Pep-1) or Pep-1-labeled mitochondria (Pep-1-Mito). www.nature.com/scientificreports/ Figure 5. Mitochondrial morphology, mitochondria-shaping proteins and mitochondrial biogenesis in rescued cells after 4 days of treatment. (A) Morphology of mitochondria stained with MitoTracker Green observed by confocal microscopy. (B) Quantification of tubular and fragmented mitochondria was further analyzed using a semi-automatic system. Images from three independent areas containing approximately 200–300 mitochondria from about 6–8 cells each were analyzed from each group (n = 3). (C) Western blot analysis of mitochondrial fusion proteins OPA1 (including long and short OPA1 isoforms) and MFN2, fission protein DRP1 and mitochondrial marker proteins Tom20 and Tim23. (D) Protein expression was quantified by densitometry and normalized to GAPDH (n = 3). (E) Mitochondrial amount analyzed by NAO staining and flow cytometry. (F) Expression of mitochondrial biogenetic genes PGC-1α, NRF-1 and Tfam analyzed by RT-PCR. Relative expression levels were determined relative to β-actin. p < 0.05, compared to control group; +p < 0.05, compared to MELAS group; #p < 0.05, compared to Pep-1 group; control cybrid cells (Con); MELAS cybrid cells (MELAS); MELAS cybrid cells treated with Pep-1 alone (Pep-1) or Pep-1-labeled mitochondria (Pep-1-Mito). cybrid cells, by increasing the amounts of IL-1β, IL-8, IL-12 (p70), and VEGF, and reducing the amounts of IL-7 and GM-CSF, compared to untreated MELAS cybrid cells. These effects only partially reflect the cytokine profile in control cells. Treatment with Pep-1 protein alone had only minor effects on the cytokine profile. Delivery of uncoated mitochondria caused similar effects to PMD. These data suggest that PMD has moderate effects on the cytokine profile in MELAS cybrid cells, but this does not seem to be linked to the rescuing effects on mitochon- drial function. cybrid cells, by increasing the amounts of IL-1β, IL-8, IL-12 (p70), and VEGF, and reducing the amounts of IL-7 and GM-CSF, compared to untreated MELAS cybrid cells. These effects only partially reflect the cytokine profile in control cells. Treatment with Pep-1 protein alone had only minor effects on the cytokine profile. Delivery of uncoated mitochondria caused similar effects to PMD. PMD alters expression of mitochondria shaping proteins and mitochondrial biogenesis in These data suggest that PMD has moderate effects on the cytokine profile in MELAS cybrid cells, but this does not seem to be linked to the rescuing effects on mitochon- drial function. 8 SCieNTiFiC REPOrTS \| 7: 10710 \| DOI:10.1038/s41598-017-10870-5 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 6. Cell viability and mitochondrial morphology after oxidation induced by hydrogen peroxide. (A) Double staining of live/dead cells with calcein-AM/PI was used to assess cell survival after 2 h treatment with 200 μM hydrogen peroxide. (B) Apoptotic cells quantified by counting PI-positive cells in images at the same magnification. (C) Morphology of mitochondria stained with MitoTracker Red observed by confocal microscopy of surviving cells under oxidative stress. (D) Quantification of tubular and fragmented mitochondria was further analyzed using a semi-automatic system. Images from three independent areas containing approximately 100–150 mitochondria from about 6–8 cells were analyzed from each group (n = 3). Control cybrid cells (Con); MELAS cybrid cells (MELAS); MELAS cybrid cells treated with Pep-1 alone (Pep-1) or Pep-1-labeled mitochondria (Pep-1-Mito). p < 0.05, compared to control group; +p < 0.05, compared to MELAS group; #p < 0.05, compared to Pep-1 group. Figure 6. Cell viability and mitochondrial morphology after oxidation induced by hydrogen peroxide. (A) Figure 6. Cell viability and mitochondrial morphology after oxidation induced by hydrogen peroxide. (A) Double staining of live/dead cells with calcein-AM/PI was used to assess cell survival after 2 h treatment with 200 μM hydrogen peroxide. (B) Apoptotic cells quantified by counting PI-positive cells in images at the same magnification. (C) Morphology of mitochondria stained with MitoTracker Red observed by confocal microscopy of surviving cells under oxidative stress. (D) Quantification of tubular and fragmented mitochondria was further analyzed using a semi-automatic system. Images from three independent areas containing approximately 100–150 mitochondria from about 6–8 cells were analyzed from each group (n = 3). Control cybrid cells (Con); MELAS cybrid cells (MELAS); MELAS cybrid cells treated with Pep-1 alone (Pep-1) or Pep-1-labeled mitochondria (Pep-1-Mito). p < 0.05, compared to control group; +p < 0.05, compared to MELAS group; #p < 0.05, compared to Pep-1 group. g y p gyt y y g p ( ) Double staining of live/dead cells with calcein-AM/PI was used to assess cell survival after 2 h treatment with 200 μM hydrogen peroxide. (B) Apoptotic cells quantified by counting PI-positive cells in images at the same magnification. PMD alters expression of mitochondria shaping proteins and mitochondrial biogenesis in (C) Morphology of mitochondria stained with MitoTracker Red observed by confocal microscopy of surviving cells under oxidative stress. (D) Quantification of tubular and fragmented mitochondria was further analyzed using a semi-automatic system. Images from three independent areas containing approximately 100–150 mitochondria from about 6–8 cells were analyzed from each group (n = 3). Control cybrid cells (Con); MELAS cybrid cells (MELAS); MELAS cybrid cells treated with Pep-1 alone (Pep-1) or Pep-1-labeled mitochondria (Pep-1-Mito). p < 0.05, compared to control group; +p < 0.05, compared to MELAS group; #p < 0.05, compared to Pep-1 group. Cytokines (pg/ml) Control MELAS Pep-1-Mito Pep-1 Mito IL-1β 11.5 ± 0.35 15.1 ± 1.06* 22.6 ± 1.49& 17.6 ± 1.42 29.9 ± 2.89&+ IL-8 566.8 ± 18.46 558.6 ± 32.23 795.8 ± 74.34& 469.3 ± 14.84& 858.8 ± 0.42&+ IL-7 34.4 ± 2.63 60.4 ± 1.04* 54.7 ± 2.07& 63.9 ± 5.52 66.2 ± 4.80+ IL-12(p70) 110.0 ± 5.06 26.4 ± 4.19* 37.6 ± 5.85& 33.8 ± 14.76 46.9 ± 8.85&+ GM-CSF 666.3 ± 7.30 713.0 ± 18.67* 658.0 ± 13.24& 733.0 ± 28.21 675.9 ± 33.22 VEGF 657.8 ± 9.40 157.1 ± 16.50* 205.7 ± 7.02& 144.3 ± 41.34 187.8 ± 26.88 Cytokines (pg/ml) Control MELAS Pep-1-Mito Pep-1 Mito IL-1β 11.5 ± 0.35 15.1 ± 1.06* 22.6 ± 1.49& 17.6 ± 1.42 29.9 ± 2.89&+ IL-8 566.8 ± 18.46 558.6 ± 32.23 795.8 ± 74.34& 469.3 ± 14.84& 858.8 ± 0.42&+ IL-7 34.4 ± 2.63 60.4 ± 1.04* 54.7 ± 2.07& 63.9 ± 5.52 66.2 ± 4.80+ IL-12(p70) 110.0 ± 5.06 26.4 ± 4.19* 37.6 ± 5.85& 33.8 ± 14.76 46.9 ± 8.85&+ GM-CSF 666.3 ± 7.30 713.0 ± 18.67* 658.0 ± 13.24& 733.0 ± 28.21 675.9 ± 33.22 VEGF 657.8 ± 9.40 157.1 ± 16.50* 205.7 ± 7.02& 144.3 ± 41.34 187.8 ± 26.88 Table 1. Cytokine levels in cell lysates after 3 days of treatment. *p < 0.05, compared to control group; &p < 0.05, compared to MELAS group; p < 0.05, compared to Pep-1-Mito group. Interleukin (IL)-1β, -7, -8, -12; Granulocyte-macrophage colony-stimulating factor (GM-CSF); Vascular endothelial growth factor (VEGF). All pooled data represent mean ± SD n = 3. Table 1. Cytokine levels in cell lysates after 3 days of treatment. p < 0.05, compared to control group; &p < 0.05, compared to MELAS group; p < 0.05, compared to Pep-1-Mito group. PMD alters expression of mitochondria shaping proteins and mitochondrial biogenesis in Interleukin (IL)-1β, -7, -8, -12; Granulocyte-macrophage colony-stimulating factor (GM-CSF); Vascular endothelial growth factor (VEGF). All pooled data represent mean ± SD n = 3. y yt y p p g p &p < 0.05, compared to MELAS group; p < 0.05, compared to Pep-1-Mito group. Interleukin (IL)-1β, -7, -8, -12; Granulocyte-macrophage colony-stimulating factor (GM-CSF); Vascular endothelial growth factor (VEGF). All pooled data represent mean ± SD n = 3. www.nature.com/scientificreports/ as shown by sustained cell survival and morphological elongation of the mitochondrial network in response to oxidative injury. The cytokine profile of the MELAS cybrid cells did not appear to be linked to the rescuing effects of PMD on mitochondrial function. During normal growth conditions, consistent changes in mitochondrial morphology and mitochondrial shaping proteins OPA1, MFN2, and DRP1 were detected only after PMD. OPA1 and MFN2 are central to mito- chondrial fusion, while DRP1 is a key player in mitochondrial fission. PMD not only dramatically increased mitochondrial elongation but also upregulated MFN2 and OPA1 expression, while DRP1 was significantly down- regulated. These results suggest that a combination of increased mitochondrial fusion following a decrease in fission may help to improve resistance to oxidative stress-induced apoptosis32, 33. Indeed, increased cell survival against oxidative stress and a sustained mitochondrial tubular morphology was observed in cells following PMD, compared to untreated MELAS cells. OPA1-induced mitochondrial fusion is also associated with increased OXPHOS activity34. Furthermore, fusion of damaged and functional mitochondria results in the dilution of mutant mtDNA and compensation of mitochondrial components to preserve mitochondrial function. Our data also indicate that mitochondrial mass and biogenesis were consistently increased in MEALS cybrid cells after PMD, as shown by increased Tim23 and Tom20 expression and upregulation of mitochondrial biogenetic genes. Increased mitochondrial biogenesis and mitochondrial mass may support functional recovery of the cells, and could result from successful and efficient delivery of donor mitochondria and/or increased mitochondrial biogen- esis. Thus, our data show that PMD improves cellular viability during oxidative stress, which is likely linked to its protective effects on mitochondrial dynamics and function. Moreover, we suggested that the improved mitochon- drial quality could contribute to the beneficial effects of PMD, because the quality control process is intimately linked to the dynamic behavior of mitochondria, which undergo cycles of fusion and fission and communicate in a number of ways with their cellular environment35. An increase in respiration, number, and elongated morphol- ogy of mitochondria, without a dynamic balance between mitochondrial fusion and fission, was not enough to protect cells from oxidative stress-induced cell death, which was seen following Pep-1 treatment. Stimulating the quality control pathway to drive mitochondrial turnover has recently been considered as a therapeutic target for mitochondrial disorders36. www.nature.com/scientificreports/ We are beginning to explore the regulatory role of selective mitochondrial macroau- tophagy (mitophagy) in PMD therapy.if gy gy y In MELAS, significant effects on respiration and ATP production have been shown to occur when the level of A3243G point mutation reaches above a threshold of 90% to 94%37. In our study, the presence of donor wild-type mtDNA in MELAS recipient cells was confirmed by genotyping. Since fluorescent mitochondria are due to fusion of GFP with the import sequence of the mitochondrial cytochrome c oxidase subunit 8 (COX8) rather than direct labeling with mtDNA, the amount of GFP signal does not accurately reflect the proportion of foreign mtDNA, especially after isolation from cells. A relatively high level of heteroplasmy (percentage of wild-type mtDNA) was observed immediately following PMD treatment (from 4.6 ± 4.6% to 34.6 ± 3.2%) and 11-days post treatment (from 9.0 ± 2.8% to 14.5 ± 2.9%) of stable culture. The proportion of normal mtDNA cannot be maintained at levels as high as at the start of treatment. We postulated that this could be related with mtDNA dilution with the deletion in the rapidly dividing cells38, or an increase in mtDNA replication within the cells39. The level of heter- oplasmy has been reported to affect the transcription of genes involved glycolysis and OXPHOS-related genes in MELAS cybrid cells40 and determined the clinically relevant biochemical defect of diseases41. That could explain why mitochondrial respiration of MELAS cybrid cells was worse after delivery of Pep-1-Mito3243 in this study, due to a probable increase of the mutant proportion of mtDNA. Mitochondrial dysfunction in mitochondrial disease not only depends on the mtDNA heteroplasmy37 but also involved complex regulations such as nuclear gene mutations causing OXPHOS deficiency41. Thus, it is reasonable that the clinical expression threshold of mtDNA proportion can occur over a narrow range (generally a few percent) and various approaches have been used to induce a heteroplasmic shifting to below the mutation threshold41. Furthermore, the absolute amount of mtDNA in cybrid cells harboring both MELAS and wild-type mtDNA was found to correlate with increased respiration, even when the wild-type to mutant ratio remained low42. The increased content of non-mutated mtDNA in the MELAS cells seen after PMD in our study is consistent with the observed improvement in mitochondrial res- piratory function. SCieNTiFiC REPOrTS \| 7: 10710 \| DOI:10.1038/s41598-017-10870-5 Discussion I h In the present study, PMD was shown to comprehensively improve mitochondrial function in a MELAS cybrid model to compare with the Pep-1 treatment which only useful aspect of mitochondrial respiration and biogenesis. Pep-1-Mito entered the MELAS cybrid cells 1 h after treatment, which resulted in increased numbers of both for- eign and innate mitochondria within the cells. 2 days after treatment, clear expression of Pep-1-MitoGFP could still be observed within the cells, and normal mtDNA was increased by approximately 6%, relative to untreated cells. A significant increase of normal mtDNA was observed immediately and 11-days post treatment in the cells. After 4 days of PMD treatment, mitochondrial function was restored, and was associated with enhanced mitochondrial mass, mitochondrial biogenesis and mitochondrial fusion versus fission. Recovery regulation depends on the function of the delivered mitochondria. Pep-1 treatment showed similar regulation to PMD, except for the mito- chondrial dynamic balance. Only PMD treatment was able to effectively improve mitochondrial functionality, SCieNTiFiC REPOrTS \| 7: 10710 \| DOI:10.1038/s41598-017-10870-5 9 www.nature.com/scientificreports/ Materials and Methods ll l Cell culture. Human 143B osteosacroma cybrid cells harboring wild-type (control cybrid) or A3243G mutant mtDNA (MELAS cybrid) were kindly donated by Prof. Y.H. Wei, as previously described51, with permission from the Institutional Review Board of National Yang-Ming University (approval No: IRB-1000030). All experimental procedures were approved by the Institutional Biosafety Committee of Changhua Christian Hospital (approval No: BS-T-004) and were carried out in accordance with the approved relevant guidelines and regulations. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, high glucose) (Gibco/Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, GeneDireX, Las Vegas, NV, USA), 100 mg/mL sodium pyruvate (Gibco) and 1% PS (100 U/L penicillin G sodium, 100 mg/L streptomycin sulfate) (Gibco). For starva- tion experiments, cells were cultured in low glucose (1 g/L) DMEM (Gibco) supplemented with 2% FBS and 1% PS. Cells were cultured at 37 °C in a humidified incubator with 5% CO2. Mitochondrial labeling with fluorescent proteins. Donor (143B osteosarcoma cybrid) cells were transfected with a vector carrying a mitochondrial targeting fluorescent protein (GFP gene from Aequorea coer- ulescens, Ac-GFP). At 80% confluency, cells were transfected with 40 μg plasmid DNA encoding mitochondrial matrix-localized GFP (import targeting sequence of cytochrome c oxidase subunit 8, COX8) (Clontech, Palo Alto, CA, USA) using 60 μL PureFection transfection reagent (System Bioscience, SBI, Mountain View, CA, USA) according the manufacturer’s instructions. After 36 h, cells were transferred to normal growth medium for 4 h. Following G418 selection, cells containing GFP-tagged mitochondria (MitoGFP) were prepared for mitochondrial isolation. Mitochondria in MELAS cybrid cells were labeled with red fluorescence by transfecting with a plasmid encoding mitochondrial matrix-localized far-red fluorescent protein HcRed1 (MitoRFP) fused to the mitochon- drial targeting sequence of COX8 (Clontech)52, as described above. Mitochondrial labeling with fluorescent dyes. Mitochondria were stained while within donor cells, prior to their isolation, using a MitoTracker dye (100 nM final concentration; MitoTracker Red CMXRos or MitoTracker Green FM; Invitrogen-Molecular Probes, Eugene, OR, USA) for 20 min in a 37 °C incubator. Cells were then washed twice with PBS to remove any remaining dye. Mitochondrial isolation and Pep-1-mediated delivery. Purification of mitochondria, Pep-1 conju- gation and isolation of mitochondria were conducted as previously described30, 31. Donor mitochondria were isolated from human 143B osteosacroma cybrid cells harboring either wild-type or A3243G mutant mtDNA, according to experimental needs. www.nature.com/scientificreports/ Furthermore, although mitochondrial function was not examined after long-term culture, we suggest that PMD could support the restoration of mitochondria in MELAS cells as well as in MERRF31, because normal mtDNA did not decline over time, but rather increased after 11 days of treatment. ,t y Unlike other CPPs, such as Tat or penetratin, which are internalized through a form of endocytosis43, Pep-1 has a high affinity for lipidic membranes such that it can insert itself into the membrane bilayer and induce local destabilization to facilitate cell uptake44. Hence, it is not surprising that Pep-1 was able to alter the structure of the mitochondrial membrane in a dose-dependent manner. Pep-1 treatment not only induced fusion of two lipid bilayers of isolated mitochondria, but also increased tubular network formation in mitochondria within the cells, without affecting mitochondria-shaping proteins. To date, the effect of Pep-1 on mitochondrial function whether in vitro or in vivo, remains unclear. Recently, novel CPP targeting mitochondria (mtCPP) were developed to prevent mitochondrial damage by oxidative stress, which relied on their own antioxidant properties45. This study also demonstrated no cytotoxicity, even at high concentrations (100 µM)45. Thus, we suggest that mem- brane fusion caused by appropriate concentrations of Pep-1 rather improved function of mitochondria in vitro or in vivo. Moreover, we found that mitochondrial respiration as well as mitochondrial biogenesis and morpho- logical elongation were increased in Pep-1-treated MELAS cybrid cells, although it failed to sustain cell survival after oxidative damage. We suggested that invalid regulation of mitochondrial dynamic in Pep-1 treatment could cause cells to lose the balance of mitochondrial fusion-fission to resist environmental stress via mitochondrial turnover46. Our previous study invalidated treatment with Pep-1 alone for neuroprotection in Parkinson’s disease47, which is in agreement with Meloni et al. who showed that the neuroprotective efficacy of CPPs is dependent on the diversity of CPPs and neuronal diseases48. Nonetheless, how dose Pep-1 regulate the genes and proteins involved in mitochondrial biogenesis and mitochondrial respiration are still worthy of further study. SCieNTiFiC REPOrTS \| 7: 10710 \| DOI:10.1038/s41598-017-10870-5 10 www.nature.com/scientificreports/ Cellular stress due to mitochondrial function may affect immune responses, as observed in patients suf- fering from primary mitochondrial disorders49. We observed changes in the release of cytokines and chemok- ines, including IL-1β, -7,-12, GM-CSF and VEGF, from MELAS cybrid cells compared to control cybrid cells. www.nature.com/scientificreports/ Delivery of functional mitochondria by PMD partly attenuated the effects on IL-7, IL-12, GM-CSF and VEGF. However, it remains unclear whether mitochondrial transplantation has the potential to counteract some of the immunological aberrations in MELAS cells. There is also a potential for an inflammatory reaction against donor mitochondria in vivo. The presence of donor mitochondria caused increased levels of IL-1β, an important proinflammatory cytokine that is also involved in inflammatory disease50. Pep-1 alone did not induce significant changes in cytokine production in MELAS cybrid cells, and coating mitochondria with Pep-1 resulted in weaker upregulation of IL-1β, −8 and −12 compared to uncoated mitochondria. This could suggest that coating donor mitochondria with Pep-1 produces a weaker inflammatory reaction compared to uncoated donor mitochon- dria. A recent study using autologous mitochondrial transplantation in an animal model of ischemia-reperfusion injury found a decrease in inflammatory markers such as CRP, IL-6 and TNFα after transplantation compared to vehicle-only controls. In this study, CPP coating was not used to facilitate mitochondrial internalization, and only isolated mitochondria were injected into the target tissue22. Additionally, there was no development of anti-mitochondrial antibodies one month after transplantation, suggesting that at least autologous mitochondria are non-immunogenic in vivo22. g In conclusion, our results suggest that PMD treatment can partly repair mitochondrial defects and improve cellular stress tolerance in cultured MELAS cells via regulation of mitochondrial quality, and are consistent with our previous studies using a MERRF cybrid model30. Future studies should be aimed at investigating the potential efficacy of PMD in animal models of mitochondrial disease. SCieNTiFiC REPOrTS \| 7: 10710 \| DOI:10.1038/s41598-017-10870-5 Materials and Methods ll l Mitochondria isolated from mouse liver (5 mg/mL) were conjugated with 5 (Mito/Pep-1, 339:1), 25 (Mito/Pep-1, 68:1) or 50 μM (Mito/Pep-1, 34:1) Pep-1 to observe mitochondrial ultrastructure. Mixtures were centrifuged at 9000 × g for 2 min. Pellets were then fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 4 h at room temperature as around 20 to 25 °C. After three rinses in 0.1 M phosphate buffer for 15 min each, samples were dehydrated through a graded ethanol series (30%, 50%, 70%, 85%, 90% and 100%) for 20 min each. Samples were infiltrated with LR white resin in a gelatin capsule and stored at 4 °C for 48 h. Capsules were polymerized at 60 °C for 16 h, before cutting 70 nm ultrathin sections (Leica EM UC7, Germany). Sections were viewed using a transmission electron microscope (TEM) (Hitachi H-7000, Japan). The proportion of membrane-fused mitochondria was calculated by counting the number of mitochondria with any one side in contact with another within the total number of mitochondria in each image. Approximately 200 mitochondria were counted in an individual image. Confocal microscopy. For visualization of mitochondria, cells were seeded onto a chamber slide after 4 days of treatment (µ-Slide 8 well, Ibidi GmbH, Martinsried, Germany), stained with Mitotracker Green (250 nM) (Invitrogen) and placed in an incubator at 37 °C for 40 min. After staining, the remaining dye was removed, and stained cells were mounted onto a perfusion chamber in culture media and imaged at 37 °C using an Olympus FluoView FV 1200 confocal microscope (Olympus, Tokyo, Japan). p y p y p To track the internalization of Pep-1-labeled MitoGFP (Pep-1-MitoGFP) in MELAS cybrid cells expressing MitoRFP, three dimensional (3D) reconstructions were generated from confocal microscopy images combined with difference interference contrast (DIC). To determine the distribution of Pep-1-MitoGFP and innate MitoRFP within host cells, line scans through the z-axis were integrated to yield a longitudinal view, which were combined (0.55 μm z-steps) using Olympus Fluoview Viewer software. Innate MitoRFP and internalized Pep-1-MitoGFP in host cells were quantified at different time points by calculating the mean area of red and green fluorescence (pixels) per cell within the same section thickness (μm) using ImageJ Software. The expression of MitoRFP and Pep-1-MitoGFP in the incomplete cells was excluded to calculate in the z-stack images. Genotyping. DNA was extracted from cultured cells using a Qiagen DNeasy kit (Qiagen, Valencia, CA, USA). Materials and Methods ll l Presence of the MELAS A3243G mutation in mtDNA was determined using a polymerase chain reac- tion (PCR)-restriction fragment length polymorphism (RFLP) analysis with Apa I, as previously described53. A DNA fragment of 446 bp encompassing a putative mutation at nucleotide position (np) 3243 was amplified by PCR using a forward primer from np 3010 to 3029 and a reverse primer from np 3436 to 3456. Amplification was performed for 36 cycles of denaturation at 94 °C for 1 min, annealing at 54 °C for 1.5 min, and elongation at 72 °C for 1 min. DNA was then digested at 25 °C overnight in 3 μL 10 × BSA, 3 μL NEB Buffer 4 and 0.8 μL Apa I (New England Biolabs, Ipswich, UK). The mtDNA carrying the A3243G mutation was cut by Apa I resulting in digestion bands of 233 and 213 bp. In the absence of the mtDNA mutation, the PCR product would not be cut and would remain at its original size of 446 bp. Digestion of the mutant DNA did not affect wild-type DNA. Digested products were visualized by electrophoresis on a 4% agarose gel with 0.3 μg/mL ethidium bromide. The proportion of mtDNA with the A3243G mutation in each group was calculated by normalizing the mutant DNA fragments of length 233 and 213 bp to total band intensity using GelPro Analyzer (Media Cybernetics, Silver Spring, MD, USA).h The mtDNA A3243G mutation ratio was determined by sequencing the PCR products, which were randomly sampled then sequenced using an ABI 3130xl genetic analyzer and a BigDye Terminator v1.1 cycle sequencing kit (Applied Biosystems, Foster City, CA). To confirm the sequencing results, the position 3243 A to G mutation of mtDNA heteroplasmy was performed by using an automated DNA sequencer (ABI Prism 310 Genetic Analyzer, PE Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Mitochondrial respiration analysis. Mitochondrial respiration was measured using a Seahorse Xfe24 Analyzer (Agilent Technologies). After 4 days of treatment, cells were seeded onto a XF24 microplate (30,000 cells/well) in normal culture medium and cultured for 16–18 h. The culture medium was then replaced by analysis medium (DMEM, high glucose, without FBS, and sodium bicarbonate) and incubated without CO2 for 1 h before analysis according to the manufacturer’s protocol. Basal oxygen consumption rate (OCR) was measured first, then after sequential injections of three compounds that affect bioenergetics: 1 μM oligomycin (Sigma, St. Materials and Methods ll l The isolation procedure was performed using a mitochondria isolation kit for cultured cells, according to the manufacturer’s protocol (Thermo Fisher Scientific, Carlsbad, CA, USA). Briefly, donor cells were detached from the plate by trypsinization, harvested, and centrifuged at 1000 rpm for 5 min. Twenty million cells were collected for each mitochondrial isolation procedure. Cells were washed twice in ice-cold PBS before beginning the isolation procedure. Cells were homogenized on ice in isolation reagent sup- plemented with proteinase inhibitor (EMD Millipore, Billerica, MA, USA) using a glass Dounce tissue grinder. After adding an equal volume of separation buffer, the homogenate was centrifuged at 3000 rpm for 10 min at 4 °C to separate cytosol and intact cells. To exclude other organelles, the cytosol was centrifuged at 3000 × g for 15 min, the pellet was suspended in separation buffer and collected by centrifugation at 12000 × g for 3 min, at 4 °C. Protein concentration was determined using a bicinchoninic acid (BCA) assay kit (Pierce Biotechnology, Rockford, IL, USA). For delivery of mitochondria, 200,000 cells were plated onto 10-cm dishes and received 105 μg mitochondria coated with Pep-1 (Anaspec, San Jose, CA, USA) (Pep-1-Mito) at the appropriate weight ratio (Mito/Pep-1, 1.75:1). Pep-1 concentration was about 50 μM. After 2 days of exposure to Pep-1-Mito, recipient cells were washed twice with PBS before further culturing in regular medium.i g g For mitochondrial isolation from mouse liver, mice was sacrificed and perfused with 50 mL 0.9% NaCl. Li were sliced and homogenized using a Dounce homogenizer with 5 mL isolation buffer (0.25 M sucrose, 0.5 SCieNTiFiC REPOrTS \| 7: 10710 \| DOI:10.1038/s41598-017-10870-5 11 www.nature.com/scientificreports/ EGTA, 3 mM HEPES-NaOH, pH 7.2) with protease inhibitors. All operations were carried out at 0–4 °C. The homogenate was centrifuged for 15 min at 1000 × g. The supernatant was collected, layered onto a sucrose gra- dient and centrifuged for 30 min at 35000 × g. The sucrose concentration gradient was from 30%, 40% and 55% for isopycnic banding and from 40% to 55% when mitochondria were collected in a pellet. The pellet was resus- pended in isolation buffer and centrifuged twice at 13000 × g for 3 min each. Resuspended rat mitochondria were prepared for ultrastructural analysis after Pep-1 conjugation as described below. Transmission electron microscopy. Materials and Methods ll l Membranes were incubated with primary anti- bodies: anti-OPA1 (NOVUS Biologicals, Littleton, CO, USA; 1:1000), anti-MFN2 (NOVUS Biologicals, 1:1000), anti-DRP1 (Santa Cruz, CA, USA; 1:500), anti-Tom 20 (Santa Cruz; 1:1000), anti-Tim 23 (Santa Cruz; 1:1000) and anti-GAPDH (Santa Cruz; 1:1000), followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (goat anti-mouse-HRP or goat anti-rabbit; Jackson ImmunoResearch, PA, USA; 1:50000). Signal was detected using an ECL western blotting detection system (Millipore). Mitochondrial mass assay. Mitochondrial mass was measured using a nonyl acridine orange (NAO) stain. Treated cells were incubated with medium containing 100 nM NAO for 20 min at 37 °C. Cells were washed twice in PBS, centrifuged at 1200 rpm for 5 min, and fluorescence was detected using flow cytometry (Beckman Coulter, CA, USA). Data were analyzed using Flowjo software (TreeStar, OR, USA). RNA extraction and quantitative real-time RT-PCR analysis. Total RNA was purified from cells using a NucleoSpin RNA kit (Macherey-Nagel, Düren, Germany) and reverse-transcribed using a Transcriptor First Strand cDNA Synthesis kit (Roche Applied Science, Indianapolis, IN, USA). Expression of mitochondrial bioge- netic genes including peroxisomal proliferator activator receptor gamma coactivator 1a (PGC-1α, forward primer: 5′ GGAGAGGCAGAGGCAGAAGG-3′ and reverse primer: 5′-AAGCATCACAGGTATAACGGTAGG-3′), nuclear respiratory factor 1(NRF-1, 5′-CCGTGGCTGATGGAGAGGTGGAAC-3′ and forward primer: 5′-CTGATGCTTGCGTCGTCTGGATGG-3′) and mitochondrial transcriptional factor A (Tfam, forward primer: 5′-GGAGTTGTGTATTGCCAGGAG-3′ and reverse primer: 5′-CTTCGGAGAAACGCCATCG-3′) were determined by quantitative real-time RT–PCR using SYBR Green PCR Master Mix (Roche Applied Science) and an ABI Prism 7300 system (Applied Biosystems). Reaction parameters were 2 min hold at 50 °C, 10 min hold at 95 °C, followed by 40 cycles of 15 s at 95 °C, and 1 min at 60 °C. All measurements were performed in triplicate. mRNA expression was normalized to β-actin, and presented as the relative expression level. Cell viability after oxidative stress. Cells were seeded onto 24-well plates with 2000 cells per well, allowed to adhere overnight, then treated with 200 µM hydrogen peroxide for 2 h in regular medium at 37 °C. To examine cell viability after oxidative induction, cells were washed twice with PBS and double stained with 1 μM Calcein-AM (Calcein acetoxymethyl ester) (Invitrogen, South San Francisco, CA, USA) and 1 μM propidium iodide (PI) (Invitrogen) at 37 °C for 30 min. Viable and apoptotic cells were observed using an inverted fluores- cence microscope (IX81, Olympus, Tokyo, Japan). Multiplex cytokine assay. Materials and Methods ll l Louis, MO, USA), 0.5 μM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (Sigma) and 2 μM rote- none (Sigma). After completion of the analysis, protein was measured using a BCA assay (Pierce Biotechnology). Results were normalized to the protein OD value of corresponding wells. Analysis of mitochondrial morphology. For visualization of mitochondria, stained cells were mounted onto a perfusion chamber in culture media and imaged at 37 °C using an Olympus FluoView FV 1200 confocal microscope (Olympus, Tokyo, Japan). Subtyping of mitochondrial morphology was quantified using an auto- matic classification system according to Peng et al.54. After semi-automatic segmentation of cell micrographs, mitochondria were classified into six distinct subtypes (small globe, swollen globe, straight tubule, twisting tubule, branch tubule and loop) using automatic classification software. Considering the error of automatic anal- ysis caused by fuzzy staining of mitochondria due to excessive dye loading, the mitochondrial loop subtype was SCieNTiFiC REPOrTS \| 7: 10710 \| DOI:10.1038/s41598-017-10870-5 12 www.nature.com/scientificreports/ excluded. The proportion of tubular mitochondria was calculated by totaling the straight tubule, twisting tubule and branch tubule mitochondrial populations. The proportion of fragmented tubular mitochondria was calcu- lated by totaling the small globe and swollen globe mitochondrial populations. Micrographs from three inde- pendent areas per group were analyzed, and about 200–300 mitochondria from about 6–8 cells in each image was calculated. excluded. The proportion of tubular mitochondria was calculated by totaling the straight tubule, twisting tubule and branch tubule mitochondrial populations. The proportion of fragmented tubular mitochondria was calcu- lated by totaling the small globe and swollen globe mitochondrial populations. Micrographs from three inde- pendent areas per group were analyzed, and about 200–300 mitochondria from about 6–8 cells in each image was calculated. Western blot. Following incubation, treated cells were washed with PBS and collected into RIPA buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 1% NP40, 0.25% Na-deoxycholate, 1 mM PMSF) (Pierce Biotechnology) containing protein inhibitor (Sigma) and phosphatase inhibitor cocktails (Pierce Biotechnology). Cells were incubated on ice for 20 min then homogenized. Extracts were spun down at 14,000 × g for 10 min at 4 °C and supernatants analyzed using the Bradford assay. Total cell lysates were separated by SDS–PAGE, and transferred onto Immobilon-P membranes (Millipore, Bedford, MA, USA). Materials and Methods ll l Three days post-treatment, cell lysates were extracted using RIPA buffer (Millipore) containing proteinase inhibitor (Millipore) at 4 °C. Protein concentration was measured using a BCA assay kit (Pierce Biotechnology). Multiplex cytokines in cell lysates with a final protein concentration of approx- imately 2 mg/mL were analyzed using a BioplexTM Pro-human cytokine 27-plex panel (Bio-Rad Laboratory, Hercules, CA, USA), according to manufacturer’s guidelines. Briefly, 50 μL antibody-conjugated beads were added to a 96-well filter plate and adhered using vacuum filtration. Fifty microliters of pre-diluted standards, blanks or samples were individually transferred into the wells after washing. The plate was incubated at room temperature on a shaker at 850 rpm for 30 min. After washing, 50 μL pre-diluted streptavidin-conjugated PE was added and incubated at room temperature on a shaker for 10 min. After washing, 125 μL assay buffer was added to each well, and the plate was placed onto a shaker for 30 seconds. The concentration of each cytokine was deter- mined using a BioRad BioPlex 200 instrument with BioManager v6.0 software (Bio-Rad). All groups were run with triplicate samples. Statistical analysis. 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Y.W. and F.C. provided research resources. S.K. and C.L. provided research funding. F.H. and J.C. wrote the paper. Acknowledgementsh g This research was supported by the Changhua Christian Hospital (104-CCH-IST-005) and National Science Council (MOST 103-2320-B-371 -001-, MOST 104-2314-B-371 -001 -MY2). We thank Prof. Hong-Lin Su of National Chung Hsing University, Taiwan, for their effort in providing us with a TEM data for mitochondrial National Chung Hsing University, Taiwan, for their effort in providing us with a TEM data for mitochondria morphology. www.nature.com/scientificreports/ Nature reviews Molecular cell biology 15, 634–646 (2014). 0. Picard, M. et al. Progressive increase in mtDNA 3243A > G heteroplasmy causes abrupt transcriptional reprogramming. Proc Nat Acad Sci USA 111, E4033–4042, doi:10.1073/pnas.1414028111 (2014). 41. Wallace, D. C., Fan, W. & Procaccio, V. Mitochondrial energetics and therapeutics. Annual Review of Pathological Mechanical Disease 5, 297–348 (2010).i 42. Bentlage, H. A. & Attardi, G. Relationship of genotype to phenotype in fibroblast-derived transmitochondrial cell lines carrying the 3243 mutation associated with the MELAS encephalomyopathy: shift towards mutant genotype and role of mtDNA copy number. Hum Mol Genet 5, 197–205 (1996). 43. Filipovska, A., Smith, R. A. & Murphy, M. P. Cell-penetrating peptides do not cross mitochondrial membranes even when conjugated to a lipophilic cation: evidence against direct passage through phospholipid bilayers. Biochemical Journal 383, 457–468 (2004). 44. Henriques, S. T. & Castanho, M. A. R. B. Consequences of nonlytic membrane perturbation to the translocation of the cell penetrating peptide pep-1 in lipidic vesicles. Biochemistry 43, 9716–9724 (2004).h p g p p p p p y 5. Cerrato, C. P., Pirisinu, M., Vlachos, E. N. & Langel, Ü. Novel cell-penetrating peptide targeting mitochondria. The FASEB Journa 29, 4589–4599 (2015). SCieNTiFiC REPOrTS \| 7: 10710 \| DOI:10.1038/s41598-017-10870-5 14 SCieNTiFiC REPOrTS \| 7: 10710 \| DOI:10.1038/s41598-017-10870-5 Additional Informationh Competing Interests: The authors declare that they have no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. 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https://openalex.org/W3142128510	https://zenodo.org/records/3899985/files/10.25255.jss.2020.9.2.354.372.pdf	Arabic	null	The types of escape in the Holy Quran and its faith significance	Journal of social sciences	2,020	cc-by	13,664	Journal of Social Sciences (COES&RJ-JSS) ISSN (E): 2305-9249 ISSN (P): 2305-9494 Publisher: Centre of Excellence for Scientific & Research Journalism, COES&RJ LLC Online Publication Date: 1st April 2020 Online Issue: Volume 9, Number 2, April 2020 https://doi.org/ 72 3. 2.354 .9. 20 .25255/jss.20 10 The types of escape in the Holy Quran and its faith significance Dr. Marwa Mahmoud Kharma http://orcid.org/0000-0001-5112-4001 Associate Professor in creed United Arab Emirates University (UAEU), Dubai, UAE Email: marwaomarsemobas@uaeu.ac.ae Journal of Social Sciences (COES&RJ-JSS) ISSN (E): 2305-9249 ISSN (P): 2305-9494 Publisher: Centre of Excellence for Scientific & Research Journalism, COES&RJ LLC Online Publication Date: 1st April 2020 Online Issue: Volume 9, Number 2, April 2020 https://doi.org/ 72 3. 2.354 .9. 20 .25255/jss.20 10 The types of escape in the Holy Quran and its faith significance Dr. Marwa Mahmoud Kharma http://orcid.org/0000-0001-5112-4001 Associate Professor in creed United Arab Emirates University (UAEU), Dubai, UAE Email: marwaomarsemobas@uaeu.ac.ae The types of escape in the Holy Quran and its faith significance Dr. Marwa Mahmoud Kharma http://orcid.org/0000-0001-5112-4001 Associate Professor in creed United Arab Emirates University (UAEU), Dubai, UAE Email: marwaomarsemobas@uaeu.ac.ae The types of escape in the Holy Quran and its faith significance . Creative Commons Attribution 4.0 International License This work is licensed under a Abstract: This study dealt with induction the pronunciation (escape) and its derivatives in the Holy Quran, and aims to clarify the types of escape mentioned in the Holy Quran and its faith significance. The study included a preliminary and two topics: The first topic: the escape from a specific, and has two requirements; the first requirement: escape from a certain plainly (whether in the world or the hereafter), the second requirement: escape from a certain unauthorized but known from the context, and the second topic: the escape from a non- appointed. Then stamped with a conclusion in which the most important results زAmong the most prominent results was that the escape contained in the Holy Qur’an can be divided in two ways: In terms of the escape from it, and in terms of the extent of the use to escape, and Every escape mentioned in the Noble Qur’an was a definite escape from it and not beneficial to its companions. Except for fleeing to God Almighty, it is general and includes escape from everything to Him. And it is the only escape that realizes safety and contentment. Keywords: Escape, appointed and unassigned, to be escaped from Citation: Kharma, Marwa Mahmoud (2020); The types of escape in the Holy Quran and its faith significance; Journal of Social Sciences (COES&RJ-JSS), Vol.9, No.2, pp:354- 372; https://doi.org/10.25255/jss.2020.9.2.354.372. . Creative Commons Attribution 4.0 International License This work is licensed under a Journal of Social Sciences (COES&RJ-JSS), 9(2), pp.354-372 إاا م ي ع إعداد الباحثة: د.مروه محمود خرمه إاإ ر ر إ أستاذ مشارك/ قسم الشريعة والدراسات اإلسالمية/ كلية القانون/ جامعة اإلمارات العربية المتحدة الملخص تناولت هذه الدراسة استقراء لفظ ( الفرار ) ومشتقاته في القرآن الكريم، و تهدف إلى بيان أنواع الفرار المذكور في القر آ ن الكريم ودالالته اإليمانية، وقد شملت الدراسة تمهيد ا ومبحثين: المبحث األول: الفرار من مُعيَّن؛ وفيه مطلبان: المطلب األول: فرار من معين مصرح به (سواء في الدنيا او اآلخرة) ، المطلب الثاني: فرار من معين غير مصرح به لكنه معلوم من السياق، والمبحث الثاني: الفرار من غير المعين. ثم ختمت بخاتمة فيها اهم النتائ ج. التي من أبرز ه أن ها يمكن تقسيم الفرار الوارد في القرآن الكريم من حيثيتين: من حيث ذكر المهروب منه، ومن حيث مدى االنتفاع بالفرار، وكان كل فرار مذكور في القران الكريم محدد المهروب منه وغير نافع ألصحابه، إال الفرار الى هللا تعالى فهو عام يشمل الفرار من كل ما سواه إليه سبحانه ، وهو الفرار الوحيد الذي يثمر أمانا واطمئنانا. الكلمات المفتاحية : الفرار، المعين وغير المعين، المهروب منه. هلالمقدمة الحمد هلل رب العالمين والصالة والسالم على سيد المرسلين وعلى آله وصحبه أجمعين وبعد؛ فقد ورد في القرآن الكريم لفظ (الفرار) ومشتقاته في مواضع عدة ، واختلفت دالالت ذكره وفق ال سياقات و م ضمون اآليات الوارد فيها، فما بين فرار فزع وخوف في الدينا ، وبين فرار خوف في اآلخرة، وبين فرار م ما ذكر المهروب منه صراحة او ضمنيا، وبين فرار لم يذكر فيه المهروب منه، بين فرار يتبعه أمان، وفرار متوهم ال يحقق نفعا ال في الدنيا وال في االخرة، ومن هنا جاءت فكرة البحث الستقراء مفهوم الفرار في القرآن الكريم والتعرف إلى انواعه وبيان الدالالت اإليمانية لكل منها، فجاء البحث بعنوان: " أنواع الفرار في القرآن الكريم ودالالته اإليمانية"، وأسأل هللا تعالى التوفيق والقبول. ي ر : تظهر أهمية هذا البحث في استقرائه لجميع أنواع الفرار المذكورة في القرآن الكريم وتتبع سياقاتها واالجتهاد في تقسيمها من حيث ذكر المهروب منه صراحة او ضمنيا او عدم ذكره، ومن حيث ذكر النافع من أنواع الفرار من غير النافع . ت هدف الدراسة بناء على ذلك إلى: 1. بيان أنواع الفرار في القران الكريم. 2. .اتقسيم الفرار من حيث ذكر المهروب منه، ومن حيث مدى االنتفاع به أ 3. .بيان مدى انتفاع الفار من فراره وأسباب االنتفاع لله 3. .هللبيان مدى انتفاع الفار من فراره وأسباب االنتفاع 4. }توضيح سر اختصاص اية{ففروا الى هللا م بعد ذكر المهروب منه. 4. }توضيح سر اختصاص اية{ففروا الى هللا م بعد ذكر المهروب منه. لله 4. }توضيح سر اختصاص اية{ففروا الى هللا م بعد ذكر المهروب منه. 5. .}التعرف إلى آراء المفسرين في بيان المهروب منه في قوله تعالى:{ففروا الى هللا 6. إبر از الدالالت اإليمانية لما ذكر في القران الكريم من أنواع الفرار. إشكالية الدراسة: يحاول هذا البحث اإلجابة عن التساؤالت اآلتية: }لله { ح م 5. .}التعرف إلى آراء المفسرين في بيان المهروب منه في قوله تعالى:{ففروا الى هللا 6إبر القران الكريم من أنواع الفرار از الدالالت اإليمانية لما ذكر ف }لله { ح م 5. .}التعرف إلى آراء المفسرين في بيان المهروب منه في قوله تعالى:{ففروا الى هللا أإاا 5. .}ااإالتعرف إلى آراء المفسرين في بيان المهروب منه في قوله تعالى:{ففروا الى هللا 5. .}التعرف إلى آراء المفسرين في بيان المهروب منه في قوله تعالى:{ففروا الى هللا 6. إبر از الدالالت اإليمانية لما ذكر في القران الكريم من أنواع الفرار. إشكالية الدراسة: 355 355 The types of escape in the Holy Quran and its faith significance 356 3. أي الفرار كان نافعا وايها لم يكن كذلك؟ 4. متى تكون الفائدة متوهمة من الفرار ومتى تكون حقيقية؟ 5. ما سر اختصاص االية{ففروا الى هللا} بعدم ذكر المهروب منه؟ 6. ما آراء المفسرين في بيان المهروب منه في قوله تعالى:{ففروا الى هللا}؟ 7. ما الدالالت اإليمانية لما ذكر في القران الكريم من أنواع الفرار؟ الدراسات السابقة: بعد البحث واالطالع لم أجد بحثا متخصصا في موضوع هذا البحث مما يزيد من أهمية الكتابة فيه. خطة البحث : قسمت البحث الى تمهيد ومبحثين: المبحث األول: الفرار من مُ ؛ن عيَّ وفيه مطلبان: المطلب األول: فرار من معين مصرح به (سواء في الدنيا او اآلخرة) ، المطلب الثاني: فرار من معين غير مصرح به لكنه معلوم من السياق، و المبحث الثاني: ال فرار من غير ال معين. ثم ختمت بخاتمة فيها اهم النتائج، وما توفيقي اال باهلل. تمهيد : الفِرار والمفرّ لغتان، وقيل: بل المفرّ : المَهْرَب، وهو الموضع الّذي يهرب إليه. ورَ جُلٌ فَرُورٌ وفَرُورةٌ من الفِرار. ورجلٌ فَرٌّ ورَ جُالن فَرٌّ ورجال فَرٌّ ال يثنى وال يجمعوالفَرُّ : مَصْدَر فَرَ رْ تُ عن أَسْنانِ الدّابّة، أي: كَشَفْت عنها. وافْتَرَّ عن ثَغْرِ ه إذا تَبَسَّم . وفَرَّ فالنٌ عمّا في نَفْسِهِ، وفُرَّ عن هذا األمر، أي: فَتِّشْهُ. والفَرْ فَرةُ: الطَّيْش والخِ فّة، ورَ جُلٌ فَرْ فار، وامرأةٌ فَرْ فارةٌ. وما زال فُالنٌ في أُفُرَّةِ شَرٍّّ من فالن، [أي: في أوّ ل] . والفَرُّ : الرّ جلُ الفارُّ ، وأَفْرَرْ تُه: ألجأتُه إلى الفرار1. ُوالفَرّ والفِرارُ: الرَّوَ غان وَ الْهَرَب2 ُ.. فَرَّ يَفِرُّ فِرَ ارًا: هَرَبَ، وَ رَجُلٌ فَرورٌ وفَرورةٌ وفَرَّار: غَيْر كَرَّ ارٍّ ، وفَرٌّ ، وَ صْفٌ بالمصدرْ ، فَالْوَ احِ دُ وَ الْجَمْعُ فِيهِ سَوَ اءٌ 3 : ِ.و{الفَرُّ ، بالفتْح،} والفِرَ ارُ، بالكَسْر الرَّوَ غانُ وال هَرَب من شَيْءٍّ خافَه، {كالمَفَرِّ ، بالفَتْح،} والمَفِرِّ ، بكَسْر الفاءِ مَعَ فَتْح المِيم، وَ الثَّانِي يُسْتَعْمَل لَمْوضِ عِه، أَي {الفِرارِ ، أَيضاً، وَ قد} فَرَّ {يَفِرُّ فِرَ اراً: هَربَ، فَهُوَ فَرُورٌ، كصَبُور،}4 . فالفِرار: الرَوَ غَانُ والهَرَب أي (ابت عاد عما يواجِ ه بخفة وإسراع (استرسال): {كَأَنَّهُمْ حُمُرٌ مُسْتَنْفِرَ ةٌ (50 :) فَرَّتْ مِنْ قَسْوَ رَةٍّ} [المدثر50 - 51 :]،{فَفَرَ رْ تُ مِنْكُمْ لَمَّا خِ فْتُكُمْ} [الشعراء21 ْ]، {قُلْ لَن يَنْفَعَكُمُ الْفِرَ ارُ إِنْ فَرَ رْ تُمْ مِنَ الْمَوْ تِ أَوِ الْقَتْلِ } [األحزاب: 16 ]. تمهيد : هي الفِرار والمفرّ لغتان، وقيل: بل المفرّ : المَهْرَب، وهو الموضع الّذي يهرب إليه. ورَ جُلٌ فَرُورٌ وفَرُورةٌ من الفِرار. ورجلٌ فَرٌّ ورَ جُالن فَرٌّ ورجال فَرٌّ ال يثنى وال يجمعوالفَرُّ : مَصْدَر فَرَ رْ تُ عن أَسْنانِ الدّابّة، أي: كَشَفْت عنها. وافْتَرَّ عن ثَغْرِ ه إذا تَبَسَّم . وفَرَّ فالنٌ عمّا في نَفْسِهِ، وفُرَّ عن هذا األمر، أي: فَتِّشْهُ. والفَرْ فَرةُ: الطَّيْش والخِ فّة، ورَ جُلٌ فَرْ فار، وامرأةٌ فَرْ فارةٌ. وما زال فُالنٌ في أُفُرَّةِ شَرٍّّ من فالن، [أي: في أوّ ل] . والفَرُّ : الرّ جلُ الفارُّ ، وأَفْرَرْ تُه: ألجأتُه إلى الفرار1. َْ2َ َ ٌ َ ََُ َ ُُ ] ا[ ي ي ٍّ ُوالفَرّ والفِرارُ: الرَّوَ غان وَ الْهَرَب2 ُ.. فَرَّ يَفِرُّ فِرَ ارًا: هَرَبَ، وَ رَجُلٌ فَرورٌ وفَرورةٌ وفَرَّار: غَيْر كَرَّ ارٍّ ، وفَرٌّ ، وَ صْفٌ بالمصدرْ ، فَالْوَ احِ دُ وَ الْجَمْعُ فِيهِ سَوَ اءٌ 3 : ِ.و{الفَرُّ ، بالفتْح،} والفِرَ ارُ، بالكَسْر الرَّوَ غانُ وال هَرَب من شَيْءٍّ خافَه، {كالمَفَرِّ ، بالفَتْح،} والمَفِرِّ ، بكَسْر الفاءِ مَعَ فَتْح المِيم، وَ الثَّانِي يُسْتَعْمَل لَمْوضِ عِه، أَي {الفِرارِ ، أَيضاً، وَ قد} فَرَّ {يَفِرُّ فِرَ اراً: هَربَ، فَهُوَ فَرُورٌ، كصَبُور،}4 . فالفِرار: الرَوَ غَانُ والهَرَب أي (ابت عاد عما يواجِ ه بخفة وإسراع (استرسال): {كَأَنَّهُمْ حُمُرٌ مُسْتَنْفِرَ ةٌ (50 :) فَرَّتْ مِنْ قَسْوَ رَةٍّ} [المدثر50 - 51 :]،{فَفَرَ رْ تُ مِنْكُمْ لَمَّا خِ فْتُكُمْ} [الشعراء21 ْ]، {قُلْ لَن يَنْفَعَكُمُ الْفِرَ ارُ إِنْ فَرَ رْ تُمْ مِنَ الْمَوْ تِ أَوِ الْقَتْلِ } [األحزاب: 16 ]. وكل ما في القرآن من التركيب هو من الفرار المباعدة بخفة أي سرعة: {يَقُولُ اإلْ ِنْسَانُ يَوْ مَئِذٍّ أَيْنَ الْمَفَرُّ } [القيامة: 10 ] أي المكان الذي يمكن أن يَفِرّ إليه5 ُُ ] ا[ ي ي ٍّ ُوالفَرّ والفِرارُ: الرَّوَ غان وَ الْهَرَب2 ُ.. فَرَّ يَفِرُّ فِرَ ارًا: هَرَبَ، وَ رَجُلٌ فَرورٌ وفَرورةٌ وفَرَّار: غَيْر كَرَّ ارٍّ ، وفَرٌّ ، وَ صْفٌ بالمصدرْ ، فَالْوَ احِ دُ وَ الْجَمْعُ فِيهِ سَوَ اءٌ 3 : ِ.و{الفَرُّ ، بالفتْح،} والفِرَ ارُ، بالكَسْر الرَّوَ غانُ وال هَرَب من شَيْءٍّ خافَه، {كالمَفَرِّ ، بالفَتْح،} والمَفِرِّ ، بكَسْر الفاءِ مَعَ فَتْح المِيم، وَ الثَّانِي يُسْتَعْمَل لَمْوضِ عِه، أَي {الفِرارِ ، أَيضاً، وَ قد} فَرَّ {يَفِرُّ فِرَ اراً: هَربَ، فَهُوَ فَرُورٌ، كصَبُور،}4 . َ َ ُُا ٍّ ُوالفَرّ والفِرارُ: الرَّوَ غان وَ الْهَرَب2 ُ.. The types of escape in the Holy Quran and its faith significance وكل ما في القرآن من التركيب هو من الفرار المباعدة بخفة أي سرعة: {يَقُولُ اإلْ ِنْسَانُ يَوْ مَئِذٍّ أَيْنَ الْمَفَرُّ } [القيامة: 10 ] أي المكان الذي يمكن أن يَفِرّ إليه5 اما الفرار في القرآن الكريم فقد تنوع ذكره بين فرار من معين _سواء مصرح به او غير مصرح به_ أو فرار من غير معين، وكل فرار له دالالته، فهناك فرار نافع، وهناك فرار متوهم ال ينفع صاحبه بشيء، وبيان ذلك في اآلتي: المبحث األول: الفرار من معين فقد ورد في القرآن الكريم ذكر لفرارٍّ مع تعيين المهروب منه سواء بصريح العبارة أو بالمفهوم الضمني المشار إليه في السياق، وكان ذلك في العديد من اآليات، وبيانها فيما هو آت: المطلب األول: فرار من معين مصرح به: يشمل الفرار من معين المصرح به في القرآن الكريم ثالثة أصناف: فرار من معين مصرح به في الدنيا، وفرار من معين مصرح به في اآلخرة، وفرار من معين على سبي ل التصوير( فرار مجازي على سبيل التشبيه) وبيان ذلك في النقاط اآلتية: أوال: فرار من معين مصرح به في الدنيا إن الفرار من معين مصرح به في الدنيا الوارد في القران الكريم يشمل صنفين: فرار من القوم، وفرار من الموت او القتل، وذلك على النحو اآلتية: 1. :فرار من القوم 3. أي الفرار كان نافعا وايها لم يكن كذلك؟ 4. متى تكون الفائدة متوهمة من الفرار ومتى تكون حقيقية؟ 5. ما سر اختصاص االية{ففروا الى هللا} بعدم ذكر المهروب منه؟ 6. ما آراء المفسرين في بيان المهروب منه في قوله تعالى:{ففروا الى هللا}؟ 7. ما الدالالت اإليمانية لما ذكر في القران الكريم من أنواع الفرار؟ الدراسات السابقة: اا بعد البحث واالطالع لم أجد بحثا متخصصا في موضوع هذا البحث مما يزيد من أهمية الكتابة فيه. خطة البحث : أَأ بعد البحث واالطالع لم أجد بحثا متخصصا في موضوع هذا البحث مما يزيد من أهمية الكتابة فيه. خطة البحث : أَأ ب قسمت البحث الى تمهيد ومبحثين: المبحث األول: الفرار من مُ ؛ن عيَّ وفيه مطلبان: المطلب األول: فرار من معين مصرح به (سواء في الدنيا او اآلخرة) ، المطلب الثاني: فرار من معين غير مصرح به لكنه معلوم من السياق، و المبحث الثاني: ال فرار من غير ال معين. ثم ختمت بخاتمة فيها اهم النتائج، وما توفيقي اال باهلل. ت Journal of Social Sciences (COES&RJ-JSS), 9(2), pp.354-372 وعن اليهود الذين اخبر هللا عنهم انهم لن يتمنوا الموت ذكر فرارهم منه فقال تعالى: { قُلْ يَا أَيُّهَا الَّذِينَ هَادُوا إِن زَ عَمْتُمْ أَنَّكُمْ أَوْ لِيَاءُ ّلِلِ َّ ِ مِن دُونِ النَّاسِ فَتَمَنَّوُ ا الْمَوْ تَ إِن كُنتُمْ صَادِقِي ن وَ الَ يَتَمَنَّوْ نَهُ أَبَدًا بِمَا قَدَّمَتْ أَيْدِيهِ مْ ۚ وَ َّللاَّ ُ عَلِيمٌ بِالظَّالِمِينَ * قُلْ إِنَّ الْمَوْ تَ الَّذِي تَفِرُّونَ مِنْهُ فَإِنَّهُ مُالَ قِيكُمْ ۖ ثُمَّ تُرَ دُّونَ إِلَىٰ :عَالِمِ الْغَيْبِ وَ الشَّهَادَةِ فَيُنَبِّئُكُم بِمَا كُنتُمْ تَعْمَلُونَ} [الجمعة6 - 8 ] :وتفسير اآلية أي: إن كنتم تزعمون أنكم على هدى ، وأن محمدا وأصحابه على ضاللة، فادعوا بالموت على الضال من الفئتينإن كنتم صادقينفيما تزعمونه، وقال تعالى: وال يتمنونه أبدا بما قدمت أيديهم: أي : بما يعملون لهم من الكفر والظلم والفجور9. 2. فرار من الموت أو القتل : وشمل هذا الصنف من الفرار فرار المنافقين من الموت او القتل، وفرار اليهود من الموت، وتوضيح ذلك في النقطتين اآلتيتين : 2. فرار من الموت أو القتل : وشمل هذا الصنف من الفرار فرار المنافقين من الموت او القتل، وفرار اليهود من الموت، وتوضيح ذلك في النقطتين اآلتيتين : أ آ ي ح أ. في سورة األحزاب في بيان حال المنافقين الذين تخلفوا عن القتال بحجج واهية فرارا من الموت قال تعالى: {وَ إِذْ قَالَت طَّائِفَةٌ مِّنْهُمْ يَا أَهْلَ يَثْرِ بَ الَ مُقَامَ لَكُمْ فَارْ جِ عُوا ۚ وَ يَسْتَأْذِنُ فَرِ يقٌ مِّنْهُمُ النَّبِيَّ يَقُولُونَ إِنَّ بُيُوتَنَا عَوْ رَةٌ وَ مَا هِيَ بِعَوْ رَةٍّ ۖ إِن يُرِ يدُونَ إِالَّ فِرَ ارً ا * وَ لَوْ دُخِ لَتْ عَلَيْهِم مِّنْ أَقْطَارِ هَا ثُمَّ سُئِلُوا الْفِتْنَ ةَ آلَ تَوْ هَا وَ مَا تَلَبَّثُوا بِهَا إِالَّ يَسِيرً ا * وَ لَقَدْ كَانُوا عَاهَدُوا َّللاَّ َ مِن قَبْلُ الَ يُوَ لُّونَ األْ َدْبَ ارَ ۚ وَ كَانَ عَهْدُ َّللاَّ ِ مَسْئُوالً * قُل لَّن يَنفَعَكُمُ الْفِرَ ارُ إِن فَرَ رْ تُم مِّنَ الْمَوْ تِ أَوِ الْقَتْلِ وَ إِذًا الَّ تُمَتَّعُونَ إِالَّ قَلِيالً * قُلْ ِّمَن ذَا الَّذِي يَعْصِ مُكُم مِّنَ َّللاَّ ِ إِنْ أَرَ ادَ بِكُمْ سُوءًا أَوْ أَرَ ادَ بِكُمْ رَ حْمَةً ۚ وَ الَ يَجِ دُونَ لَهُم م ن دُونِ َّللاَّ ِ وَ لِيًّا وَ الَ نَصِ يرً ا} [ األحزاب: 13 - 17 ] فقد كان المنافقون يرون حاجتهم للف رار للنجاة لكنه كان مطمعا محال المنال. تمهيد : فَرَّ يَفِرُّ فِرَ ارًا: هَرَبَ، وَ رَجُلٌ فَرورٌ وفَرورةٌ وفَرَّار: غَيْر كَرَّ ارٍّ ، وفَرٌّ ، وَ صْفٌ بالمصدرْ ، فَالْوَ احِ دُ وَ الْجَمْعُ فِيهِ سَوَ اءٌ 3 : ِ.و{الفَرُّ ، بالفتْح،} والفِرَ ارُ، بالكَسْر الرَّوَ غانُ وال هَرَب من شَيْءٍّ خافَه، {كالمَفَرِّ ، بالفَتْح،} والمَفِرِّ ، بكَسْر الفاءِ مَعَ فَتْح المِيم، وَ الثَّانِي يُسْتَعْمَل لَمْوضِ عِه، أَي {الفِرارِ ، أَيضاً، وَ قد} فَرَّ {يَفِرُّ فِرَ اراً: هَربَ، فَهُوَ فَرُورٌ، كصَبُور،}4 . فالفِرار: الرَوَ غَانُ والهَرَب أي (ابت عاد عما يواجِ ه بخفة وإسراع (استرسال): {كَأَنَّهُمْ حُمُرٌ مُسْتَنْفِرَ ةٌ (50 :) فَرَّتْ مِنْ قَسْوَ رَةٍّ} [المدثر50 - 51 :]،{فَفَرَ رْ تُ مِنْكُمْ لَمَّا خِ فْتُكُمْ} [الشعراء21 ْ]، {قُلْ لَن ْ ْ أَ ْ ُ ْ ُ لأ 1. 356 356 Journal of Social Sciences (COES&RJ-JSS), 9(2), pp.354-372 Journal of Social Sciences (COES&RJ-JSS), 9(2), pp.354-372 في قصة موسى عليه السالم لما قال له فرعون:"وفعلت فعلتك" أي قتلت الذي قتله من القبط، قال له موسى :{ فَعَلْتُهَا إِذًا وَ أَنَا مِنَ الضَّال ِين* فَفَرَرْتُ مِنْكُمْ لَمَّا خِ فْتُكُمْ فَوَ هَبَ لِي رَب ِي حُكْمًا وَجَعَلَنِي مِنَ }َالْمُرْسَلِين [ الشعراء:20 - 21 ] قال: {ففررت} أي: فهربت {منكم} أي: معشر المأل من قوم فرعون، {لما خفتكم} على نفسي بالقتل أن تقتلوني بقتلي القتيل منكم {فوهب لي ربي حكما} النبوة وقيل: فهماً وعلماً {وجعلني من المرسلين} إليك وإلى قومك6. هللهلل في قصة موسى عليه السالم لما قال له فرعون:"وفعلت فعلتك" أي قتلت الذي قتله من القبط، قال له موسى :{ فَعَلْتُهَا إِذًا وَ أَنَا مِنَ الضَّال ِين* فَفَرَرْتُ مِنْكُمْ لَمَّا خِ فْتُكُمْ فَوَ هَبَ لِي رَب ِي حُكْمًا وَجَعَلَنِي مِنَ }َالْمُرْسَلِين [ الشعراء:20 - 21 ] قال: {ففررت} أي: فهربت {منكم} أي: معشر المأل من قوم فرعون، {لما خفتكم} على نفسي بالقتل أن تقتلوني بقتلي القتيل منكم {فوهب لي ربي حكما} النبوة وقيل: فهماً وعلماً {وجعلني من المرسلين} إليك وإلى قومك6. فهذا الفرار كان فرارا بدافع الخوف من فرعون وملئه، فلما كان فرارا الى هللا أمنه هللا، وأعقبه عطاءً إلهيا جزيال؛ فآتاه الحكم وهو النبوة او الفهم والعلم، وبذلك امتاز هذا الفرار عن غيره من أنواع الفرار في الدنيا بأنه أعقبه أمنا وخيرا كثيرا بخالف الفرار المذكور في اآليات التي سنذكرها تاليا. } فهذا الفرار كان فرارا بدافع الخوف من فرعون وملئه، فلما كان فرارا الى هللا أمنه هللا، وأعق عطاءً إلهيا جزيال؛ فآتاه الحكم وهو النبوة او الفهم والعلم، وبذلك امتاز هذا الفرار عن غيره م أنواع الفرار في الدنيا بأنه أعقبه أمنا وخيرا كثيرا بخالف الفرار المذكور في اآليات التي سنذكر تاليا. 357 2. فرار من الموت أو القتل : وشمل هذا الصنف من الفرار فرار المنافقين من الموت او القتل، وفرار اليهود من الموت، وتوضيح ذلك في النقطتين اآلتيتين : أ. Journal of Social Sciences (COES&RJ-JSS), 9(2), pp.354-372 في سورة األحزاب في بيان حال المنافقين الذين تخلفوا عن القتال بحجج واهية فرارا من الموت قال تعالى: {وَ إِذْ قَالَت طَّائِفَةٌ مِّنْهُمْ يَا أَهْلَ يَثْرِ بَ الَ مُقَامَ لَكُمْ فَارْ جِ عُوا ۚ وَ يَسْتَأْذِنُ فَرِ يقٌ مِّنْهُمُ النَّبِيَّ يَقُولُونَ إِنَّ بُيُوتَنَا عَوْ رَةٌ وَ مَا هِيَ بِعَوْ رَةٍّ ۖ إِن يُرِ يدُونَ إِالَّ فِرَ ارً ا * وَ لَوْ دُخِ لَتْ عَلَيْهِم مِّنْ أَقْطَارِ هَا ثُمَّ سُئِلُوا الْفِتْنَ ةَ آلَ تَوْ هَا وَ مَا تَلَبَّثُوا بِهَا إِالَّ يَسِيرً ا * وَ لَقَدْ كَانُوا عَاهَدُوا َّللاَّ َ مِن قَبْلُ الَ يُوَ لُّونَ األْ َدْبَ ارَ ۚ وَ كَانَ عَهْدُ َّللاَّ ِ مَسْئُوالً * قُل لَّن يَنفَعَكُمُ الْفِرَ ارُ إِن فَرَ رْ تُم مِّنَ الْمَوْ تِ أَوِ الْقَتْلِ وَ إِذًا الَّ تُمَتَّعُونَ إِالَّ قَلِيالً * قُلْ ِّمَن ذَا الَّذِي يَعْصِ مُكُم مِّنَ َّللاَّ ِ إِنْ أَرَ ادَ بِكُمْ سُوءًا أَوْ أَرَ ادَ بِكُمْ رَ حْمَةً ۚ وَ الَ يَجِ دُونَ لَهُم م ن دُونِ َّللاَّ ِ وَ لِيًّا وَ الَ نَصِ يرً ا} [ األحزاب: 13 - 17 ] فقد كان المنافقون يرون حاجتهم للف رار للنجاة لكنه كان مطمعا محال المنال. وفي تفسير اآلية قال الطبري: {قل} يا محمد لهؤالء الذين يستأذنوك في االنصراف عنك ويقولون {إن بيوتنا عورة}: {لن ينفعكم الفرار إن فررتم من الموت أو القتل} ألن ذلك أو ما كتب هللا منهما واصل إليكم بكل حال كرهتم أو أحببتم {و إذا ال تمتعون إال قليال}، وإذا فررتم من الموت أو القتل لم يزد فراركم ذلك في أعماركم وآجالكم، بل إنما تمتعون في هذه الدنيا إلى الوقت الذي كتب لكم، ثم يأتيكم ما كتب لكم وعليكم .... عن قتادة، " {قل لن ينفعكم الفرار إن فررتم من الموت أو القتل وإذا ال تمتعون إال قليال}: وإنما الدنيا كلها قليل.7 َّوفسرها النسفي بقوله: "{قُل لَّن يَنفَعَكُمُ الفرار إِن فَرَ رْ تُمْ مّنَ الموت أَوِ القتل وَ إِذاً الَّ تُمَتَّعُونَ إِال قَلِيالً} أي إن كان حضر أجلكم لم ينفعكم الفرار وإن لم بحضر وفررتم لم تمتعوا في الدنيا إال قليالً وهو مدة أعماركم، وذلك قليل، وعن بعض المروانية أنه مر بحائط مائل فأسرع فتليت له هذه اآلية فقال: ذلك القليل نطلب"8 ،ففرار المنافقين هنا من الموت أو القتل وهو فرار غير نافع فما قدّره هللا عليهم حاصل ال محالة وما فراراهم اال جبن وخوار. ب. The types of escape in the Holy Quran and its faith significance والبنون: لم تعلمنا ولم ترشدنا، وقيل: أول من يفر من أخيه: هابيل، ومن أبويه: إبراهيم، ومن صاحبته: نوح ولوط، ومن ابنه: نوح.13 فالفر ار: الهروب للتخلص من مخيف. وحرف (من) هنا يجوز أن يكون بمعنى التعليل الذي يعدى به فعل الفرار إلى سبب الفرار حين يقال: فر من األسد، وفر من العدو، وفر من الموت، ويجوز أن يكون بمعنى المجاوزة مثل (عن) وكون أقرب الناس لإلنسان يفر منهم يقتضي هول ذلك اليوم؛ بحيث إ ذا رأى ما يحل من العذاب بأقرب الناس إليه توهم أن الفرار منه ينجيه من الوقوع في مثله، إذ قد علم أنه كان مماثال لهم فيما ارتكبوه من األعمال فذكرت هنا أصناف من القرابة، فإن القرابة آصرة تكون لها في النفس معزة وحرص على سالمة صاحبها وكرامته. واإللف يحدث في النف س حرصا على المالزمة والمقارنة. وكال هذين الوجدانين يصد صاحبه عن المفارقة فما ظنك بهول يغشى على هذين الواجدين فال يترك لهما مجاال في النفس. ورتبت أصناف القرابة في اآلية حسب الصعود من الصنف إلى من هو أقوى منه تدرجا في تهويل ذلك اليوم. فابتدئ باألخ لشدة اتصال ه بأخيه من زمن الصبا فينشأ بذلك إلف بينهما يستمر طول الحياة، ثم ارتقي من األخ إلى األبوين وهما أشد قربا البنيهما، وقدمت األم في الذكر ألن إلف ابنها بها أقوى منه بأبيه وللرعي على الفاصلة، وانتقل إلى الزوجة والبنين وهما مجتمع عائلة اإلنسان وأشد الناس قربا به ومالزمة. وكل من هؤالء القرابة إذا قدرته هو الفار كان من ذكر معه مفرورا منه إال قوله: وصاحبته لظهور أن معناه: والمرأة من صاحبها، ففيه اكتفاء، وإنما ذكرت بوصف الصاحبة الدال على القرب والمالزمة دون وصف الزوج ألن المرأة قد تكون غير حسنة العشرة لزوجها فال يكون فراره منها " :وقال القرطبي لما ادّعت اليهود الفضيلة وقالوا : نحن أبناء هللا وأحباؤه قال هللا تعالى : إن زعمتم أنكم أولياء هلل من دون الناس فلألولياء عند هللا الكرامة فتمنوا الموت إن كنتم صادقين لتصيروا إلى ما يصير إليه أولياء هللا، وقوله تعالى : {قل إن الموت الذي تفرون منه فإنه مالق يكم ثم تردون إلى عالم الغيب والشهادة فينبئكم بما كنتم تعملون} قال الزجاج : ال يقال : إن زيدا فمنطلق ، وهاهنا قال : فإنه مالقيكم لما في معنى الذي من الشرط والجزاء ، أي إن فررتم منه فإنه مالقيكم ، ويكون مبالغة في الداللة على أنه ال ينفع الفرار منه . Journal of Social Sciences (COES&RJ-JSS), 9(2), pp.354-372 وفي تفسير اآلية قال الطبري: {قل} يا محمد لهؤالء الذين يستأذنوك في االنصراف عنك ويقولون {إن بيوتنا عورة}: {لن ينفعكم الفرار إن فررتم من الموت أو القتل} ألن ذلك أو ما كتب هللا منهما واصل إليكم بكل حال كرهتم أو أحببتم {و إذا ال تمتعون إال قليال}، وإذا فررتم من الموت أو القتل لم يزد فراركم ذلك في أعماركم وآجالكم، بل إنما تمتعون في هذه الدنيا إلى الوقت الذي كتب لكم، ثم يأتيكم ما كتب لكم وعليكم .... عن قتادة، " {قل لن ينفعكم الفرار إن فررتم من الموت أو القتل وإذا ال تمتعون إال قليال}: وإنما الدنيا كلها قليل.7 َاََا َّ اا }ا َّوفسرها النسفي بقوله: "{قُل لَّن يَنفَعَكُمُ الفرار إِن فَرَ رْ تُمْ مّنَ الموت أَوِ القتل وَ إِذاً الَّ تُمَتَّعُونَ إِال قَلِيالً} أي إن كان حضر أجلكم لم ينفعكم الفرار وإن لم بحضر وفررتم لم تمتعوا في الدنيا إال قليالً وهو مدة أعماركم، وذلك قليل، وعن بعض المروانية أنه مر بحائط مائل فأسرع فتليت له هذه اآلية فقال: ذلك القليل نطلب"8 ،ففرار المنافقين هنا من الموت أو القتل وهو فرار غير نافع فما قدّره هللا عليهم حاصل ال محالة وما فراراهم اال جبن وخوار. للهُ َ ُ ب. وعن اليهود الذين اخبر هللا عنهم انهم لن يتمنوا الموت ذكر فرارهم منه فقال تعالى: { قُلْ يَا أيُّهَا الَّذِينَ هَادُوا إِن زَ عَمْتُمْ أَنَّكُمْ أَوْ لِيَاءُ ّلِلِ َّ ِ مِن دُونِ النَّاسِ فَتَمَنَّوُ ا الْمَوْ تَ إِن كُنتُمْ صَادِقِي ن وَ الَ يَتَمَنَّوْ نَهُ أَبَدًا بِمَا قَدَّمَتْ أَيْدِيهِ مْ ۚ وَ َّللاَّ ُ عَلِيمٌ بِالظَّالِمِينَ * قُلْ إِنَّ الْمَوْ تَ الَّذِي تَفِرُّونَ مِنْهُ فَإِنَّهُ مُالَ قِيكُمْ ۖ ثُمَّ تُرَ دُّونَ إِلَىٰ :عَالِمِ الْغَيْبِ وَ الشَّهَادَةِ فَيُنَبِّئُكُم بِمَا كُنتُمْ تَعْمَلُونَ} [الجمعة6 - 8 ] :وتفسير اآلية أي: إن كنتم تزعمون أنكم على هدى ، وأن محمدا وأصحابه على ضاللة، فادعوا بالموت على الضال من الفئتينإن كنتم صادقينفيما تزعمونه، وقال تعالى: وال يتمنونه أبدا بما قدمت أيديهم: أي : بما يعملون لهم من الكفر والظلم والفجور9. 357 The types of escape in the Holy Quran and its faith significance قال زهير : عاا ي ومن هاب أسباب المنايا ينلنه ولو رام أسباب السماء بسلم" 10 م و و ر م ي ي و ن }وقد ذكر الماوردي أربعة أوجه لهذا الفرار فقال: "{قل إن الموت الذي تفرون منه فإنه مالقيكم يحتمل أربعة أوجه: أحدها: معناه تفرون من الداء بالدواء فإنه مالقيكم بانقضاء األجل. الثاني: تفرون من الجهاد ب القعود فإنه مالقيكم بالوعيد. الثالث: تفرون منه بالطيرة من ذكره حذراً من حلوله فإنه مالقيكم بالكره والرضا. الرابع: إنه الموت الذي تفرون أن تتمنوه حين قال تعالى: {فتمنوا الموت}"11 فهذا الفرار فرار متوهم ال يمكن تحقيقه اذ ال مفر من الموت مهما حرصوا على ذلك، وب التالي فهو فرار غير نافع. آ ثانيا: فرار من معين مصرح به في اآلخرة يخبرنا هللا عز وجل عن فرار مخيف يحصل يوم القيامة من شدة هول الموقف قال تعالى: {فَإِذَا جَاءَتِ الصَّاخَّةُ* يَوْ مَ يَفِرُّ الْمَرْ ءُ مِنْ أَخِ يهِ* وَ أُمِّهِ وَ أَبِيهِ* وَ صَاحِ بَتِهِ وَ بَنِيهِ * لِكُلِّ امْرِ ئٍّ مِّنْهُمْ يَوْ مَئِذٍّ :شَأْنٌ يُغْنِيهِ} [عبس33 - 37 ] {فَإِذا جاءَتِ الصَّاخَّةُ} يعني: الصيحة تصخ األسماع أي: تصمها فال يسمع إال ما يدعا به، ويقال الصاخة اسم من أسماء يوم القيامة، وكذلك الطامة والقارعة والحاقة.12 :يقال: صخ لحديثه، مثل أصاخ ل ه، فوصفت النفخة بالصاخة مجازا، ألن الناس يصخون لها يفر منهم الشتغاله بما هو مدفوع إليه، ولعلمه أنهم ال يغنون عنه شيئا، وبدأ باألخ، ثم باألبوين ألنهما أقرب منه، ثم بالصاحبة والبنين ألنهم أقرب وأحب، كأنه قال: يفر من أخيه، بل من أبويه، بل من صاحبته وبنيه. وقي ل: يفر منهم حذرا من مطالبتهم بالتبعات. يقول األخ: لم تواسنى بمالك. واألبوان: قصرت في برنا. والصاحبة: أطعمتنى الحرام وفعلت وصنعت. والبنون: لم تعلمنا ولم ترشدنا، وقيل: أول من يفر من أخيه: هابيل، ومن أبويه: إبراهيم، ومن صاحبته: نوح ولوط، ومن ابنه: نوح.13 فالفر ار: الهروب للتخلص من مخيف. وحرف (من) هنا يجوز أن يكون بمعنى التعليل الذي يعدى به فعل الفرار إلى سبب الفرار حين يقال: فر من األسد، وفر من العدو، وفر من الموت، ويجوز أن يكون بمعنى المجاوزة مثل (عن) وكون أقرب الناس لإلنسان يفر منهم يقتضي هول ذلك اليوم؛ بحيث إ ذا رأى ما يحل من العذاب بأقرب الناس إليه توهم أن الفرار منه ينجيه من الوقوع في مثله، إذ قد علم أنه كان مماثال لهم فيما ارتكبوه من األعمال فذكرت هنا أصناف من القرابة، فإن القرابة آصرة تكون لها في النفس معزة وحرص على سالمة صاحبها وكرامته. واإللف يحدث في النف س حرصا على المالزمة والمقارنة. The types of escape in the Holy Quran and its faith significance The types of escape in the Holy Quran and its faith significance 358 " :وقال القرطبي لما ادّعت اليهود الفضيلة وقالوا : نحن أبناء هللا وأحباؤه قال هللا تعالى : إن زعمتم أنكم أولياء هلل من دون الناس فلألولياء عند هللا الكرامة فتمنوا الموت إن كنتم صادقين لتصيروا إلى ما يصير إليه أولياء هللا، وقوله تعالى : {قل إن الموت الذي تفرون منه فإنه مالق يكم ثم تردون إلى عالم الغيب والشهادة فينبئكم بما كنتم تعملون} قال الزجاج : ال يقال : إن زيدا فمنطلق ، وهاهنا قال : فإنه مالقيكم لما في معنى الذي من الشرط والجزاء ، أي إن فررتم منه فإنه مالقيكم ، ويكون مبالغة في الداللة على أنه ال ينفع الفرار منه . قال زهير : ومن هاب أسباب المنايا ينلنه ولو رام أسباب السماء بسلم" 10 }وقد ذكر الماوردي أربعة أوجه لهذا الفرار فقال: "{قل إن الموت الذي تفرون منه فإنه مالقيكم يحتمل أربعة أوجه: أحدها: معناه تفرون من الداء بالدواء فإنه مالقيكم بانقضاء األجل. الثاني: تفرون من الجهاد ب القعود فإنه مالقيكم بالوعيد. الثالث: تفرون منه بالطيرة من ذكره حذراً من حلوله فإنه مالقيكم بالكره والرضا. الرابع: إنه الموت الذي تفرون أن تتمنوه حين قال تعالى: {فتمنوا الموت}"11 فهذا الفرار فرار متوهم ال يمكن تحقيقه اذ ال مفر من الموت مهما حرصوا على ذلك، وب التالي فهو فرار غير نافع. ثانيا: فرار من معين مصرح به في اآلخرة يخبرنا هللا عز وجل عن فرار مخيف يحصل يوم القيامة من شدة هول الموقف قال تعالى: {فَإِذَا جَاءَتِ الصَّاخَّةُ* يَوْ مَ يَفِرُّ الْمَرْ ءُ مِنْ أَخِ يهِ* وَ أُمِّهِ وَ أَبِيهِ* وَ صَاحِ بَتِهِ وَ بَنِيهِ * لِكُلِّ امْرِ ئٍّ مِّنْهُمْ يَوْ مَئِذٍّ :شَأْنٌ يُغْنِيهِ} [عبس33 - 37 ] {فَإِذا جاءَتِ الصَّاخَّةُ} يعني: الصيحة تصخ األسماع أي: تصمها فال يسمع إال ما يدعا به، ويقال الصاخة اسم من أسماء يوم القيامة، وكذلك الطامة والقارعة والحاقة.12 :يقال: صخ لحديثه، مثل أصاخ ل ه، فوصفت النفخة بالصاخة مجازا، ألن الناس يصخون لها يفر منهم الشتغاله بما هو مدفوع إليه، ولعلمه أنهم ال يغنون عنه شيئا، وبدأ باألخ، ثم باألبوين ألنهما أقرب منه، ثم بالصاحبة والبنين ألنهم أقرب وأحب، كأنه قال: يفر من أخيه، بل من أبويه، بل من صاحبته وبنيه. وقي ل: يفر منهم حذرا من مطالبتهم بالتبعات. يقول األخ: لم تواسنى بمالك. واألبوان: قصرت في برنا. والصاحبة: أطعمتنى الحرام وفعلت وصنعت. Journal of Social Sciences (COES&RJ-JSS), 9(2), pp.354-372 كناية عن شدة الهول فذكر بوصف الصاحبة. واألقرب أن هذا فرار المؤمن من قرابته المشركين خشية أن يؤاخذ بتبعتهم إذ بقوا على الكفر. وتعليق جار األقرباء بفعل: يفر المرء يقتضي أنهم قد وقعوا في عذاب يخشون تعديه إلى من يتصل بهم. وقد اجتمع في قوله: يوم يفر المرء من أخيه إلى آخره أبلغ ما يفيد هول ذلك اليوم بحيث ال يترك هوله للمرء بقية من رشده فإن نفس الفرار للخائف مسبة فيما تعارفوه لداللته على جبن صاحبه وهم يتعيرون بالجبن وكونه يترك أعز األعزة عليه مسبة عظمى. وجملة: لكل امرئ منهم يومئذ شأن يغنيه مستأنفة استئ نافا ابتدائيا لزيادة تهويل اليوم، وتنوين شأن للتعظيم. وحيث كان فرار المرء من األقرباء الخمسة يقتضي فرار كل قريب من أولئك من مثله كان االستئناف جامعا للجميع تصريحا بذلك المقتضى، فقال: لكل امرئ منهم يومئذ شأن يغنيه أي عن االشتغال بغيره من المذكورات بله االشت غال عمن هو دون أولئك في القرابة والصحبة.14 مما يدل على الرهبة والخوف الشديد، و{يغنيه}: يصرفه عن غيره.15 فهذا الفرار فرار فزع وخوف وتبري عن السوى، وهو غير نافع ألحد ألبتة، فمهما فر من اآلخرين فإنه لن يفر من حسابه وعقابه. كناية عن شدة الهول فذكر بوصف الصاحبة. واألقرب أن هذا فرار المؤمن من قرابته المشركين خشية أن يؤاخذ بتبعتهم إذ بقوا على الكفر. وتعليق جار األقرباء بفعل: يفر المرء يقتضي أنهم قد وقعوا في عذاب يخشون تعديه إلى من يتصل بهم. ل كا ل لك ل أ ل آ ل أ ل م ي وقد اجتمع في قوله: يوم يفر المرء من أخيه إلى آخره أبلغ ما يفيد هول ذلك اليوم بحيث ال يترك هوله للمرء بقية من رشده فإن نفس الفرار للخائف مسبة فيما تعارفوه لداللته على جبن صاحبه وهم يتعيرون بالجبن وكونه يترك أعز األعزة عليه مسبة عظمى. وجملة: لكل امرئ منهم يومئذ شأن يغنيه مستأنفة استئ نافا ابتدائيا لزيادة تهويل اليوم، وتنوين شأن للتعظيم. وحيث كان فرار المرء من األقرباء الخمسة يقتضي فرار كل قريب من أولئك من مثله كان االستئناف جامعا للجميع تصريحا بذلك المقتضى، فقال: لكل امرئ منهم يومئذ شأن يغنيه أي عن االشتغال بغيره من المذكورات بله االشت غال عمن هو دون أولئك في القرابة والصحبة.14 مما يدل على الرهبة والخوف الشديد، و{يغنيه}: يصرفه عن غيره.15 فهذا الفرار فرار فزع وخوف وتبري عن السوى، وهو غير نافع ألحد ألبتة، فمهما فر من اآلخرين فإنه لن يفر من حسابه وعقابه. The types of escape in the Holy Quran and its faith significance وكال هذين الوجدانين يصد صاحبه عن المفارقة فما ظنك بهول يغشى على هذين الواجدين فال يترك لهما مجاال في النفس. ورتبت أصناف القرابة في اآلية حسب الصعود من الصنف إلى من هو أقوى منه تدرجا في تهويل ذلك اليوم. فابتدئ باألخ لشدة اتصال ه بأخيه من زمن الصبا فينشأ بذلك إلف بينهما يستمر طول الحياة، ثم ارتقي من األخ إلى األبوين وهما أشد قربا البنيهما، وقدمت األم في الذكر ألن إلف ابنها بها أقوى منه بأبيه وللرعي على الفاصلة، وانتقل إلى الزوجة والبنين وهما مجتمع عائلة اإلنسان وأشد الناس قربا به ومالزمة. وكل من هؤالء القرابة إذا قدرته هو الفار كان من ذكر معه مفرورا منه إال قوله: وصاحبته لظهور أن معناه: والمرأة من صاحبها، ففيه اكتفاء، وإنما ذكرت بوصف الصاحبة الدال على القرب والمالزمة دون وصف الزوج ألن المرأة قد تكون غير حسنة العشرة لزوجها فال يكون فراره منها آ ي ح يخبرنا هللا عز وجل عن فرار مخيف يحصل يوم القيامة من شدة هول الموقف قال تعالى: {فَإِذَا جَاءَتِ الصَّاخَّةُ* يَوْ مَ يَفِرُّ الْمَرْ ءُ مِنْ أَخِ يهِ* وَ أُمِّهِ وَ أَبِيهِ* وَ صَاحِ بَتِهِ وَ بَنِيهِ * لِكُلِّ امْرِ ئٍّ مِّنْهُمْ يَوْ مَئِذٍّ ْ أْ ٌ {فَإِذا جاءَتِ الصَّاخَّة} يعني: الصيحة تصخ األسماع أي: تصمها فال يسمع إال ما يدعا به، ويقال الصاخة اسم من أسماء يوم القيامة، وكذلك الطامة والقارعة والحاقة.12 :يقال: صخ لحديثه، مثل أصاخ ل ه، فوصفت النفخة بالصاخة مجازا، ألن الناس يصخون لها يفر منهم الشتغاله بما هو مدفوع إليه، ولعلمه أنهم ال يغنون عنه شيئا، وبدأ باألخ، ثم باألبوين ألنهما أقرب منه، ثم بالصاحبة والبنين ألنهم أقرب وأحب، كأنه قال: يفر من أخيه، بل من أبويه، بل من صاحبته وبنيه. وقي ل: يفر منهم حذرا من مطالبتهم بالتبعات. يقول األخ: لم تواسنى بمالك. واألبوان: قصرت في برنا. والصاحبة: أطعمتنى الحرام وفعلت وصنعت. والبنون: لم تعلمنا ولم ترشدنا، وقيل: أول من يفر من أخيه: هابيل، ومن أبويه: إبراهيم، ومن صاحبته: نوح ولوط، ومن ابنه: نوح13 358 Journal of Social Sciences (COES&RJ-JSS), 9(2), pp.354-372 The types of escape in the Holy Quran and its faith significance م قال ابن قتيبة: أصل العورة ما ذهب عنه الستر والحفظ، وكأن الرجال ستر وحفظ للبيوت، فإذا ذهبوا عنها أعورت البيوت، تقول العرب: أعور منزلك إذا ذهب سترها وسقط جدارها، وأعور الفارس إذا بدا فيه موضع خلل للضرب والطعن، وقال مجا هد والحسن ومقاتل: قالوا بيوتنا ضائعة نخشى عليها السرق، وقال قتادة: قالوا: بيوتنا مما يلي العدو، وال نأمن على أهالينا، وقال الكلبي: بيوتنا عورة أي خالء يعني من الرجال، وقال أبو إسحاق: فكذبهم هللا وأعلم أن قصدهم الهرب والفرار فقال: {وما هي بعورة إن يريدون إال فرارا} قال مقاتل: ما يريدون إال فرارا من القتال ونصرة النبي - .صلى هللا عليه وسلم24 ي ر و هللي و م ى وفي أوضح التفاسير: "{يقولون إن بيوتنا عورة} أي مكشوفة، ينالها العدو لعدم تحصينها، {إن يريدون} ما يريدون بزعمهم هذا {إال فرارا} من الجهاد؛ لكفرهم وجبنهم25 . فالفرار هنا فرار ت هرب المنافقين من طاعة هللا والقتال في سبيله، وهو فرار غير منجي من الموت، فالموت الذي يخافون منه ويفرون منه فهو مالقيهم أينما كانوا، وما فرارهم من القتال اال مدعاة ي ر ملله ى وفي أوضح التفاسير: "{يقولون إن بيوتنا عورة} أي مكشوفة، ينالها العدو لعدم تحصينها، {إن يريدون} ما يريدون بزعمهم هذا {إال فرارا} من الجهاد؛ لكفرهم وجبنهم25 . لله م م }ا { م } فالفرار هنا فرار ت هرب المنافقين من طاعة هللا والقتال في سبيله، وهو فرار غير منجي من الموت، فالموت الذي يخافون منه ويفرون منه فهو مالقيهم أينما كانوا، وما فرارهم من القتال اال مدعاة لغضب هللا عليهم وعقابه لهم. فالفرار في هذا الموضع في قوله تعالى{إن يريدون إال فرارا} وإن لم ي صرح فيه بالمهروب منه اال ان السياق يدل عليه وهو مصرح به في االية بعد التالية فو قوله تعالى: {قُل لَّن يَنفَعَكُمُ الْفِرَ ارُ إِن فَرَ رْ تُم مِّنَ الْمَوْ تِ أَوِ الْقَتْلِ وَ إِذًا الَّ تُمَتَّعُونَ إِالَّ قَلِيالً }، وهذا فرار متوهم ان فيه النفع، لكنه في الحق يقة غير نافع فال مفر من الموت. Journal of Social Sciences (COES&RJ-JSS), 9(2), pp.354-372 ثالثا: فرار من معين على سبيل التصوير(فر ار مجازي) ذكر الفرار على سبيل المجاز لبيان صورة الخوف والفزع وشدة الفرار في موضعين في القران الكريم؛ فرار من أهل الكهف ، وفرار من التذكرة كأنه فرار من قسورة، وبيانه اآلتي: 1. ْالفرار من أصحب الكهف بتصوير المشهد المخيف المستدعي للفرار، قال تعالى: {لَوِ اطَّلَع تَ عَلَيْهمْ لَوَ لَّيْتَ مِنْهُمْ فِرَارًا وَلَمُلِئْتَ مِنْهُمْ رُعْبًا} [الكهف: 18 ] 1. قوله {لو اطلعت عليهم لوليت منهم فرارا} أي: لو اطلعت عليهم في رقدتهم التي رقدوها في كهفهم ألدبرت عنهم هاربا منهم فارا ولملئت منهم رعبا ، ولملئت نفسك من اطالعك عليهم فزعا ل ما كان هللا ألبسهم من الهيبة كي ال يصل إليهم واصل وال تلمسهم يد المس حتى يبلغ الكتاب فيهم أجله، وتوقظهم من رقدتهم قدرته وسلطانه في الوقت الذي أراد أن يجعلهم عبرة لمن شاء من خلقه، وآية لمن أراد االحتجاج بهم عليه من عباده، ليعلموا أن وعد هللا حق، وأن الساعة آتية ال ريب فيها16 . قال النسفي: "{لَوِ اطلعت عَلَيْهِمْ} لو أشرفت عليهم فنظرت إليهم {لَوْ لَّيْتَ مِنْهُمْ} ألعرضت عنهم وهربت منهم {فِرَ اراً} منصوب على المصدر ألن معنى وليت منهم فررت منهم {ولملئت منهم} وبتشديد الالم حجازي للمبالغة {رُعْبًا} تمييز وبضم العين شامي وعلي وهو الخوف الذي يرعب الصدر أي يمأله وذلك لما ألبسهم هللا من الهيبة أو لطول أظفارهم وشعورهم وعظم أجرامهم"17 .فقد منعهم هللا بالرعب من دخول أحد عليهم18 359 ور ن ر ب رر ي ي و يهم ب ي رب ن ي 2. ْالفرار من التذكرة كأنه فرار من قسورة: قال تعالى: {فَمَا لَهُمْ عَنِ التَّذْكِرَةِ مُعْرِ ضِ ينَ * كَأَنَّهُم حُمُرٌ مُّسْتَنفِرَةٌ * فَرَّتْ مِن قَسْوَ رَةٍ} [المدثر: 49 - 51 ] قال الطبري: وقوله: ( فَمَا لَهُمْ عَنِ التَّذْكِرَةِ مُعْرِ ضِ ينَ ) يقول: فما لهؤالء المشركين عن تذكرة هللا إياهم بهذا القرآن معرضين، ال يستمعون لها فيتعظوا ويعتبروا.19 ْ( كَأَنَّهُمْ حُمُرٌ مُس تَنْفِرَةٌ* فَرَّتْ مِنْ قَسْوَ رَةٍّ ) أي: كأنهم في نفارهم عن الحق، وإعراضهم عنه حُمُر من حمر الوحش إذا فرت ممن يريد صيدها من أسد.20 وقد ذكر المفسرون عدة أقوال في معنى (القسورة) منها: الرماة والقنّص وجماعة الرجال وصوت الرجال واألسد21.وعلى هذا فهو تشبيه مبتكر لحالة إعراض مخلوط برغب مما تضمنته قوارع القرآن فاجتمع في هذه الجملة تمثيالن.22 .فهذا فرار الخوف والفزع، وهو ال يورث أمانا بل هم في خوف دائم، ففرارهم غير نافع ألبتة 2.ُ 359 The types of escape in the Holy Quran and its faith significance The types of escape in the Holy Quran and its faith significance The types of escape in the Holy Quran and its faith significance المطلب الثاني: فرار من معين غير مصرح به لكنه معلوم من السياق تعرفنا في المطلب السابق إلى ما ذكر في القران الكريم من أصناف فرار من معين (مصرح به)، وهنا نتعرف إلى فرار من معين (غير مصرح به) لكنه معلوم من السياق بشكل واضح، وهو مذكور في ثالثة مواضع: فرار من القتال، وفرار من االستجابة الى دعوة الحق، وفرار من الموقف الشديد يوم القيامة، وبيان ذلك كله ف ي اآلتي: أوال: الفرار من القتال ا أخبرنا هللا تعالى عن المنافقين انهم يتهربون من القتال في سبيل هللا ويعتذرون بأعذار واهية وما يريدون بها اال الفرار من القتال، قال تعالى :{وَ إِذْ قَالَت طَّائِفَةٌ مِّنْهُمْ يَا أَهْلَ يَثْرِ بَ الَ مُقَامَ لَكُمْ فَارْ جِ عُوا ۚ وَ يَسْتَأْذِنُ فَرِ يقٌ مِّنْهُمُ النَّبِيَّ يَقُولُونَ إِنَّ بُيُوتَنَا عَوْ رَةٌ وَ مَا هِيَ بِعَوْ رَةٍّ ۖ إِن يُرِ يدُونَ إِالَّ فِرَ ارً ا * َوَ لَوْ دُخِ لَتْ عَلَيْهِم مِّنْ أَقْطَارِ هَا ثُمَّ سُئِلُوا الْفِتْنَةَ آلَ تَوْ هَا وَ مَا تَلَبَّثُوا بِهَا إِالَّ ي سِيرً ا* وَ لَقَدْ كَانُوا عَاهَدُوا َّللاَّ َ مِن قَبْلُ الَ يُوَ لُّونَ األْ َدْبَارَ ۚ وَ كَانَ عَهْدُ َّللاَّ ِ مَسْئُوالً قُل لَّن يَنفَعَكُمُ الْفِرَ ارُ إِن فَرَ رْ تُم مِّ نَ الْمَوْ تِ أَوِ الْقَتْلِ وَ إِذًا الَّ تُمَتَّعُونَ إِالَّ قَلِيالً قُلْ مَن ذَا الَّ ذِي يَعْصِ مُكُم مِّنَ َّللاَّ ِ إِنْ أَرَ ادَ بِكُمْ سُوءًا أَوْ أَرَ ادَ بِكُمْ رَ حْمَةً ۚ وَ الَ يَجِ دُونَ لَهُم مِّن دُونِ َّللاَّ ِ وَ لِيًّا وَ الَ نَصِ يرً ا} [ األحزاب: 13 - 17 ] ذ ل ل لل أ ل ل أ وقول المنافقين عن بيوتهم انها عورة أي: عورة فيها خلل يخاف منه العدو والسارق، يعتذرو ن أن بيوتهم معرضة للعدو وممكنة للسراق23 . The types of escape in the Holy Quran and its faith significance ا ي ع ما ع ثانيا: الفرار من االستجابة لدعوة الحق ذكر لنا هللا تعالى في قصة نوح عليه السالم ودعوته التي استخدم فيها شتى الطرق لهداية قومه اال ان النتيجة كانت كما قال: {فَلَمْ يَزِ دْهُمْ دُعَائِي إِال فِرَارً ا} [نوح:6 ] أي: مما دعوتهم إليه وإسناد الزيادة إلى الدعاء لسببيته كما في قوله تعالى زادتهم إيمانا26 ومعنى {فلم يزدهم دعائي إال فرارا}: أن دعائي لهم بأن يعبدوا هللا وبطاعتهم لي لم يزدهم ما دعوتهم إليه إال بعدا منه، فالفرار مستعار لقوة اإلعراض، أي فلم يزدهم دعائي إياهم قربا مما أدعوهم إليه . واستثناء الفرار من عموم الزيادات استثناء منقطع. والتقدير: فلم يزدهم دعائي قربا من الهدى لكن زادهم فرارا كما في قوله تعالى حكاية عن صالح عليه السالم فما تزيدونني غير تخسير [هود: 63 ] . وإسناد زيادة الفرار إلى الدعاء مجاز ألن دعاءه إياهم كان سببا في تزايد إ عراضهم وقوة تمسكهم بشركهم. وهذا من األسلوب المسمى في علم البديع تأكيد المدح بما يشبه 360 Journal of Social Sciences (COES&RJ-JSS), 9(2), pp.354-372 Journal of Social Sciences (COES&RJ-JSS), 9(2), pp.354-372 ار ر ز ي قال ابن كثير: "وقوله: ( يَقُولُ اإلنْسَانُ يَوْ مَئِذٍّ أَيْنَ الْمَفَرُّ ) أي: إذا عاين ابنُ آدم هذه األهوال يوم القيامة، حينئذ يريد أن يفر ويقول: أين المفر؟ أ ي: هل من ملجأ أو موئل ؟قال هللا تعالى : ( كال ال وزر إلى ربك يومئذ المستقر) قال ابن مسعود وابن عباس وسعيد بن جبير ، وغير واحد من السلف : أي ال نجاة .وهذه كقوله : (ما لكم من ملجإ يومئذ وما لكم من نكير) [ الشورى : 47 ] أي : ليس لكم مكان تتنكرون فيه ، وكذا قا ل هاهنا ( ال وزر) أي : ليس لكم مكان تعتصمون فيه الى ربك يومئذ المستقر: أي المرجع والمصيرثم قال تعالى : ( ينبأ اإلنسان يومئذ بما قدم وأخر) أي : يخبر بجميع أعماله قديمها وحديثها ، أولها وآخرها ، صغيرها وكبيرها ، كما قال تعالى : (ووجدوا ما عملوا حاضرا وال يظل م ربك أحدا ) [ الكهف : 49 "] 30 ،فهذا طلب للفرار من هول الموقف لشدة الخوف والفزع، لكنه لن يجد مهربا ينجيه من ذلك اليوم فهو محاسب ال محالة، ومجزي بأعماله كلها، وال يظلم ربنا أحدا، فهذا فرار متوهم ال نفع فيه وال نجاة ألهله. أ المبحث الثاني: ال فرار من غير معين تعرفنا في المبحث السابق إلى مواضع الفرار المذكور في القران الكريم مما فيه ذكرٌ للمهروب منه؛ وذلك سواء ببيان المهروب منه بصريح العبارة(إن في الدنيا أو في اآلخرة أو بالتشبيه) أو سواء بما لم يصرح به لكن السياق دل داللة قطعية على بيان المهروب منه، وفي هذا المب حث سنتعرف إلى فرار ذكر في القران الكريم لكنه لم يذكر المهروب منه، مما أدى الى اختالف آراء المفسرين في المقصود به ومن أي شيء يكون هذا الفرار. Journal of Social Sciences (COES&RJ-JSS), 9(2), pp.354-372 قال ابن كثير: "وقوله: ( يَقُولُ اإلنْسَانُ يَوْ مَئِذٍّ أَيْنَ الْمَفَرُّ ) أي: إذا عاين ابنُ آدم هذه األهوال يوم القيامة، حينئذ يريد أن يفر ويقول: أين المفر؟ أ ي: هل من ملجأ أو موئل ؟قال هللا تعالى : ( كال ال وزر إلى ربك يومئذ المستقر) قال ابن مسعود وابن عباس وسعيد بن جبير ، وغير واحد من السلف : أي ال نجاة .وهذه كقوله : (ما لكم من ملجإ يومئذ وما لكم من نكير) [ الشورى : 47 ] أي : ليس لكم مكان تتنكرون فيه ، وكذا قا ل هاهنا ( ال وزر) أي : ليس لكم مكان تعتصمون فيه الى ربك يومئذ المستقر: أي المرجع والمصيرثم قال تعالى : ( ينبأ اإلنسان يومئذ بما قدم وأخر) أي : يخبر بجميع أعماله قديمها وحديثها ، أولها وآخرها ، صغيرها وكبيرها ، كما قال تعالى : (ووجدوا ما عملوا حاضرا وال يظل م ربك أحدا ) [ الكهف : 49 "] 30 ،فهذا طلب للفرار من هول الموقف لشدة الخوف والفزع، لكنه لن يجد مهربا ينجيه من ذلك اليوم فهو محاسب ال محالة، ومجزي بأعماله كلها، وال يظلم ربنا أحدا، فهذا فرار متوهم ال نفع فيه وال نجاة ألهله. ي فهذا فرار الجحود واالستكبار، وهو فرار من الحق الى الباطل، يظنون انهم بفرارهم من دعوة نوح عليه السالم سينجون، فجاء الطوفان فأغرقهم وكانوا هم الخاسرون، فهو فرار متوهم ان فيه النفع ولكنه لم ينفع أهله بل كان سبب هالكم والعيا ذ باهلل. ي و : ر ر ن جاء في سورة القيامة عمن ينكرون ذلك اليوم: {يَسْأَلُ أَيَّانَ يَوْ مُ الْقِيَامَةِ * فَإِذَا بَرِ قَ الْبَصَرُ * وَ خَسَفَ الْقَمَرُ * وَ جُمِعَ الشَّمْسُ وَ الْقَمَرُ يَقُولُ اإلْ ِنسَانُ يَوْ مَئِذٍّ أَيْنَ الْمَفَرُّ كَ الَّ الَ وَ زَ رَ * إِلَىٰ رَ بِّكَ :يَوْ مَئِذٍّ الْمُسْتَقَرُّ* يُنَبَّأُ اإلْ ِنسَانُ يَوْ مَئِذٍّ بِمَا قَدَّمَ وَ أَخَّرَ }[القيامة6 - 13 ] ( يَقُولُ اإلنْسَانُ) أي الكافر المكذب ( يَوْ مَئِذٍّ أَيْنَ الْمَفَرُّ) أي: المهرب وهو موضع الفرار. وقيل: هو مصدر، أي: أين ال فرار28 أي: يقول اإلنسان يوم يعاين أهوال يوم القيامة: أين المفرّ من هول هذا الذي قد نـزل، وال فرار29 . Journal of Social Sciences (COES&RJ-JSS), 9(2), pp.354-372 الذم، أو تأكيد الشيء بما يشبه ضده، وهو هنا تأكيد إعراضهم المشبه باالبتعاد بصورة تشبه ضد اإلعراض. ولما كان فرارهم من التوحيد ثابتا لهم من قبل كان قوله: فلم يزدهم دعائي إال فرارا من تأكيد الشيء بما يشبه ضده.27 الذم، أو تأكيد الشيء بما يشبه ضده، وهو هنا تأكيد إعراضهم المشبه باالبتعاد بصورة تشبه ضد اإلعراض. ولما كان فرارهم من التوحيد ثابتا لهم من قبل كان قوله: فلم يزدهم دعائي إال فرارا من تأكيد الشيء بما يشبه ضده.27 فهذا فرار الجحود واالستكبار، وهو فرار من الحق الى الباطل، يظنون انهم بفرارهم من دعوة نوح عليه السالم سينجون، فجاء الطوفان فأغرقهم وكانوا هم الخاسرون، فهو فرار متوهم ان فيه النفع ولكنه لم ينفع أهله بل كان سبب هالكم والعيا ذ باهلل. ثالثا: الفرار من الموقف الشديد جاء في سورة القيامة عمن ينكرون ذلك اليوم: {يَسْأَلُ أَيَّانَ يَوْ مُ الْقِيَامَةِ * فَإِذَا بَرِ قَ الْبَصَرُ * وَ خَسَفَ الْقَمَرُ * وَ جُمِعَ الشَّمْسُ وَ الْقَمَرُ يَقُولُ اإلْ ِنسَانُ يَوْ مَئِذٍّ أَيْنَ الْمَفَرُّ كَ الَّ الَ وَ زَ رَ * إِلَىٰ رَ بِّكَ :يَوْ مَئِذٍّ الْمُسْتَقَرُّ* يُنَبَّأُ اإلْ ِنسَانُ يَوْ مَئِذٍّ بِمَا قَدَّمَ وَ أَخَّرَ }[القيامة6 - 13 ] ( يَقُولُ اإلنْسَانُ) أي الكافر المكذب ( يَوْ مَئِذٍّ أَيْنَ الْمَفَرُّ) أي: المهرب وهو موضع الفرار. وقيل: هو مصدر، أي: أين ال فرار28 أي: يقول اإلنسان يوم يعاين أهوال يوم القيامة: أين المفرّ من هول هذا الذي قد نـزل، وال فرار29 . Journal of Social Sciences (COES&RJ-JSS), 9(2), pp.354-372 فقد جاء في سورة الذاريات وبعد أن اخبرنا هللا تعالى ببعض قصص األنبياء وكفر أقوامهم بهم ثم ذكر لنا من مظاهر قدرته في خلق السماء واألرض وخلق الزوجين من كل شيء قال تعالى: { فَفِرُّوا إِلَى َّللاَّ ِ إِنِّي لَكُمْ مِنْهُ نَذِيرٌ مُبِينٌ}[الذاريات:50 ] ،فاآلية الكريمة لم تذكر المهروب منه، فقد دعت الى الفرار اليه تعالى، دون تعيين ما الذي نفر منه ويمكننا تقسيم آراء المفسرين في تفس يرهم لهذا الفرار الى ثالثة آراء؛ رأي يعمم الفرار اليه سبحانه دون ذكر للمهروب منه، ورأي يحدد المهروب منه ويقيده، ورأي يوسع دائرة المهروب منه، وبيان ذلك في اآلتي: الرأي األول: في تعميم الفرار إليه تعالى دون ذكر المهروب منه ذهب ابن كثير الى تعميم الدعوة الى الفرارالى هللا تعالى، فلم يذكر مم هو هذا الفرار، بل اكتفى بأن {" :بين انه فرار الى هللا والتجاء الى هللا وحده قال فَفِرُّوا إِلَى َّللاَّ ِ} أي: الجئوا إليه، واعتمدوا في ."أموركم عليه31 الرأي األول: في تعميم الفرار إليه تعالى دون ذكر المهروب منه ذهب ابن كثير الى تعميم الدعوة الى الفرارالى هللا تعالى، فلم يذكر مم هو هذا الفرار، بل اكتفى بأن {" :بين انه فرار الى هللا والتجاء الى هللا وحده قال فَفِرُّوا إِلَى َّللاَّ ِ} أي: الجئوا إليه، واعتمدوا في ."أموركم عليه31 361 The types of escape in the Holy Quran and its faith significance وبنحوه فسرها المراغي قائال: "(ففروا إلى هللا) أي فالجئوا إلى هللا واعتمدو ا عليه فى جميع أموركم، واتبعوا أوامره، واعملوا على طاعته.32 وهذا ابن الجوزي يكتفي في تفسير الفرار بأنه بالتوبة دون ذكر المهروب منه قال: "{ففروا إلى هللا} بالتوبة".33 وكذا قال الماوردي: {ففروا إلى هللا} أي فتوبوا إلى هللا34 الرأي الثاني: في تقييد المهروب منه فسر الطبري قوله تعالى:{ففروا الى هللا} أي : فاهربوا أيها الناس من عقاب هللا إلى رحمته باإليمان ,به, واتباع أمره, والعمل بطاعته ( إِنِّي لَكُمْ مِنْهُ نَذِيرٌ ) يقول: إني لكم من هللا نذير أنذركم عقابه وأخوّ فكم عذابه الذي أحله بهؤالء األمم الذي قصّ عليكم قصصه م, والذي هو مذيقهم في اآلخرة35 .وبنحوه فسرها البيضاوي فقال: {ففروا إلى هللا}: من عقابه باإليمان والتوحيد ومالزمة الطاعة إني لكم منه أي من عذابه المعد لمن أشرك أو عصى. نذير مبين بين كونه منذرا من هللا بالمعجزات، أو مبين ما يجب أن يحذر عنه36 . األله وفسرها ابن عثيمين بقوله: والفرار إلى هللا يكون بالقيام بطاعته واجتناب نواهيه، ألنه ال ينقذك من عذاب هللا، إال أن تقوم بطاعة هللا، فكأن اإلنسان إذا قام بطاعة هللا عز وجل كأنه فر من عدو، أرأيت لو أن واديا عرما يهدر، أقبل عليك فإنك لن تقف أمامه، بل تهرب منه وتفر منه، كذلك لو أن حريقا ملتهبا أقبل إليك فإنك لن تقف بل تفر، كذلك نار جهنم أشد وأعظم وأولى بالفرار منها، ولهذا قال: {ففروا إلى هللا} ، أي: من عذاب هللا37 هروب ر ي و يع ر ي في حين قصر بعض المفسرين معنى الفرار بفرار من اشياء محددة كالعقاب والعذاب نجد مفسرين آخرين وسعوا دائرة الفرار لتشمل اشياء كثيرة بل لتتسع عند بعضهم اكثر فتشمل الفرار من كل ما سوى هللا اليه سبحانه. ومن أولئك المفسرين: ا لله القرطبي الذي فسر الفرار بأنه فرار من المعاصي الى الطاعات لكنه نقل اقواال أشمل من ذلك فقال: "قوله تعالى:{ففروا إلى هللا إني لكم منه نذير مبين} لما تقدم ما جرى من تكذيب أممهم ألنبيائهم وإهالكهم، لذلك قال هللا تعالى: لنبيه صلى هللا عليه وسلم قل لهم يا محمد، أي قل لقومك: (ففروا إلى هللا إني لكم منه نذير مبين) أي فروا من معاصيه إلى طاعته. وقال ابن عباس: فروا إلى هللا بالتوبة من ذنوبكم. وعنه فروا منه إليه واعملوا بطاعته. وقال محمد بن عبد هللا بن عمرو بن عثمان بن عفان: (ففروا إلى هللا) اخرجوا إلى مكة. وقال الحسينبن الفضل: احترزوا من كل شي دون هللا فمن فر إلى غيره لم يمتنع منه38 . Journal of Social Sciences (COES&RJ-JSS), 9(2), pp.354-372 وبنحوه ذكر النسفي فقال: {ففروا إل ى هللا} أي من الشرك إلى اإليمان باهلل أو من طاعة الشيطان }إلى طاعة الرحمن أو مما سواه إليه {إنى لكم منه نذير مبين42 :وقد اشار ابن عطية الى لطيفة من لطائف التعبير بالفرار دون غيره من المصطلحات فقال "وقوله: فَفِرُّوا أمر بالدخول في اإليمان وطاعة هللا، وجعل ا ألمر بذلك بلفظ الفرار لينبه على أن وراء الناس عقابا وعذابا وأمرا حقه أن يفر منه، فجمعت لفظة « فروا » بين التحذير واالستدعاء، وينظر إلى هذا المعنى قول النبي صلى هللا عليه وسلم: « ال ملجأ وال منجى منك إال إليك » الحديث43 وبنحوه ذكر النسفي فقال: {ففروا إل ى هللا} أي من الشرك إلى اإليمان باهلل أو من طاعة الشيطان }إلى طاعة الرحمن أو مما سواه إليه {إنى لكم منه نذير مبين42 :وقد اشار ابن عطية الى لطيفة من لطائف التعبير بالفرار دون غيره من المصطلحات فقال "وقوله: فَفِرُّوا أمر بالدخول في اإليمان وطاعة هللا، وجعل ا ألمر بذلك بلفظ الفرار لينبه على أن وراء الناس عقابا وعذابا وأمرا حقه أن يفر منه، فجمعت لفظة « فروا » بين التحذير واالستدعاء، وينظر إلى هذا المعنى قول النبي صلى هللا عليه وسلم: « ال ملجأ وال منجى منك إال إليك » الحديث43 ي اما الرازي فذكر لطائف عديدة في هذه االية ا لكريمة فقال: أمر بالتوحيد، وفيه لطائف األولى: قوله تعالى: ففروا ينبئ عن سرعة اإلهالك كأنه يقول اإلهالك والعذاب أسرع وأقرب من أن يحتمل الحال اإلبطاء في الرجوع، فافزعوا إلى هللا سريعا وفروا الثانية: قوله تعالى: إلى هللا بيان المهروب إليه ولم يذكر الذي منه ال هرب ألحد وجهين، إما لكونه معلوما وهو هول العذاب أو الشيطان الذي قال فيه إن الشيطان لكم عدو فاتخذوه عدوا [فاطر: 6 ] وإما ليكون عاما كأنه يقول: كل ما عدا هللا عدوكم ففروا إليه من كل ما عداه، وبيانه وهو أن كل ما عداه فإنه يتلف عليك رأس مالك الذي هو العمر، ويفو ت عليك ما هو الحق والخير، ومتلف رأس المال مفوت الكمال عدو، وأما إذا فررت إلى هللا وأقبلت على هللا فهو يأخذ عمرك ولكن يرفع أمرك ويعطيك بقاء ال فناء معه والثالثة: الفاء للترتيب معناه إذا ثبت أن خالق الزوجين فرد ففروا إليه واتركوا غيره تركا مؤبدا الرابعة: في تنوع الكالم فائدة وبيانها هو أن هللا تعالى قال: والسماء بنيناها [الذاريات: 47 ] واألرض فرشناها [الذاريات: 48 :] ومن كل شيء خلقنا [الذاريات49 ] ثم جعل الكالم للنبي عليه السالم وقال: ففروا إلى هللا إني لكم منه نذير مبين ولم يقل ففروا إلينا، وذلك ألن الختالف ال كالم تأثيرا، وكذلك الختالف المتكلمين تأثيرا، ولهذا يكثر اإلنسان من النصائح مع ولده الذي حاد عن الجادة، ويجعل الكالم مختلفا، نوعا ترغيبا ونوعا ترهيبا، وتنبيها بالحكاية، ثم يقول لغيره تكلم معه لعل كالمك ينفع، لما في أذهان الناس أن اختالف المتكلمين واختالف ال كالم كالهما مؤثر، وهللا تعالى ذكر أنواعا من الكالم وكثيرا من االستدالالت واآليات وذكر طرفا صالحا من الحكايات، ثم ذكر كالما من متكلم آخر هو النبي صلى هللا عليه وسلم، ومن المفسرين من يقول تقديره فقل لهم ففروا وقوله .إني لكم منه نذير إشارة إلى الرسالة44 ااآاا م ي وهذا ا لشعرواي يلفت االنتباه الى ضرورة االستعداد لليوم اآلخر اذ ال مفر منه اال اليه سبحانه فيقول: أي أن الحق جل جالله يريدنا أن نعرف يقينا أننا ال نستطيع أن نفر من علمه. The types of escape in the Holy Quran and its faith significance آ ع م وهذا السعدي يوسع دائرة المهروب منه فيقول: "لما دعا العباد إلى النظر آلياته الموجبة لخشيته واإلنابة إليه، أمر بما هو المقصود من ذلك، وهو الفرار إليه أي: الفرار مما يكرهه هللا ظاهرًا وباطنًا، إلى ما يحبه، ظاهرً ا وباطنًا، فرار من الجهل إلى العلم، ومن الكفر إل ى اإليمان، ومن المعصية إلى الطاعة، و من الغفلة إلى ذكر هللا فمن استكمل هذه األمور، فقد استكمل الدين كله وقد .زال عنه المرهوب، وحصل له، نهاية المراد والمطلوب39 للهلله ع م وهذا السعدي يوسع دائرة المهروب منه فيقول: "لما دعا العباد إلى النظر آلياته الموجبة لخشيته واإلنابة إليه، أمر بما هو المقصود من ذلك، وهو الفرار إليه أي: الفرار مما يكرهه هللا ظاهرًا وباطنًا، إلى ما يحبه، ظاهرً ا وباطنًا، فرار من الجهل إلى العلم، ومن الكفر إل ى اإليمان، ومن المعصية إلى الطاعة، و من الغفلة إلى ذكر هللا فمن استكمل هذه األمور، فقد استكمل الدين كله وقد .زال عنه المرهوب، وحصل له، نهاية المراد والمطلوب39 ،ويبين السعدي تميز الفرار الى هللا عن غيره من الفرار فيقول: "وسمى هللا الرجوع إليه، فرارَ ا ألن في الرجوع لغيره، أنواع المخاوف والمكاره، وفي الرجوع إليه، أنواع المحاب واألمن، والسرور والسعادة والفوز، فيفر العبد من قضائه وقدره، إلى قضائه وقدره، وكل من خفت منه .فررت منه إال هللا تعالى، فإنه بحسب الخوف منه، يكون الفرار إليه40 " وفسرها البغوي بأنها فرار من العذاب الى الثواب ونقل اقواال اخرى اعم واشمل فقال: { فَفِرُّوا إِلَى َّللاَّ ِ}: فاهربوا من عذاب هللا إلى ثوابه, باإليمان والطاعة. قال ابن عباس: فروا منه إليه واعملوا ,بطاعته. وقال سهل بن عبد هللا: فروا مما سوى هللا إلى هللا41 ،ويبين السعدي تميز الفرار الى هللا عن غيره من الفرار فيقول: "وسمى هللا الرجوع إليه، فرارَ ا ألن في الرجوع لغيره، أنواع المخاوف والمكاره، وفي الرجوع إليه، أنواع المحاب واألمن، والسرور والسعادة والفوز، فيفر العبد من قضائه وقدره، إلى قضائه وقدره، وكل من خفت منه .فررت منه إال هللا تعالى، فإنه بحسب الخوف منه، يكون الفرار إليه40 " وفسرها البغوي بأنها فرار من العذاب الى الثواب ونقل اقواال اخرى اعم واشمل فقال: { فَفِرُّوا إِلَى َّللاَّ ِ}: فاهربوا من عذاب هللا إلى ثوابه, باإليمان والطاعة. قال ابن عباس: فروا منه إليه واعملوا ,بطاعته. وقال سهل بن عبد هللا: فروا مما سوى هللا إلى هللا41 362 Journal of Social Sciences (COES&RJ-JSS), 9(2), pp.354-372 Journal of Social Sciences (COES&RJ-JSS), 9(2), pp.354-372 وال من قدره وال من عذابه . . وأن الطريق الوحيد المفتوح أمامنا هو أن نفر إلى هللا. . وأنه ال منجاة من هللا إال إليه. . ولذلك ال يظن كافر أو عاص أنه سيفلت من هللا. . وال يظن أنه لن يكون موجودا يوم القيامة أو أنه لن يحاسب أو أنه يستطيع أن يختفي.إن غرور الدنيا قد يركب بعض الناس فيظنون أنهم في منعة من هللا وأنهم لن يالقوه. . نقول لهم إنكم ستفاجأون في اآلخرة حين تعرفون أن الحساب حق والجنة حق والنار حق. ستفاجأون بما سيحدث لكم. . ومن لم يؤمن ولم يسارع إلى الخير سيلقى الخزي والعذاب األليم. . إن هللا ينصحنا أن نؤمن وأن نسارع في الخيرات لننجو ا من عذابه، ويقول لنا لن يفلت واحد منكم وال ذرة من ذرات جسده من الوقوف بين يدي هللا للحساب. . ولذلك ختم هللا هذه اآلية الكريمة بقوله: {إن هللا على كل شيء قدير} . . أي أن هللا سبحانه وتعالى ال يعجزه شيء وال يخرج عن طاعته شيء. . إنه سبحانه على كل شيء قدير45 . 363 :ويمكننا جمع اقوال المفسرين في تفسيرهم للفرار بأنه يشمل الفرار .من مكة الى المدينة _وهذا معنى محدود وخاص جدا وال ينبغي قصر المقصود به من ذنوبكم، ومن معاصيكم الى طاعته سبحانه . من عقاب هللا إلى رحمته باإليمان به, واتباع أمره, والعمل بطاعته . 363 363 The types of escape in the Holy Quran and its faith significance من هول العذاب او من عذاب هللا إلى ثوابه, باإليمان والطاعة. ومن عقابه وعذابه الى الدخول في اإليمان وطاعة هللا . من الشيطان الى الرحمن . لله مما يكرهه هللا ظاهرً ا وباطنًا، إلى ما يحبه، ظاهرً ا وباطنًا . م من الغفلة إلى ذكر هللا . من الغفلة إلى ذكر هللا . .هللمنه إليه .هللمنه إليه إ ي مما سوى هللا إلى هللا, و كل ما عدا هللا عدوكم ففروا إليه من كل ما عداه. وهذا فرار _ وهو الفرار من كل ما سوى هللا اليه وحده سبحانه _ هو الفرار الذي يعقبه األمان ، اذ ال خوف يستولي على المرء في هروبه وفراره بل هي طمأنينة بالفرار اليه اذ قال تعالى: {اال بذكر هللا تطمئن القلوب}. نسأل هللا أن يشغلنا به عمن سواه وأن نتحقق بالفرار إليه وحده إنه على كل شيء قدير.والحمد هلل رب العالمين. مما سوى هللا إلى هللا, و كل ما عدا هللا عدوكم ففروا إليه من كل ما عداه. وهذا فرار _ وهو الفرار من كل ما سوى هللا اليه وحده سبحانه _ هو الفرار الذي يعقبه األمان ، اذ ال خوف يستولي على المرء في هروبه وفراره بل هي طمأنينة بالفرار اليه اذ قال تعالى: {اال بذكر هللا تطمئن القلوب}. لهلله خاتمة: الحمد هلل الذي أعانني على كتا بة هذا البحث، والذي توصلت من خالله الى نتائج عدة،كان من أبرزها: : الحمد هلل الذي أعانني على كتا بة هذا البحث، والذي توصلت من خالله الى نتائج عدة،كان م أبرزها: اا نال حظ ان كل أنواع الفرار في القران الكريم كانت نابعة عن خوف ويغلفها الخوف وال تثمر أ مانا، اال الفرار الى هللا تعالى، ويشمل فرار موسى عليه السالم الذي نبع عن خوف لكنه لما لجأ الى هللا كان فرار ه الى هللا ؛ لذا اعقبه امانا ووهبه الحكم والنبوة . لله م إن الفرار المذكور في القران الكريم هو:في الدنيا: فرار المنافقين واليهود من الموت او القتل ، وفرار من الحق كالفار من قسورة ، وفرار من اهل الكهف الذين لو اطلعت عليهم لوليت منهم فرارا ولنتج عنه خوف كبير (ولملئت منهم رعبا) ، وفرار من االستجابة لدعوة نوح عليه السالم وهو فرار خوف على مصالحهم التي تتعارض مع دعوة الحق ، وفي االخرة فرار من األخ واالهل وخوف شديد في البحث عن المفر المنجي . في حين الفرار الى هللا تعالى هو فرار من كل ما يش غل عن هللا ، فهو فرار امان ويثمر امانا وعطاء الهيا عظيما . The types of escape in the Holy Quran and its faith significance لله ن ن وي ر و ر ر ي ي و ويمكننا تقسيم انوع الفرار المذكورة في القران الكريم من حيثيتين: ا أ أا أ ويمكننا تقسيم انوع الفرار المذكورة في القران الكريم من حيثيتين: أوال: ٌأقسام أنواع الفرار المذكورة في القرآن الكريم من حيث االنتفاع بالفرار: فالفرار في القران الكريم من حيث االنتفاع به ينقسم الى قسمين: ا ويمكننا تقسيم انوع الفرار المذكورة في القران الكريم من حيثيتين: أوال: ٌأقسام أنواع الفرار المذكورة في القرآن الكريم من حيث االنتفاع بالفرار: ا ريم ن ي ي ين: ور ي ر ن يم وع ر ر وي أوال: ٌأقسام أنواع الفرار المذكورة في القرآن الكريم من حيث االنتفاع بالفرار: ا ا م ي ع ما فالفرار في القران الكريم من حيث االنتفاع به ينقسم الى قسمين: ا 1. .فرار ضار أو متوهم وال يتبعه أمان 2 . فرار نافع يتعبه أمان اما الفرار الضار او المتوهم فهو: أوال: فرار في الدنيا: فرار المنافقين واليهود من الموت ومن القتل، وفرار الكافرين من الحق كالفار من قسورة ، والفرار من اهل الكهف، والفرار من االستجابة لدعوة نوح عليه السالم، وثانيا: فرار في اال خرة:وهو الفرار من األخ واألم واألب والصاحبة والبنين،وبحث عن المفر المنجي يوم القيامة بسبب الخوف الشديد. لله ي و ي ي يوم ر ن ين و و وأماالفرار النافع المذكور في القران الكريم فهو الفرار الى هللا تعالى سواء كان فرارا من عقاب هللا او عذابه او الشرك او الشيطان او طاعة الشيطان او من كل ما سوى هللا تعالى، فالفرار عام وشامل وغير معين؛ ليشمل كل ما سوى هللا، ويدخل فيه أيضا فرار موسى عليه السالم من قومه لما خافهم، لكنه لما فر الى هللا أتبعه أمانا وآتاه الحكم والنبوة، فكان فرارا نافعا الرتباطه باهلل تعالى. ثانيا: أقسام أنواع الفرار المذكورة ف ي القرآن الكريم من حيث بيان المهروب منه: 364 Journal of Social Sciences (COES&RJ-JSS), 9(2), pp.354-372 :فالفرار في القران الكريم من حيث بيان المهروب منه ينقسم الى قسمين 1. فرار من معين ، سواء من معين: أ . مصرح به في الدنيا او االخرة على سبيل الحقيقة او المجاز والتشبيه. ب . غير مصرح به لكنه معلوم من السياق. أما الفرار من معين مصرح به في الدنيا فهو فرار من القوم ومن الموت او القتل ، والفرار المصرح به في االخرة هو فرار من األهل، والفرار من معين على سبيل التصوير وهو الفرار المعبر عن نفار الكافرين عن الحق، وإعراضهم عنه كأنه فرار من قسورة، ومن اهل الكهف، واما الفر ار من غير المصرح به لكنه معلوم من السياق فهو الفرار من القتل ومن االستجابة للدعوة ومن الموقف الشديد والبحث يوم القيامة عن المفر من العذاب. 2. أما الفرار من غير المعين ؛ فهو الفرار الى هللا تعالى، وفي عدم ذكر المهروب منه الى هللا إشارة إلى شمول الفرار ا لهروب من كل ما سواه عز وجل إ ليه سبحانه. وقد اختلفت آراء المفسرين في بيان المهروب منه في تفسيرهم لقوله تعالى:{ففروا الى هللا} بين معمم للفرار دون ذكر المهروب منه، وبين مقيد للمهروب منه بأشياء معينة، وبين موسع لدائرة المهروب منه ليشمل كل ما سوى هللا، فالمؤمن يفر م ن كل ما سوى هللا الى هللا عز وجل؛ اذ ال ملجأ إال اليه وال أمان اال بمعيته سبحانه. فكل فرار مذكور في القران الكريم كان محدداللمهروب منه وغير نافع ألصحابه، إال الفرار الى هللا تعالى فهو عام يشمل الفرار من كل ما سواه إليه سبحانه وهو الفرار الوحيد الذي يثمر أم انا واطمئنانا. هذا ما فتح هللا علي من تعرف إلى أنواع الفرار في القرآن الكريم ودالالتها اإليمانية، نسأل هللا تعالى القبول، فما كان من صواب فبمحض فضله سبحانه، وما كان من خطأ في االجتهاد والفهم فأسأل هللا المغفرة والهداية الى ما يحب ويرضى، وآخر دعوانا ان صل ا للهم على نبيك محمد وعلى آله وصحبه أجمعين، والحمد هلل رب العالمين. ر ر أ . مصرح به في الدنيا او االخرة على سبيل الحقيقة او المجاز والتشبيه. أما الفرار من معين مصرح به في الدنيا فهو فرار من القوم ومن الموت او القتل ، والفرار المصرح به في االخرة هو فرار من األهل، والفرار من معين على سبيل التصوير وهو الفرار المعبر عن نفار الكافرين عن الحق، وإعراضهم عنه كأنه فرار من قسورة، ومن اهل الكهف، واما الفر ار من غير المصرح به لكنه معلوم من السياق فهو الفرار من القتل ومن االستجابة للدعوة ومن الموقف الشديد والبحث يوم القيامة عن المفر من العذاب. المراجع - ،إبراهيم بن إسماعيل األبياري الموسوعة القرآنية، د.ط، مؤسسة سجل العرب، 1405 ه - أحمد بن مصطفى المراغي ، تفسر المراغي، ط1، شركة مكتبة ومطبعة مصطفى الباب وأوالده بمصر، 1365 هـ- 1946 م أ يل أبي ري إبر يم بن إ ر ي . Journal of Social Sciences (COES&RJ-JSS), 9(2), pp.354-372 للهلله م 2. أما الفرار من غير المعين ؛ فهو الفرار الى هللا تعالى، وفي عدم ذكر المهروب منه الى هللا إشارة إلى شمول الفرار ا لهروب من كل ما سواه عز وجل إ ليه سبحانه. لله 2. 2. أما الفرار من غير المعين ؛ فهو الفرار الى هللا تعالى، وفي عدم ذكر المهروب منه الى هللا إشارة إلى شمول الفرار ا لهروب من كل ما سواه عز وجل إ ليه سبحانه. وقد اختلفت آراء المفسرين في بيان المهروب منه في تفسيرهم لقوله تعالى:{ففروا الى هللا} بين معمم للفرار دون ذكر المهروب منه، وبين مقيد للمهروب منه بأشياء معينة، وبين موسع لدائرة المهروب منه ليشمل كل ما سوى هللا، فالمؤمن يفر م ن كل ما سوى هللا الى هللا عز وجل؛ اذ ال ملجأ إال اليه وال أمان اال بمعيته سبحانه. وقد اختلفت آراء المفسرين في بيان المهروب منه في تفسيرهم لقوله تعالى:{ففروا الى هللا} بين معمم للفرار دون ذكر المهروب منه، وبين مقيد للمهروب منه بأشياء معينة، وبين موسع لدائرة المهروب منه ليشمل كل ما سوى هللا، فالمؤمن يفر م ن كل ما سوى هللا الى هللا عز وجل؛ اذ ال ملجأ إال اليه وال أمان اال بمعيته سبحانه. ااا فكل فرار مذكور في القران الكريم كان محدداللمهروب منه وغير نافع ألصحابه، إال الفرار الى هللا تعالى فهو عام يشمل الفرار من كل ما سواه إليه سبحانه وهو الفرار الوحيد الذي يثمر أم انا واطمئنانا. هذا ما فتح هللا علي من تعرف إلى أنواع الفرار في القرآن الكريم ودالالتها اإليمانية، نسأل هللا تعالى القبول، فما كان من صواب فبمحض فضله سبحانه، وما كان من خطأ في االجتهاد والفهم فأسأل هللا المغفرة والهداية الى ما يحب ويرضى، وآخر دعوانا ان صل ا للهم على نبيك محمد وعلى آله وصحبه أجمعين، والحمد هلل رب العالمين. المراجع و و رب جل ؤ1405 - أحمد بن مصطفى المراغي ، تفسر المراغي، ط1 ، شركة مكتبة ومطبعة مصطفى البابى الحلبي وأوالده بمصر، 1365 هـ- 1946 م ا م - أبو السعود العمادي محمد بن محمد، إرشاد العقل السليم إلى مزايا الكتاب الكريم، د.ط، دار إحياء التراث العربي، بيروت، د.ت - أبو السعود العمادي محمد بن محمد، إرشاد العقل السليم إلى مزايا الكتاب الكريم، د.ط، دار إحياء التراث العربي، بيروت، د.ت التراث العربي، بيروت، د.ت - إسماعيل بن عمر بن كثير، تفسير القرآن العظيم ، تحقيق: محمد حسين شمس الدين، ط1 ، دار الكتب العلمية، منشورات محمد علي بيضون، بيروت، 1419 هـ - ،جالل الدين المحلي، وجالل الدين السيوطي تفسير الجاللين، ط1 ، ،دار الحديث، القاهرة د.ت الق آ ال ف األ ذك ال ا ال ال أ ال ال - إسماعيل بن عمر بن كثير، تفسير القرآن العظيم ، تحقيق: محمد حسين شمس الدين، ط1 ، دار الكتب العلمية، منشورات محمد علي بيضون، بيروت، 1419ااا هـ - إسماعيل بن عمر بن كثير، تفسير القرآن العظيم ، تحقيق: محمد حسين شمس الدين، ط1 ، دار الكتب العلمية، منشورات محمد علي بيضون، بيروت، 1419 هـ - ،جالل الدين المحلي، وجالل الدين السيوطي تفسير الجاللين، ط1 ، ،دار الحديث، القاهرة د.ت - جمال الدين أبو الفرج عبد الرحمن ابن الجوزي، تذكرة األريب في تفسير الغريب (غريب القرآن الكريم) تحقيق: طارق فتحي السيد، ط1 ، دار الكتب العلمية، بيروت – لبنان، 1425 هـ- 2004 م - الحسين بن مسعود بن الفراء البغوي، معالم التنزيل في تفسير القرآن، تحقيق: عبد الرزاق المهدي ، ط1 ،، دار إحياء التراث العرب ، بيروت1420ه - ،جالل الدين المحلي، وجالل الدين السيوطي تفسير الجاللين، ط1 ، ،دار الحديث، القاهرة د.ت - جمال الدين أبو الفرج عبد الرحمن ابن الجوزي، تذكرة األريب في تفسير الغريب (غريب القرآن الكريم) تحقيق: طارق فتحي السيد، ط1 ، دار الكتب العلمية، بيروت – لبنان، 1425 هـ- 2004 م - الحسين بن مسعود بن الفراء البغوي، معالم التنزيل في تفسير القرآن، تحقيق: عبد الرزاق المهدي ، ط1 ،، دار إحياء التراث العربي، بيروت1420هـ - ،جالل الدين المحلي، وجالل الدين السيوطي تفسير الجاللين، ط1 ، ،دار الحديث، القاهرة د.ت - جمال الدين أبو الفرج عبد الرحمن ابن الجوزي، تذكرة األريب في تفسير الغريب (غريب القرآن الكريم) تحقيق: طارق فتحي السيد، ط1 ، دار الكتب العلمية، بيروت – لبنان، 1425 هـ- 2004 م آ - الحسين بن مسعود بن الفراء البغوي، معالم التنزيل في تفسير القرآن، تحقيق: عبد الرزاق المهدي ، ط1 ،، دار إحياء التراث العربي، بيروت1420 هـ ي - ،الخليل بن أحمد الفراهيدي، كتاب العين، تحقيق: د مهدي المخزومي، د إبراهيم السامرائي، د.ط دار ومكتبة الهالل ، د.ت ي - الخليل بن أحمد الفراهيدي، كتاب العين، تحقيق: د مهدي المخزومي، د إبراهيم السامرائي، د.ط دار ومكتبة الهالل ، د.ت أ ا - عبد الحق بن غالب بن عطية األندلسي، المحرر الوجيز في تفسير الكتاب العزيز، تحقيق: عبد السالم عبد الشافي ، ط1 ،، دار الكتب العلمية، بيروت1422 .ه ا - عبد الحق بن غالب بن عطية األندلسي، المحرر الوجيز في تفسير الكتاب العزيز، تحقيق: عبد السالم عبد الشافي ، ط1 ،، دار الكتب العلمية، بيروت1422 .ه 365 The types of escape in the Holy Quran and its faith significance رير و ور بن ر بن 1984 - محمد بن أحمد القرطبي، الجامع ألحكام القرآن، تحقيق: أحمد البردوني وإب راهيم أطفيش، ط2 ،دار الكتب المصرية، القاهرة1384 هـ- 1964 م - محمد بن أحمد القرطبي، الجامع ألحكام القرآن، تحقيق: أحمد البردوني وإب راهيم أطفيش، ط2 ، ،دار الكتب المصرية، القاهرة1384 هـ- 1964 م م - محمد بن جرير الطبري، جامع البيان في تأويل القرآن، تحقيق: أحمد محمد شاكر، ط1 ، مؤسسة الرسالة، 1420 هـ- 2000م م - محمد بن جرير الطبري، جامع البيان في تأويل القرآن، تحقيق: أحمد محمد شاكر، ط1 ، مؤسسة الرسالة، 1420 هـ- 2000م م - محمد بن صالح ابن العثيمين، تفسير الحجرات – الحديد، ط1 ،، دار الثريا للنشر والتوزيع الرياض، 1425 هـ- 2004 م - محمد بن صالح ابن العثيمين، تفسير الحجرات – الحديد، ط1 ،، دار الثريا للنشر والتوزيع الرياض، 1425 هـ- 2004 م م - محمد بن عمر الرازي، مفاتيح الغيب، ط3 ،، دار إحياء التراث العربي، بيروت1420 هـ َّ - محمد بن عمر الرازي، مفاتيح الغيب، ط3 ،، دار إحياء التراث العربي، بيروت1420 هـ - محمّد بن محمّد مرتضى الزَّبيدي، تاج العروس من جواهر القاموس، د.ط، دار الهداية، د.ت ط ل ا ال ظ ا ك ا ا - محمد بن عمر الرازي، مفاتيح الغيب، ط3 ،، دار إحياء التراث العربي، بيروت1420 هـ الزَّبيدي، تاج العروس من جواهر القاموس، د ط، دار الهداية، د ت محمّد بن محمّد مرتض - محمد بن عمر الرازي، مفاتيح الغيب، ط3 ،، دار إحياء التراث العربي، بيروت1420 هـ - محمّد بن محمّد مرتضى الزَّبيدي، تاج العروس من جواهر القاموس، د.ط، دار الهداية، د.ت - محمد بن مكرم ابن منظور، لسان العرب، ط3 ، دار صادر – بيروت، 1414 هـ - محمّد بن محمّد مرتضى الزَّبيدي، تاج العروس من جواهر القاموس، د.ط، دار الهداية، د.ت - محمد بن مكرم ابن منظور، لسان العرب، ط3 ، دار صادر – بيروت، 1414 هـ م - محمد بن يعقوب الفيروزآبادى، القاموس المحيط، تحقيق: مكتب تحقيق التراث في مؤسس الرسالة، ط8 ، مؤسسة الرسالة للطباعة والنشر والتوزيع، بيروت- ،لبنان1426 هـ- 2005م - محمد بن يعقوب الفيروزآبادى، القاموس المحيط، تحقيق: مكتب تحقيق التراث في مؤسسة الرسالة، ط8 ، مؤسسة الرسالة للطباعة والنشر والتوزيع، بيروت- ،لبنان1426 هـ- 2005م - محمد بن يعقوب الفيروزآبادي، تنوير المقباس من تفسير ابن عباس، د.ط، دار الكتب العلمية – لبنان، د.ت - م حمد حسن جبل، المعجم االشتقاقي المؤصل أللفاظ القرآن الكريم، ط1 ، مكتبة اآلداب- ،القاهرة 2010م - محمد عبد اللطيف بن الخطيب، أوضح التفاسير، ط6 ، ، المطبعة المصرية ومكتبتها1383 هـ- 1964م م - محمد متولي الشعراوي، تفسير الشعراوي، مطابع أخبار اليوم1997 م - محمود بن عمر و الزمخشري، الكشاف عن حقائق غوامض التنزيل، ط3 ، دار الكتاب العربي – بيروت، 1407 .هـ وبذيله: االنتصاف فيما تضمنه الكشاف، البن المنير اإلسكندري - نصر بن محمد السمرقندي، بحر العلوم، ط1 ،، دار الكتب العلمية1413 - 1993 - وهبة بن مصطفى الزحيلي، التفسير المنير، ط2، دا ر الفكر المعاصر، دمشق، 1418 The types of escape in the Holy Quran and its faith significance - عبد الرحمن بن أبي بكر جالل الدين السيوطي، الدر المنثور، د.ط، دار الفكر، بيروت، د.ت - عبد الرحمن بن ناصر السعدي، تيسير الكريم الرحمن في تفسير كالم المنان،تحقيق: عبد الرحمن بن معال اللويحق، ط1 ، ، مؤسسة الرسالة1420 هـ- 2000 .م - ،عبد هللا بن أحمد النسفي، مدارك التنزيل وحقائق التأويل، حققه وخرج أحاديثه: يوسف علي بديوي ط1 ،، دار الكلم الطيب، بيروت1419 هـ- 1998م لله - عبد الرحمن بن أبي بكر جالل الدين السيوطي، الدر المنثور، د.ط، دار الفكر، بيروت، د.ت - عبد الرحمن بن ناصر السعدي، تيسير الكريم الرحمن في تفسير كالم المنان،تحقيق: عبد الرحم بن معال اللويحق، ط1 ، ، مؤسسة الرسالة1420 هـ- 2000 .م لله - اعبد الرحمن بن أبي بكر جالل الدين السيوطي، الدر المنثور، د.ط، دار الفكر، بي يا ي - عبد الرحمن بن ناصر السعدي، تيسير الكريم الرحمن في تفسير كالم المنان،تحقيق: عبد الر بن معال اللويحق، ط1 ، ، مؤسسة الرسالة1420 هـ- 2000 .م لله بن معال اللويحق، ط1 ، ، مؤسسة الرسالة1420 هـ- 2000 .م - ،عبد هللا بن أحمد النسفي، مدارك التنزيل وحقائق التأويل، حققه وخرج أحاديثه: يوسف علي بديوي ط1 ،، دار الكلم الطيب، بيروت1419 هـ- 1998م لله ا م - 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Wahbah bin Mostafah al Zohaily, Enlightening interpretation, p 2, Dar Alfikr al Moasir, Damascus, 1418 h. 369 1 كتاب العين، الخليل بن أحمد الفراهيدي، ص255 - 256 . 2 لسان العرب، ابن منظور، ج5 ص50.القاموس المحيط، الفيروز ابادي، ج1 ص455 3 لسان العرب، ابن منظور، ج5 ص50 . 4 تاج العروس، المرتضى الزبيدي، ج17 ص311 . 5 المعجم االشتقاقي المؤصل، محمد حسن جبل، ج1 ص1647 .و1648 6انظر: جامع البيان، الطبري، ج17 ص559 ص307 . تنوير ال مقباس من تفسير ابن عباس، الفيروزابادي، ج10 ص429 .، الموسوعة القرآنية 7 جامع البيان، الطبري، ج19، ص47 - 48 8 مدارك التنزيل وحقائق التأويل، النسفي، ج3، ص23 . 9تفسير القرآن العظيم، ابن كثير، ج8، ص118 . 10جامع البيان، القرطبي، ج18، ص96 . 11 ،النكت والعيون الماوردي،ج6، ص8. 12 بحر العلوم،السمرقندي،ص549 13 الكشاف،الزمخشري، ج4، ص705 . 14 التحرير والتنوير، ابن عاشور، ج30، ص135 - 136 . 15 التفسير المنير، الزحيلي،ج30، ص74 . 16 جامع البيان، الطبري، ج15، ص194 . 17 مدارك التنزيل وحقائق التأويل،النسفي،ج2، ص291 . 18 تفسير الجاللين، السيوطي والمحلي،ص382 . 19جامع البيان، الطبري، ج23، ص454 . 20تفسير القرآن العظيم، ابن كثير، ج8، ص273 21 :انظر جامع البيان، الطبري ج23، ص454 . ،تفسير القران العظيم ابن كثير، ج8، ص273 - 274 - الدر المنثور في التفسير بالمأثور، السيوطي، ج8 ، 339 . 22 التحري ر والتنوير، ابن عاشور، ج29 ، ص330 . 23 ،الموسوعة القرآنية إبراهيم بن إسماعيل األبياري ، ج10، ص544 . 24التفسير البسيط، الواحدي، ج18، ص197 - 198 . 25 أوضح التفاسير، محمد عبد اللطيف بن الخطيب ج1 ، ص509 . 26 إرشاد العقل السليم،أبو السعود، ج9، ص37 . 27 التحرير والتنوير، اب ن عاشور، ج29 ، ص194 . 28معالم التنزيل، البغوي، ج7، ص437 29 جامع البيان، الطبري، ج22 ، ص608 30 تفسير القران العظيم، ابن كثير، ج7 ، ص486 2 لسان العرب، ابن منظور، ج5 ص50.القاموس المحيط، الفيروز ابادي، ج1 ص455 4 تاج العروس، المرتضى الزبيدي، ج17 ص311 . 5ا 5 المعجم االشتقاقي المؤصل، محمد حسن جبل، ج1 ص1647 .و1648 6انظر: جامع البيان، الطبري، ج17 ص559 ص307 . تنوير ال مقباس من تفسير ابن عباس، الفيروزابادي، ج10 ص429 .، الموسوعة القرآنية 7 جامع البيان، الطبري، ج19، ص47 - 48 8 مدارك التنزيل وحقائق التأويل، النسفي، ج3، ص23 . 8 مدارك التنزيل وحقائق التأويل، النسفي، ج3، ص23 . 9تفسير القرآن العظيم، ابن كثير، ج8، ص118 . 10جامع البيان، القرطبي، ج18، ص96 . 5 المعجم االشتقاقي المؤصل، محمد حسن جبل، ج1 ص1647 .و1648 6انظر: جامع البيان، الطبري، ج17 ص559 ص307 . تنوير ال مقباس م آ 11 ،النكت والعيون الماوردي،ج6، ص8. 26 إرشاد العقل السليم،أبو السعود، ج9، ص37 . الفيروزابادي، ج10 ص429 .، الموسوعة القرآنية 12 بحر العلوم،السمرقندي،ص549 24التفسير البسيط، الواحدي، ج18، ص197 - 198 . 22 التحري ر والتنوير، ابن عاشور، ج29 ، ص330 . 23لأ 7 جامع البيان، الطبري، ج19، ص47 - 48 Journal of Social Sciences (COES&RJ-JSS), 9(2), pp.354-372 11 11 ،النكت والعيون الماوردي،ج6، ص8. 12 11 ،النكت والعيون الماوردي،ج6، ص8. 12 12 بحر العلوم،السمرقندي،ص549 13 الكشاف،الزمخشري، ج4، ص705 . 14 التحرير والتنوير، ابن عاشور، ج30، ص135 - 136 . 15 14 التحرير والتنوير، ابن عاشور، ج30، ص135 - 136 . 15 التفسير المنير، الزحيلي،ج30، ص74 . 15 التفسير المنير، الزحيلي،ج30، ص74 . 16 جامع البيان، الطبري، ج15، ص194 . 16 جامع البيان، الطبري، ج15، ص194 . 17 مدارك التنزيل وحقائق التأويل،النسفي،ج2، ص291 . 18ا 18 تفسير الجاللين، السيوطي والمحلي،ص382 . 19 19جامع البيان، الطبري، ج23، ص454 . 20 20تفسير القرآن العظيم، ابن كثير، ج8، ص273 21 :انظر جامع البيان، الطبري ج23، ص454 . ،تفسير القران العظيم ابن كثير، ج8، ص273 - 274 - الدر المنثور في التفسير بالمأثور، السيوطي، ج8 ، 339 . 20تفسير القرآن العظيم، ابن كثير، ج8، ص273 21 :انظر جامع البيان، الطبري ج23، ص454 . ،تفسير القران العظيم ابن كثير، ج8، ص273 - أ 20تفسير القرآن العظيم، ابن كثير، ج8، ص273 21 :انظر جامع البيان، الطبري ج23، ص454 . ،تفسير القران العظيم ابن كثير، ج8، ص273 - 274 - الدر المنثور في التفسير بالمأثور، السيوطي، ج8 ، 339 . 274 - الدر المنثور في التفسير بالمأثور، السيوطي، ج8 ، 339 . 22 التحري ر والتنوير، ابن عاشور، ج29 ، ص330 . 23أ 22 التحري ر والتنوير، ابن عاشور، ج29 ، ص330 . 23لأ 23 ،الموسوعة القرآنية إبراهيم بن إسماعيل األبياري ، ج10، ص544 . 24 23 ،الموسوعة القرآنية إبراهيم بن إسماعيل األبياري ، ج10، ص544 . 24 24التفسير البسيط، الواحدي، ج18، ص197 - 198 . 24التفسير البسيط، الواحدي، ج18، ص197 - 198 . 25 أوضح التفاسير، محمد عبد اللطيف بن الخطيب ج1 ، ص509 . 25 أوضح التفاسير، محمد عبد اللطيف بن الخطيب ج1 ، ص509 . 26أ 25 أوضح التفاسير، محمد عبد اللطيف بن الخطيب ج1 ، ص509 . 26 إرشاد العقل السليم،أبو السعود، ج9، ص37 . 26 إرشاد العقل السليم،أبو السعود، ج9، ص37 . 27 التحرير والتنوير، اب ن عاشور، ج29 ، ص194 . 28 The types of escape in the Holy Quran and its faith significance 31 تفسير القران العظيم، ابن كثير، ج7، ص144 32تفسير المراغي، أحمد المراغي، ج27، ص10 33 ،تذكرة األريب في تفسير الغريب،ابن الجوزي ص370 34النكت والعيون، الماوردي، ج5، ص374 35 جامع البيان، الطبري، ج21 ، ص385 36 أنوار التنزيل وأسرار التأويل، البيضاوي، ج5 ، ص150 37 تفسير الحجرات – الحديد، ابن العثيمين، ص162 38الجامع ألحكام القران، القرطبي،ج17 ، ص53 . Journal of Social Sciences (COES&RJ-JSS), 9(2), pp.354-372 39تيسير الكريم الرحمن في تفسير كالم المنان، ع بد الرحمن بن ناصر السعدي، ص811 40 .المرجع ذاته نفس الموضع 41 معالم التنزيل، البغوي، ج7 ، 149 42 تفسير مدارك التنزيل وحقائق التأويل، النسفي،ج3، ص379 . 43 المحرر الوجيز في تفسير الكتاب العزيز، ابن عطية، ج5 ، ص181 . 44 مفاتيح الغيب، الرازي، ج28 ، ص189 . 45 تفسير الشعر اوي،ج1، ص639 . The types of escape in the Holy Quran and its faith significance 370
https://openalex.org/W4376058055	https://www.acquaintpublications.com/get/1-2-JMCRCS2022032801 Galley_Proof-1654924637.pdf	English	null	Strangulation in a premature infant with an inguinal hernia in the neonatal intensive care unit	Journal of medical case reports and case series	2,022	cc-by	1,787	Introduction Incarceration is an important complication of inguinal hernias in children. The bowel, ovaries, or fallopian tubes are the organs that are most commonly incarcerated [4]. If the hernia does not regress spontaneously, the risk of incarceration is greatly increased. An incarcerated hernia can progress rapidly towards strangulation, a condition with vascular compromise and infarction of the incarcerated contents. Therefore, if an incarcerated hernia cannot be reduced manually, urgent inguinal exploration is required [4]. Pediatric inguinal hernias are congenital and classified as indirect hernias based on the pathophysiology of the persistent processus vaginalis, which is associated with an increased risk of inguinal hernias in preterms. The incidence of inguinal hernia in children is 1- 5 %, and this rate may increase up to 30 % in premature babies. Approximately 60 % of inguinal hernias are right-sided, 30% are left- sided, and 10 % are bilateral. Boys are much more likely to have a hernia, with male-female ratios between 3:1 and 10:1 [1-3]. Abstract Inguinal hernia is the most common surgical condition in childhood. Incarcerated inguinal hernia and strangulation of the sac contents are the most important and feared complications. Prematurity is estimated to be a risk factor for incarceration. In this study, a very premature baby weighing 1750 gms who was operated on for incarcerated inguinal hernia and strangulation is presented. Keywords: Incarcerated inguinal hernia, prematurity, low-birthweight infant. Strangulation in a premature infant with an inguinal hernia in the neonatal intensive care unit Şenay Kurtulus MD Strangulation in a premature infant with an inguinal hernia in the neonatal intensive care unit Şenay Kurtulus MD ediatric Surgery, Çanakkale Onsekiz Mart University Faculty of Medicine, Çanakkale, Turkey. Department of Pediatric Surgery, Çanakkale Onsekiz Mart University Faculty of Medicine, Çanakkale, Turkey. *Corresponding Author: Şenay Kurtulus MD, Department of Pediatric Surgery, Çanakkale Onsekiz Mart University Faculty of Medicine, Çanakkale, Turkey. Received date: 28 March 2022; Accepted date: 30 April 2022; Published date: 03 May 2022 Cit ti K t l S (2022) St l ti i t i f t ith i i l h i i th t l i t i it J M d C R C Received date: 28 March 2022; Accepted date: 30 April 2022; Published date: 03 May 2022 Citation: Kurtulus S (2022) Strangulation in a premature infant with an inguinal hernia in the neonatal intensive care unit. J Med Case Rep Case Series 3(07): https://doi.org/10.38207/JMCRCS/2022/JUL03070251 Copyright: © 2022 Şenay Kurtulus MD. 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. Volume 3 Issue 07 Journal of Medical Case Reports and Case Series ISSN: 2692-9880 Volume 3 Issue 07 Journal of Medical Case Reports and Case Series ISSN: 2692-9880 Case Report Discussıon prematurity [6]. On the other hand, it is known that more than half of the premature with an inguinal hernia have been incarcerated, and the risk of incarceration doubles when the repair is delayed beyond 40 weeks of postconceptional age [6,7]. The increase in survival rates of preterm infants with the contribution of advanced neonatal intensive care units is one of the reasons why inguinal hernia is more common in premature infants. Inguinal hernias often occur in the first year of life, and inguinal hernia repair is the most common surgical procedure performed by pediatric surgeons, and approximately one-third of children are younger than 6 months of age when they are operated on [1]. Those with very low birth weights have a 3-fold greater risk of requiring urgent procedures, resulting in higher recurrence rates and more complications, therefore elective hernia repair is recommended, especially in very low birth weight premature infants [8]. Management of inguinal hernia and timing of surgery is difficult in newborns with a hernia, especially in premature [5]. Hernia repair in premature infants is usually delayed until a certain weight or age is achieved in order to minimize the complications that may develop due to anesthesia, surgical technical difficulties, and comorbidities due to Although most pediatric surgeons prefer inguinal hernia repair before discharge, we think that even waiting until discharge from the hospital may increase the risk of unnecessary incarceration and therefore it should be operated on as soon as the diagnosis is confirmed [9,10]. Case Reports Figure 2: The purple appearance of the ischemic testis during the operation (white arrows). Journal of Medical Case Repor Journal of Medical Case Reports and Case Series ISSN: 2692-9880 Journal of Medical Case Reports and Case Series ISSN: 2692-9880 Journal of Medical Case Reports and Case Series ISSN: 2692-9880 Figure 1: Intraoperative view of the strangulated small intestine after inguinal exploration (white arrows). Journal of Medical Case Repor Figure 1: Intraoperative view of the strangulated small intestine after inguinal exploration (white arrows). Figure 1: Intraoperative view of the strangulated small intestine after inguinal exploration (white arrows). Figure 2: The purple appearance of the ischemic testis during the operation (white arrows). Figure 2: The purple appearance of the ischemic testis during the operation (white arrows). Case Reports observed in the intestine, and the proximal intestinal loops were dilated, thus these findings suggested strangulation. While the preterm baby was being followed up in the neonatal intensive care unit, he was consulted to the pediatric surgery department because of painful swelling in the right scrotum. It was stated that there was no swelling suggestive of a hernia since birth in the patient who was born at 28 weeks of gestation and had a very low birth weight (1000 gms). At the presentation, the baby with a postconceptional age of 36+2 weeks and a bodyweight of 1750 grams was restless and crying constantly. On physical examination, the right scrotum was edematous and tender. With the diagnosis of an incarcerated hernia, he was taken to emergency surgery after the manual hernia reduction attempt failed. Thus, there was intestinal ischemia (Figure 1), but intestinal resection was not necessary, and the right testis circulation was also impaired (Figure 2). Following the application of warm saline, the intestinal circulation was restored and herniated loop was replaced in the abdomen along with high ligation of the herniated sac. The patient recovered completely after the operation. In the control ultrasonography 3 weeks later, the volume, shape, and parenchymal echogenicity of the affected right testis were similar to the contralateral normal testis, indicating that testicular atrophy did not develop. In the ultrasonographic examination performed with the preliminary diagnosis of incarcerated inguinal hernia, herniated bowel loops were seen through a 4 mm defect from the inguinal canal to the right scrotal sac, and there was no peristaltic activity with increased echogenicity of the intestinal wall. With color Doppler, no obvious vascularity was 022) Strangulation in a premature infant with an inguinal hernia in the neonatal intensive care unit. J Med Case Rep Case Series 3(07): https://doi.org/10.38207/JMCRCS/2022/JUL0307025 Citation: Kurtulus S (2022) Strangulation in a premature infant with an inguinal hernia in the neonatal intensive care unit. J Med Case Rep Case Series 3(07): https://doi.org/1 Figure 1: Intraoperative view of the strangulated small intestine after inguinal exploration (white arrows). Figure 2: The purple appearance of the ischemic testis during the operation (white arrows). Discussıon Journal of Medical Case Reports and Case Series ISSN: 2692-9880 Figure 1: Intraoperative view of the strangulated small intestine after inguinal exploration (white arrows). Journal of Medical Case Reports and Case Se Figure 1: Intraoperative view of the strangulated small intestine after inguinal exploration (white arrows). Conclusion addition, it should be kept in mind that the first appearance of an inguinal hernia may be by incarceration and should be considered in the differential diagnosis of sudden onset painful scrotal masses. The optimal timing for inguinal hernia repair in premature infants is important in balancing the risks of surgery and respiratory complications against complications such as strangulation. In Acknowledgments: None declared Journal of Medical Case Reports and Case Series ISSN: 2692-9880 Acknowledgments: None declared Journal of Medical Case Reports and Case Series ISSN: 2692-9880 Consent for publication: Official consent received from the patinet. Citation: Kurtulus S (2022) Strangulation in a premature infant with an inguinal hernia in the neonatal intensive care unit. J Med Case Rep Case Series 3(07): https://doi.org/10.38207/JMC Citation: Kurtulus S (2022) Strangulation in a premature infant with an inguinal hernia in the neonatal intensive care unit. J Med Case Rep Case Series 3(07): https://doi.org/10.38207/JMCRCS/2022/JUL03070251 trangulation in a premature infant with an inguinal hernia in the neonatal intensive care unit. J Med Case Rep Case Series 3(07): https://doi.org/10.38207/JMCRCS/2022/JUL03070251 References 7. Lautz TB, Raval MV, Reynolds M (2011) Does timing matter? A national perspective on the risk of incarceration in premature neonates with inguinal hernia. J Pediatr. 158(4): 573-577. 1. Grosfeld JL (1989) Current concepts in inguinal hernia in infants and children. World J Surg. 13(5): 506-515. 1. Grosfeld JL (1989) Current concepts in inguinal hernia in infants and children. World J Surg. 13(5): 506-515. 2. Erdoğan D, Karaman I, Aslan MK, Karaman A, Cavuşoğlu YH (2013) Analysis of 3,776 pediatric inguinal hernia and hydrocele cases in a tertiary center. J Pediatr Surg. 48(8): 1767-1772. 8. de Goede B, Verhelst J, van Kempen BJ, Baartmans MG, Langeveld HR, et al. (2015) Very low birth weight is an independent risk factor for emergency surgery in premature infants with inguinal hernia. J Am Coll Surg. 220(3): 347-352. 3. Morini F, Dreuning KMA, Janssen Lok MJH, Wester T, Derikx JPM, et al. (2021) Surgical Management of Pediatric Inguinal Hernia: A Systematic Review and Guideline from the European Pediatric Surgeons' Association Evidence and Guideline Committee. Eur J Pediatr Surg. 3. Morini F, Dreuning KMA, Janssen Lok MJH, Wester T, Derikx JPM, et al. (2021) Surgical Management of Pediatric Inguinal Hernia: A Systematic Review and Guideline from the European Pediatric Surgeons' Association Evidence and Guideline Committee. Eur J Pediatr Surg. 9. Crankson SJ, Al Tawil K, Al Namshan M, Al Jadaan S, Baylon BJ, et al. (2015) Management of inguinal hernia in premature infants: 10-year experience. J Indian Assoc Pediatr Surg. 20(1): 21-24. 4. Meena D, Jhuria R, Saxena S, Saini U (2017) Inguinoscrotal hernia in infants: Three case reports in ultrasound diagnosis. Indian J Radiol Imaging. 27(1): 78-81. 10. Verhelst J, de Goede B, van Kempen BJ, Langeveld HR, Poley MJ, et al. (2016) Emergency repair of inguinal hernia in the premature infant is associated with high direct medical costs. Hernia. 20(4): 571-577. 5. Misra D (2001) Inguinal hernias in premature babies: wait or operate?. Acta Paediatr. 90(4): 370-371. 5. Misra D (2001) Inguinal hernias in premature babies: wait or operate?. Acta Paediatr. 90(4): 370-371. 6. Sulkowski JP, Cooper JN, Duggan EM, Balci O, Anandalwar SP, et al. (2015) Does timing of neonatal inguinal hernia repair affect outcomes?. J Pediatr Surg. 50(1): 171-176.
https://openalex.org/W4252023444	https://figshare.com/articles/preprint/Performance_Analysis_for_Discrete-Time_Linear_Systems_with_Saturated_Linear_Feedback_a_Nonlinear_Saturation-Dependent_Gain-Scheduling_Approach/16832881/1/files/31130146.pdf	English	null	Performance Analysis for Discrete-Time Linear Systems with Saturated Linear Feedback: a Nonlinear Saturation-Dependent Gain-Scheduling Approach	null	2,021	cc-by	9,289	CC BY 4.0 SUBMISSION DATE / POSTED DATE 19-10-2021 / 21-10-2021 CITATION Hoai Nam, Nguyen (2021): Performance Analysis for Discrete-Time Linear Systems with Saturated Linear Feedback: a Nonlinear Saturation-Dependent Gain-Scheduling Approach. TechRxiv. Preprint. https://doi.org/10.36227/techrxiv.16832881.v1 Hoai Nam, Nguyen (2021): Performance Analysis for Discrete-Time Linear Systems with Saturated Linear Feedback: a Nonlinear Saturation-Dependent Gain-Scheduling Approach. TechRxiv. Preprint. https://doi.org/10.36227/techrxiv.16832881.v1 DOI 10.36227/techrxiv.16832881.v1 Abstract In this paper, we propose a new technique for the performance analysis of discrete- time linear systems controlled by a saturated linear control law. Two performance indices, the computation of invariant sets and the L2 performance, are considered. The main contributions of the paper are the following: i) a new linear parameter varying system framework is presented to model the saturated system, ii) a nonlin- ear saturation-dependent auxiliary feedback matrix is considered, iii) new sufficient conditions for the performance analysis are proposed. It is shown that the conditions can be expressed as a set of linear matrix inequalities. Furthermore, it is shown that the conditions are guaranteed to be less conservative than existing solutions in the literature. Three numerical examples are presented to illustrate the effectiveness of the proposed method. Key words: Discrete-Time Linear System, Actuator Saturation, LPV Modeling, Invariant Set, L2 Gain Analysis, Linear Matrix Inequalities (LMI) Key words: Discrete-Time Linear System, Actuator Saturation, LPV Modeling, Invariant Set, L2 Gain Analysis, Linear Matrix Inequalities (LMI) Performance Analysis for Discrete-Time Linear Systems with Saturated Linear Feedback: a Nonlinear Saturation-Dependent Gain-Scheduling Approach Hoai-Nam Nguyen a,b aIP Paris - Institute Polytechnique de Paris, 19 rue Marguerite Perey. 91120 Palaiseau, France bElectronics and Physics Department, Telecom-SudParis, 9 Rue Charles Fourier, 91000 Evry, France Hoai-Nam Nguyen a,b Hoai-Nam Nguyen a,b aIP Paris - Institute Polytechnique de Paris, 19 rue Marguerite Perey. 91120 Palaiseau, France bElectronics and Physics Department, Telecom-SudParis, 9 Rue Charles Fourier, 91000 Evry, France aIP Paris - Institute Polytechnique de Paris, 19 rue Marguerite Perey. 91120 Palaiseau, France bElectronics and Physics Department, Telecom-SudParis, 9 Rue Charles Fourier, 91000 Evry, France Preprint submitted to Elsevier Science Key words: Discrete-Time Linear System, Actuator Saturation, LPV Modeling, Invariant Set, L2 Gain Analysis, Linear Matrix Inequalities (LMI) 1 Introduction Saturation is probably the most commonly encountered non-linearity in con- trol engineering. Analyzing the system performance that can be achieved un- Email address: hoai-nam.nguyen@telecom-sudparis.eu (Hoai-Nam Nguyen). Preprint submitted to Elsevier Science Preprint submitted to Elsevier Science 19 October 2021 der the input saturation is of great importance, and has received the attention of many researchers, for example [10], [15], [22], [26], and references therein. With the absolute stability analysis tools, such as the circle and Popov criteria, several methods have been proposed for the performance analysis of saturated systems such as the estimation of the domain of attraction [9], [16], L2 and/or L∞disturbance rejection [21]. In [14], a piecewise quadratic estimate of the domain of attraction for a continuous-time saturated system is considered. The main idea is to use an initial ellipsoidal estimation obtained by means of the circle criterion. One of the most relevant methods to the performance analysis of saturated systems is based on a linear difference inclusion (LDI) framework. The idea is to express the saturated linear feedback law as a convex hull of a group of 2m linear feedback laws, where m is the control input dimension. Using this frame- work, in [11], [4], [2], an estimation of the domain of attraction is obtained, and in [6], [24], the problem of L2 gain analysis is addressed. In conjunction with the LDI framework, various Lyapunov functions were developed, for exam- ple, quadratic Lyapunov functions [11], [12], saturation-dependent Lyapunov function [4], [24], composite Lyapunov function and max quadratic Lyapunov function [13]. However all the existing results were obtained by using a linear saturation-independent auxiliary feedback law. In this paper we present an approach to the performance analysis of discrete- time saturated linear systems using the LDI framework. Given a system with m saturated control inputs, we show how to select an LDI in such a way the performance is optimized. Our idea is to use a nonlinear saturation-dependent auxiliary feedback law, whereby the real-time information on the severity of saturation is fully exploited. The linear parameter varying modeling frame- work is used to model the resulting system. The obtained conditions are con- verted into linear matrix inequalities (LMI) constraints. The conference contribution [20] touches on the contents of this paper. The paper is structured as follows. Section 2 describes the problem formulation and some preliminaries. Section 3 is dedicated to the main results of the paper. 2 Problem Formulation and Preliminaries 2 Problem Formulation and Preliminaries 2 Problem Formulation and Preliminaries 2.1 Problem Formulation 1 Introduction Three numerical examples with comparison to earlier solutions are evaluated in Section 4, before drawing the conclusions in Section 5. Notation: A positive-definite (semi-definite) matrix P is denoted by P ≻0 (P ⪰0). 0, I, 1 are, respectively, the zero matrix, the identity matrix, and the all-ones vector of appropriate dimensions. For a given P ⪰0, E(P) represents the following ellipsoid E(P) = {x ∈Rn : xTP −1x ≤1} 2 For a given matrix H of appropriate dimension, L(H) is used to denote the following symmetric polyhedron L(H) = {x ∈Rn : −1 ≤Hx ≤1} The inequalities are to be interpreted element-wise. The inequalities are to be interpreted element-wise. For symmetric matrices, the symbol (∗) denotes each of its symmetric block. 2.1 Problem Formulation Consider the following discrete-time linear system Consider the following discrete-time linear system      x(k + 1) = Ax(k) + Bsat(u(k)) + Ew(k) z(k) = Cx(k) + Dw(k) (1) (1) where x ∈Rn is the measured state, u ∈Rm is the control input, w ∈Rq is the disturbance, z ∈Rp is the performance output, A ∈Rn×n, B ∈Rn×m, E ∈Rn×q, C ∈Rp×n, and D ∈Rp×q. The saturation function sat(u) : Rm →Rm is defined as The saturation function sat(u) : Rm →Rm is defined as The saturation function sat(u) : Rm →Rm is defined as sat(u) = [sat(u1) sat(u2) . . . sat(um)]T, (2) sat(uj) =              −1, if uj ≤−1 uj, if −1 ≤uj ≤1, ∀j = 1, m 1, if uj ≥1 (2) The objective of this paper is to carry out systematically an analysis of system (1), under a given linear state feedback law The objective of this paper is to carry out systematically an analysis of system (1), under a given linear state feedback law u(k) = Kx(k) (3) u(k) = Kx(k) (3) (3) The following two problems will be considered The following two problems will be considered (1) In the absence of w, we would like to compute an invariant set Ωas large as possible so that if x(k) ∈Ω, we have x(k + 1) ∈Ω, ∀k ≥0. (2) With a given bound on the L2 norm of w, i.e., ∥w∥2 2 ≤β, we would like to determine a number α > 0 as small as possible, so that under the condition x(0) = 0, we have ∥z∥2 ≤α∥w∥2. Performing this analysis for each β ∈(0, ∞), we obtain an estimate of the nonlinear L2 gain. 3 It is assumed that all eigenvalues of A + BK are in the interior of the unit circle. It is assumed that all eigenvalues of A + BK are in the interior of the unit circle. 2.2 Previous Works: LDI Modeling Framework In the following we recall the linear differential inclusion (LDI) modeling framework, which was proposed in [1], [11], [12]. This framework can be con- sidered as a generalization of the circle criterion [16] for the saturation non- linearity. Define M = {1, 2, . . . 2.1 Problem Formulation For example, if m = 2 then Define D as the set of m × m diagonal matrices whose diagonal elements are either 1 or 0. For example, if m = 2 then D =        1 0 0 1  ,   1 0 0 0  ,   0 0 0 1  ,   0 0 0 0        There are 2m elements in D. Define El, l = 1, 2m as an element in D. Then V = {El, l = 1, 2m}. Define also E− l = Im −El. Clearly, E− l is also an element of D. There are 2m elements in D. Define El, l = 1, 2m as an element in D. Then V = {El, l = 1, 2m}. Define also E− l = Im −El. Clearly, E− l is also an element of D. In Lemma 1, if we select vl(j) = vj = Hjx, l = 1, 2m, j = 1, m, then we obtain the following result, which is proposed in [11] In Lemma 1, if we select vl(j) = vj = Hjx, l = 1, 2m, j = 1, m, then we obtain the following result, which is proposed in [11] Lemma 2: [11] Let K, H ∈Rm×n. For all x ∈L(H), one has Lemma 2: [11] Let K, H ∈Rm×n. For all x ∈L(H), one has sat(Kx) = 2m X l=1 λl(ElK + E− l H)x (6) (6) where 2m P l=1 λl = 1, λl ≥0 where 2m P l=1 λl = 1, λl ≥0 For the single input case, Lemma 1 and Lemma 2 are the same if a linear auxiliary feedback gain is chosen for v2(1). For the multi-input case, Lemma 1 provides an extra degree of freedom. Hence for the performance analysis, con- ditions based on Lemma 1 are less conservative than that are based on Lemma 2. However, this comes with a cost of a higher computational complexity. Substituting (6) in (1), one obtains x(k + 1) = A + 2m X l=1 λl(ElK + E− l H) ! x(k) + Ew(k) (7) (7) Hence (1) can be modeled as an uncertain time-varying system ∀x : −1 ≤ Hx ≤1, whereby the parameters λl, l = 1, 2m are unknown and time-varying. 2.1 Problem Formulation , m}, and V as the set of all subsets of M, i.e., V = {S : S ⊆M}. Note that the empty set belongs to V. Define also Sc as the complementary of S in M, i.e., Sc = {i ∈M : i /∈S}. For example, if m = 2, then M = {1, 2} and V = {S1, S2, S3, S4} with S1 = ∅, S2 = {1}, S3 = {2}, S4 = {1, 2}, Sc 1 = {1, 2}, Sc 2 = {2}, Sc 3 = {1}, Sc 4 = ∅ Denote ej as the jth standard basis of Rm, i.e., ej = [0 . . . 0 1 \|{z} jth element 0 . . . 0]T Associated to Sl, ∀l = 1, 2m, consider the following scalars vl(j), ∀j = 1, m,      −1 ≤vl(j) ≤1, if j ∈Sl vl(j) = 0, if j /∈Sl (4) (4) The following lemma is taken from [22]. It has been proposed originally in [1]. It will be used to model the saturation non-linearity (2). Lemma 1: [22] With vs(j) defined as in (4), the following equation holds sat(Kx) = 2m X l=1 λl  X j∈Sc l ejKjx + X j∈Sl ejvl(j)   (5) (5) where Kj denotes the jth row of K, j = 1, m. where Kj denotes the jth row of K, j = 1, m. For example, if m = 1, we have For example, if m = 1, we have sat(Kx) = λ1Kx + λ2v2(1) 4 with λ1 + λ2 = 1, λl ≥0, l = 1, 2. If m = 2, we have   sat(K1x) sat(K2x)  = λ1   K1x K2x  + λ2   v2(1) K2x  + λ3   K1x v3(2)  + λ4   v4(1) v4(2)   with 4P l=1 λl = 1, λl ≥0, l = 1, 4. with 4P l=1 λl = 1, λl ≥0, l = 1, 4. Define D as the set of m × m diagonal matrices whose diagonal elements are either 1 or 0. For example, if m = 2 then Define D as the set of m × m diagonal matrices whose diagonal elements are either 1 or 0. 2.1 Problem Formulation It was shown in [11] that for the estimation of the domain of attraction, conditions using (7) are less conservative than that are based on the circle criterion or the vertex analysis. Furthermore, as it is proved in [11], Lemma 2 5 provides a necessary and sufficient conditions for an ellipsoid to be invariant for the single input case, i.e., m = 1. In [4], it was noticed that the parameters λl, l = 1, 2m reflects the severity of the saturation function. Consequently, λl, l = 1, 2m are functions of x. To see this, consider the case m = 1, and assume H = 0. In this case, ∀x, −1 ≤Hx = 0 ≤1. Using (6), one obtains sat(Kx) = λ1Kx If −1 ≤Kx ≤1, then the saturation function becomes If −1 ≤Kx ≤1, then the saturation function becomes sat(Kx) = Kx therefore λ1 = 1 and λ2 = 0. If Kx = 3, then therefore λ1 = 1 and λ2 = 0. If Kx = 3, then sat(Kx) = 1 = 1 3Kx sat(Kx) = 1 = 1 3Kx therefore λ1 = 1 3, λ2 = 2 3. therefore λ1 = 1 3, λ2 = 2 3. Similarly, if we assume H = 1 2K, then using (6), one gets, Similarly, if we assume H = 1 2K, then using (6), one gets, sat(Kx) = (λ1 + 1 2λ2)Kx for all x such that −1 ≤1 2Kx ≤1. If −1 ≤Kx ≤1, we have λ1 = 1, λ2 = 0. If Kx = 3 2, then λ1 = 1 3, λ2 = 2 3. for all x such that −1 ≤1 2Kx ≤1. If −1 ≤Kx ≤1, we have λ1 = 1, λ2 = 0. If Kx = 3 2, then λ1 = 1 3, λ2 = 2 3. Using the available information of λl, l = 1, 2m, a saturation-dependent Lya- punov function was proposed in [4] to estimate the domain of attraction, and in [24] to estimate the L2 gain. The conditions are proved to be less conservative than that are based on quadratic Lypanov function. The auxiliary feedback matrix H in [4], [11], [24] is a decision variable. It can be optimized to provide a less conservative estimation of the domain of attraction and/or of the L2 gain. 2.1 Problem Formulation However, in [11], [4], [24], H is a constant matrix, and does not depend on λl, l = 1, 2m. Hence, the real-time information on the severity of saturation is not exploited. The main objective of this paper is to show that by selecting a nonlinear saturation-dependent auxiliary feed- back matrix H, a significant improvement in the performance analysis can be obtained. For this purpose, some preliminary results are recalled in the next section. 6 2.3 Preliminaries The following double sum positivity problem of the form xT( 2m X l1=1 2m X l2=1 λl1λl2Γl1l2)x ≥0 (8) (8) will be dealt several times in this paper, where the coefficients λl satisfy 2m X l=1 λl = 1, λl ≥0, ∀l = 1, 2m 2m X l=1 λl = 1, λl ≥0, ∀l = 1, 2m Lemma 3: The double sum (8) is positive, if, see [25], Lemma 3: The double sum (8) is positive, if, see [25],      Γll ⪰0, l = 1, 2m Γl1l2 + Γl2l1 ⪰0, ∀l1, l2 = 1, 2m, l1 < l2 (9) (9) Lemma 4: The double sum (8) is positive, if, see [23], Lemma 4: The double sum (8) is positive, if, see [23],      Γll ⪰0, l = 1, 2m 2 2m−1Γl1l1 + Γl1l2 + Γl2l1 ⪰0, ∀l1, l2 = 1, 2m, l1 ̸= l2 (10) (10) Remark 1: It is well known [23] that Lemma 4 is less conservative Lemma 3, i.e., if there exist matrices Γl1l2 satisfying (10), they also satisfy (9). The main advantage of (9) with respect to (10) is that (9) has a fewer number of LMI constraints than (10). Hence the computational complexity is reduced. Remark 1: It is well known [23] that Lemma 4 is less conservative Lemma 3, i.e., if there exist matrices Γl1l2 satisfying (10), they also satisfy (9). The main advantage of (9) with respect to (10) is that (9) has a fewer number of LMI constraints than (10). Hence the computational complexity is reduced. Lemma 5: Given matrices P, G of appropriate dimension with P ≻0. 2.1 Problem Formulation Then, see [5] (G −P)TP −1(G −P) ⪰0 ⇔GTP −1G ⪰GT + G −P (11) 6: For given matrices F ∈Rnf×n, and P ⪰0, E(P, 1) ⊆L(F) if and e [10] (G −P)TP −1(G −P) ⪰0 ⇔GTP −1G ⪰GT + G −P (11) 6: For given matrices F ∈Rnf×n, and P ⪰0, E(P, 1) ⊆L(F) if and (G −P)TP −1(G −P) ⪰0 ⇔GTP −1G ⪰GT + G −P (11) (11) (G P) P (G P) ⪰0 ⇔G P G ⪰G + G P (11) Lemma 6: For given matrices F ∈Rnf×n, and P ⪰0, E(P, 1) ⊆L(F) if and only if, see [10] Lemma 6: For given matrices F ∈Rnf×n, and P ⪰0, E(P, 1) ⊆L(F) if and only if, see [10] 1 −ejFPF TeT j ⪰0, j = 1, nf (12) (12) where ej is the jth standard basic of Rnf. where ej is the jth standard basic of Rnf. Concerning the LMIs, we will make use of the following results. and M11, M22 being square matrices. Then, see [3] M ⪰0 ⇔      M11 ⪰0, M22 −M T 12M −1 11 M12 ⪰0 ⇔      M22 ⪰0, M11 −M12M −1 22 M T 12 ⪰0 (14) (14) ⇔   M11 −M12M −1 22 M T 12 ⪰0 Concerning the LMIs, we will make use of the following results. Concerning the LMIs, we will make use of the following results. Property 1 (Congruence): Let P and Q are matrices of appropriate di- mension, where P = P T, and Q is a full rank matrix. It holds that P ⪰0 ⇔QTPQ ⪰0 (13) (13) 7 Property 2: (Schur complements): Consider a matrix M, with M =   M11 M12 M T 12 M22   M =   M11 M12 M T 12 M22   M =   M11 M12 M T 12 M22   and M11, M22 being square matrices. Then, see [3] 3.1 LPV Modeling In this section, we present an LPV framework to model the saturated sys- tem (1). As will be shown, our LPV model allows to consider a nonlinear saturation-dependent auxiliary feedback matrix in contrast to the LPV model (7) in [4], [24]. ( ) [ ], [ ] Substituting (3) to (1), one obtains Substituting (3) to (1), one obtains Substituting (3) to (1), one obtains x(k + 1) = Ax(k) + Bsat(Kx(k)) + Ew(k) (15) (15) Using Lemma 1, there exist λl(k) and vl(j)(k), l = 1, 2m, j = 1, m such that Using Lemma 1, there exist λl(k) and vl(j)(k), l = 1, 2m, j = 1, m such that              2m P l=1 λl(k) = 1, λl(k) ≥0, l = 1, 2m      −1 ≤vl(j)(k) ≤1, if j ∈Sl vl(j)(k) = 0, if j /∈Sl and and and x(k + 1) = Ax(k) + 2m X l=1 λl(k)  X j∈Sc l BjKjx(k) + X j∈Sl Bjvl(j)(k)  + Ew(k) x(k + 1) = Ax(k) + 2m X l=1 λl(k)  X j∈Sc l BjKjx(k) + X j∈Sl Bjvl(j)(k)  + Ew(k) 8 where Bj is the jth column of B, j = 1, m. Thus, with A = 2m P l=1 λlA where Bj is the jth column of B, j = 1, m. Thus, with A = 2m P l=1 λlA x(k + 1) = 2m X l=1 λl(k)   (A + X j∈Sc l BjKj)x(k) + X j∈Sl Bjvl(j)(k)   + Ew(k) (16) Define        Al = A + P j∈Sc l BjKj, Bl = 0 Bj 0 j∈Sl , v = [v2(1) v3(2) . . . v2m(m)]T (17) (17) Note that v ∈Rm2m−1×1. For example, if m = 1, then v = v2(1) and Note that v ∈Rm2m−1×1. For example, if m = 1, then v = v2(1) and Note that v ∈Rm2m−1×1. 3.1 LPV Modeling For example, if m = 1, then v = v2(1) and A1 = A + BK, A2 = A, B1 = 0n×1, B2 = B If m = 2, then v = [v2(1) v3(2) v4(1) v4(2)]T and A1 = A + BK, A2 = A, B1 = 0n×1, B2 = B If m = 2, then v = [v2(1) v3(2) v4(1) v4(2)]T and If m = 2, then v = [v2(1) v3(2) v4(1) v4(2)]T and If m = 2, then v = [v2(1) v3(2) v4(1) v4(2)]T and      A1 = A + BK, A2 = A + B2K2, A3 = A + B1K1, A4 = A B1 = 0n×4, B2 = B1 0n×3 , B3 = 0n×1 B2 0n×2 , B4 = 0n×2 B Using (17), (16) can be rewritten as Using (17), (16) can be rewritten as x(k + 1) = 2m X l=1 λl(k)Al ! x(k) + 2m X l=1 λl(k)Bl ! v(k) + Ew(k) (18) (18) The auxiliary variable v(k) can be considered as a control input for the system (18). Hence the problem of carrying out the performance analysis for the system (15) becomes the problem of selecting the optimal input v(k) to obtain the best performance for (18). The auxiliary variable v(k) can be considered as a control input for the system (18). Hence the problem of carrying out the performance analysis for the system (15) becomes the problem of selecting the optimal input v(k) to obtain the best performance for (18). Remark 2: If Lemma 2 is used to model the saturation non-linearity instead of Lemma 1, then (18) can still be used to model (15) but with the following matrices Al, Bl, and v      Al = A + BElK, Bl = BE− l , l = 1, 2m v = [v1 v2 . . . vm]T (19) (19) Note that the dimension of v in (19) is m. 3.1 LPV Modeling For example if m = 2 then v = [v1 v2]T and      A1 = A + BK, A2 = A + B2K2, A3 = A + B1K1, A4 = A B1 = 0n×2, B2 = B1 0n×1 , B3 = 0n×1 B2 , B4 = B 9 Consider the following auxiliary feedback law Consider the following auxiliary feedback law Consider the following auxiliary feedback law v(k) = F(λ(k))G(λ(k))−1x(k) (20) (20) with with F(λ(k)) = 2m X l=1 λ(k)Fl, G(λ(k)) = 2m X l=1 λ(k)Gl (21) (21) where Fl, Gl are unknown matrices that will be treated as decision variables. Remark 3: In the literature [11], [24], [18], only a linear saturation-independent control law v(k) = Hx(k) was considered with H = FG−1. Clearly, this is a particular case of (20) with Fl = F and Gl = G, ∀l = 1, 2m. The nonlinear saturation dependent control law (20) takes the real time information of the saturation into account. As will be shown in the examples, a less conservative estimate of the performance is obtained. Substituting (20) into (16), one obtains the following closed-loop system x(k + 1) = Ac(λ(k))x(k) + Ew(k) (22) (22) where Ac(λ(k)) = A(λ(k)) + B(λ(k))F(λ(k))G(λ(k))−1 It should be stressed that system (15) can be modeled as (22) only for x such that It should be stressed that system (15) can be modeled as (22) only for x such that −1 ≤F(λ(k))G(λ(k))−1x(k) ≤1 (23) (23) Define the following saturation-dependent Lyapunov function V (k, x(k)) = x(k)T(P(λ(k)))−1x(k) (24) (24) with P(λ(k)) = 2m X l=1 λl(k)Pl (25) (25) where Pl ⪰0, l = 1, 2m are unknown matrices that will be treated as decision variables. With slight abuse of notation, Ac(k), A(k), B(k), G(k), P(k) are used to denote Ac(λ(k)), A(λ(k)), B(λ(k)), G(λ(k)), P(λ(k)). With slight abuse of notation, Ac(k), A(k), B(k), G(k), P(k) are used to denote Ac(λ(k)), A(λ(k)), B(λ(k)), G(λ(k)), P(λ(k)). 2 Invariant Set Computation 3.2 Invariant Set Computation 3.2 Invariant Set Computation In this section, we are interested in computing an invariant set as large as possible in the absence of w. For this purpose, the following definitions are recalled [19]. 10 Definition 1 (Domain of Attraction): A set Ωis said to be inside the domain of attraction for system (15), if for any initial condition x(0) ∈Ω, one has lim k→∞x(k) = 0. Definition 2 (Invariant Set): A set Ωis said to be invariant for system (15), if ∀x(k) ∈Ω, one has x(k + 1) ∈Ω, ∀k ≥0, and lim k→∞x(k) = 0. Clearly from Definition 2, if a set is invariant, then it contains the origin in its interior. It is also clear that if a set is invariant then it is inside the domain of attraction, but not the other way around, i.e., if a set is inside the domain of attraction, then it is not necessarily invariant. The following theorem provides the theoretical support of the algorithm pro- posed to calculate an invariant set for the system (15). Theorem 1: Consider the system (15). Assume that there exist matrices Pl ⪰0, Fl, Gl, l = 1, 2m satisfying the following matrix inequalities   1 ejF(k) F(k)TeT j G(k) + G(k)T −P(k)  ⪰0, j = 1, m2m−1 (26) (26)   P(k + 1) A(k)G(k) + B(k)F(k) (∗) G(k) + G(k)T −P(k)  ⪰0 (27) (27) then, the set V (k, x(k)) ≤1 is invariant. Proof: It was showing that for all x satisfying (23), (22) can be used to model (15). where Using Lemma 6, condition (23) is equivalent 1 −ejF(k)G(k)−1P(k)(G(k)−1)TF(k)TeT j ≥0, ∀j = 1, m2m−1 Recall that ej is the jth standard basic of Rm2m−1. With Schur complement, one gets, ∀j = 1, m2m−1   1 ejF(k) F(k)TeT j G(k)TP(k)−1G(k)  ≥0 (28) (28) Using Lemma 5, condition (28) is satisfied if   1 ejF(k) F(k)TeT j G(k) + G(k)T −P(k)  ≥0, ∀j = 1, m2m−1 (29) (29) Note that (29) is (26). In remains to show that V (k, x(k +1)) ≤1 is invariant. 11 It is required that x(k + 1)TP(k + 1)−1x(k + 1) −x(k)TP(k)−1x(k) ≤0 Using (22), one obtains Using (22), one obtains Ac(k)TP(k + 1)−1Ac(k) −P(k)−1 ⪯0 or equivalently or equivalently (A(k)G(k)+B(k)F(k))TP(k+1)−1(A(k)G(k)+B(k)F(k))−G(k)TP(k)−1G(k) ⪯0 Using Schur complement, one gets (A(k)G(k)+B(k)F(k))TP(k+1)−1(A(k)G(k)+B(k)F(k))−G(k)TP(k)−1G(k) ⪯0 Using Schur complement, one gets (A(k)G(k)+B(k)F(k))TP(k+1)−1(A(k)G(k)+B(k)F(k))−G(k)TP(k)−1G(k) ⪯0 Using Schur complement, one gets Using Schur complement, one gets   P(k + 1) (A(k)G(k) + B(k)F(k)) (A(k)G(k) + B(k)F(k))T G(k)TP(k)−1G(k)  ⪰0 (30) (30) Thus, using Lemma 5, (30) is satisfied if Thus, using Lemma 5, (30) is satisfied if Thus, using Lemma 5, (30) is satisfied if   P(k + 1) (A(k)G(k) + B(k)F(k)) (A(k)G(k) + B(k)F(k))T G(k)T + G(k) −P(k)  ⪰0 (31) (31) 2 The proof is complete. 2 The proof is complete. 2 The proof is complete. The proof is complete. In Theorem 1, if Lemma 2 is used to model the saturation non-linearity, and by setting Fl = F, Gl = G, ∀l = 1, 2m, then Theorem 1 in [4] is recovered. This implies that Theorem 1 in [4] is a particular case of ours. Theorem 1 gives a condition for the set Theorem 1 gives a condition for the set V (k, x(k)) = {x(k) : x(k)TP(k)−1x(k) ≤1} to be invariant. Note that this set might be non-convex. If we are interested in a convex characterization of the invariant set, then the intersection of the ellipsoids 2m T l=1 E(Pl) can be used. However in this case, using Theorem 1, we can only guarantee that 2m T l=1 E(Pl) is inside the domain of attraction. In the following we prove that 2m T l=1 E(Pl) is also invariant. to be invariant. Note that this set might be non-convex. If we are interested in a convex characterization of the invariant set, then the intersection of the ellipsoids 2m T l=1 E(Pl) can be used. where However in this case, using Theorem 1, we can only guarantee that 2m T l=1 E(Pl) is inside the domain of attraction. In the following we prove that 2m T l=1 E(Pl) is also invariant. Theorem 2: If there exist Pl ⪰0, Fl, Gl satisfying (26), (27), then the set 2m T l=1 E(Pl) is invariant. Theorem 2: If there exist Pl ⪰0, Fl, Gl satisfying (26), (27), then the set 2m T l=1 E(Pl) is invariant. 12 Proof: Note that ∀x(0) ∈ 2m T l=1 E(Pl), one has lim k→∞x(k) = 0 lim k→∞x(k) = 0 since 2m T l=1 E(Pl) is contained in the domain of attraction. It remains to prove that ∀x(k) ∈ 2m T l=1 E(Pl), one has x(k + 1) ∈ 2m T l=1 E(Pl), ∀k ≥0. since 2m T l=1 E(Pl) is contained in the domain of attraction. It remains to prove that ∀x(k) ∈ 2m T l=1 E(Pl), one has x(k + 1) ∈ 2m T l=1 E(Pl), ∀k ≥0. At time k, without loss of generality, assume that λl0(k) = 1, then λl(k) = 0, ∀l = 1, 2m, l ̸= l0. Since x(k) ∈ 2m T l=1 E(Pl) one has x(k) ∈E(Pl0). Following Theorem 1, one has x(k + 1)TP(k + 1)−1x(k + 1) ≤x(k)TP −1 l0 x(k) ≤1 (32) + 1) = Ac(k)x(k) with Ac(k) = Al0 + Bl0Fl0G−1 l0 . (32) In Theorem 1, no assumption about λl(k + 1) were made, except that In Theorem 1, no assumption about λl(k + 1) were made, except that 2m X l=1 λl(k + 1) = 1, λl(k + 1) ≥0 2m X l=1 λl(k + 1) = 1, λl(k + 1) ≥0 Hence P(k+1) can be any matrices Pl, l = 1, 2m and their convex combination. Using (32), it follows that x(k + 1) ∈E(Pl), ∀l = 1, 2m. In other words, Hence P(k+1) can be any matrices Pl, l = 1, 2m and their convex combination. Using (32), it follows that x(k + 1) ∈E(Pl), ∀l = 1, 2m. In other words, Hence P(k+1) can be any matrices Pl, l = 1, 2m and their convex combination. Using (32), it follows that x(k + 1) ∈E(Pl), ∀l = 1, 2m. In other words, Hence P(k+1) can be any matrices Pl, l = 1, 2m and their convex combination. where Using (32), it follows that x(k + 1) ∈E(Pl), ∀l = 1, 2m. In other words, x(k + 1) ∈ 2m \ l=1 E(Pl) x(k + 1) ∈ 2m \ l=1 E(Pl) or the set 2m T l=1 E(Pl) is invariant. 2 or the set 2m T l=1 E(Pl) is invariant. 2 or the set 2m T l=1 E(Pl) is invariant. 2 2 Using (21), (25), condition (26) holds if and only if Using (21), (25), condition (26) holds if and only if Using (21), (25), condition (26) holds if and only if   1 Flej F T l ej Gl + GT l −Pl  ⪰0, ∀l = 1, 2m, ∀j = 1, m2m−1 (33) (33) Rewrite (27) as 2m X l1=1 2m X l2=1 λl1λllΓr l1l2 ⪰0 (34) 2m X l1=1 2m X l2=1 λl1λllΓr l1l2 ⪰0 (34) with r = 1, 2m, and (34) with r = 1, 2m, and with r = 1, 2m, and Γr l1l2 =   Pr (Al1Gl2 + Bl1Fl2) (Al1Gl2 + Bl1Fl2)T (Gl2 + Gl2 −Pl2)   (35) (35) 13 Combining (33), (34), relaxed LMI conditions can be formulated using Lem- mas 3, or 4 as follows Combining (33), (34), relaxed LMI conditions can be formulated using Lem- mas 3, or 4 as follows Corollary 1: If there exist matrices Pl ⪰0, Gl, Fl, l = 1, 2m such that (33) hold and  (33) hold and      Γr ll ⪰0, ∀r, ∀l = 1, 2m Γr l1l2 + Γr l2l1 ⪰0, ∀r, l1, l2 = 1, 2m, l1 < l2 (36) (36) or or      Γr ll ⪰0, ∀r, ∀l = 1, 2m 2Γr l1l1 + Γr l1l2 + Γr l2l1 ⪰0, ∀m, l1, l2 = 1, 2m, l1 ̸= l2 (37) (37) then the set 2m T l=1 E(Pl) is invariant. then the set 2m T l=1 E(Pl) is invariant. The proof of corollary 1 is straightforward. In the interest of the size of the domain of attraction, which is proportional logdet(Pl), the set 2m T l=1 E(Pl) should be maximized. This can be done by solving the following SDP problem max Pl,Fl,Gl ( 2m P l=1 logdet(Pl) ) s.t. (33), (36) (38) max Pl,Fl,Gl ( 2m P l=1 logdet(Pl) ) s.t. (33), (36) (38) (38) or max Pl,Fl,Gl ( 2m P l=1 logdet(Pl) ) s.t. where (33), (37) (39) or max Pl,Fl,Gl ( 2m P l=1 logdet(Pl) ) s.t. (33), (37) (39) (39) Since (38) and/or (39) are a convex SDP problem, they can be solved effi- ciently using free available LMI parser such as CVX [8], or Yalmip [17]. In the following, we refer to the optimization problems (38), and (39), respectively, as algorithm 1 and algorithm 2. Since (38) and/or (39) are a convex SDP problem, they can be solved effi- ciently using free available LMI parser such as CVX [8], or Yalmip [17]. In the following, we refer to the optimization problems (38), and (39), respectively, as algorithm 1 and algorithm 2. Remark 4: Note that the number of LMIs in (38) and (39) increases expo- nentially as the number of the system input m increases. 3.3 L2 Performance Analysis In this section, we are interested in estimating the L2 gain for the system (1), (3). This L2 gain is defined as follows. 14 Definition 3 (L2 gain): For a given γ > 0, the system (1), (3) is said to be with a L2 gain less than γ, if for the zero initial condition, one has ∞ X k=0 z(k)Tz(k) −γ2 ∞ X k=0 w(k)Tw(k) ≤0 (40) (40) for all nonzero w ∈W. Recall that the set W is w ∈W = {w ∈Rq : ∥w∥2 2 ≤β} w ∈W = {w ∈Rq : ∥w∥2 2 ≤β} where β > 0 is a given constant. where β > 0 is a given constant. where β > 0 is a given constant. where β > 0 is a given constant. The following theorem establishes a sufficient condition to estimate the L2 gain for the system (1). Theorem 3: Consider the system (1). where For given scalars α > 0, β > 0, if there exist matrices Pl ⪰0, Fl, Gl, l = 1, 2m and a scalar γ > 0 such that the following matrix inequalities   P(k + 1) A(k)G(k) + B(k)F(k) E 0 (∗) G(k) + G(k)T −P(k) 0 G(k)TCT (∗) (∗) I DT (∗) (∗) (∗) γ2I   ⪰0 (41)   P(k + 1) A(k)G(k) + B(k)F(k) E 0 (∗) G(k) + G(k)T −P(k) 0 G(k)TCT (∗) (∗) I DT (∗) (∗) (∗) γ2I   ⪰0 (41)   1 α+β ejF(k) F(k)TeT j G(k) + G(k)T −P(k)  ⪰0, ∀j = 1, m2m−1 (42) (41)   1 α+β ejF(k) F(k)TeT j G(k) + G(k)T −P(k)  ⪰0, ∀j = 1, m2m−1 (42) (42) hold, then, for all x(0) such that x(0)TP(0)−1x(0) ≤α, one has, ∀k ≥1 x(k)TP(k)−1x(k) ≤α + β hold, then, for all x(0) such that x(0)TP(0)−1x(0) ≤α, one has, ∀k ≥1 x(k)TP(k)−1x(k) ≤α + β x(k)TP(k)−1x(k) ≤α + β x(k)TP(k)−1x(k) ≤α + β and the following inequality holds and the following inequality holds ∞ X k=0 zT(k)z(k) ≤γ2 ∞ X k=0 wT(k)w(k) + α. (43) (43) Proof: Consider the saturation-dependent Lyapunov function (24). For the L2 gain, it is required that Proof: Consider the saturation-dependent Lyapunov function (24). For the L2 gain, it is required that V (k + 1, x(k + 1)) −V (k, x(k)) ≤−1 γ2z(k)Tz(k) + w(k)Tw(k) (44) ∀x(k), ∀x(k + 1) satisfying (22), and ∀w(k) ∈W. (44) 15 44) holds, then it follows that If (44) holds, then it follows that If (44) holds, then it follows that V (∞, x(∞)) −V (0, x(0)) ≤−1 γ2 ∞ P k=0 z(k)Tz(k) + ∞ P k=0 w(k)Tw(k) (45) (45) Note that (22) is asymptotically stable for states near the origin. It follows that lim k→∞x(k) = 0. Hence lim k→∞V (k, x(k)) = 0. With the zero initial condition, condition (45) becomes 0 ≤−1 γ2 ∞ X k=0 z(k)Tz(k) + ∞ X k=0 w(k)Tw(k) or equivalently ∞ X k=0 z(k)Tz(k) ≤γ2 ∞ X k=0 w(k)Tw(k) It is concluded that the system (1) has L2 gain performance γ. where Rewrite the left hand side of (44) as Rewrite the left hand side of (44) as V (k + 1, x(k + 1)) −V (k, x(k)) = = x(k + 1)TP(k + 1)−1x(k + 1) −x(k)TP(k)−1x(k) = x(k)T w(k)T   Ac(k)T ET  P(k + 1)−1 Ac(k) E   x(k) w(k)   − x(k)T w(k)T   P(k)−1 0 0 0     x(k) w(k)   (46) (46) and the right hand side of (44) as and the right hand side of (44) as −1 γ2 ∞ P k=0 z(k)Tz(k) + ∞ P k=0 w(k)Tw(k) = = −1 γ2 x(k)T w(k)T   CT DT   C D   x(k) w(k)  + + x(k)T w(k)T   0 0 0 I     x(k) w(k)   (47) (47) Combining (46), (47), one obtains   P(k)−1 0 0 0  −   Ac(k)T ET  P(k + 1)−1 Ac(k) E ⪰ 1 γ2   CT DT   C D −   0 0 0 I   16 or equivalently or equivalently   P(k)−1 −1 γ2CTC −1 γ2CTD −1 γ2DTC −1 γ2DTD + I  −   Ac(k)T ET  P(k + 1)−1 Ac(k) E ⪰0 Thus, with Schur complement, one gets Thus, with Schur complement, one gets   P(k + 1) Ac(k) E (∗) P(k)−1 −1 γ2CTC −1 γ2CTD (∗) (∗) −1 γ2DTD + I   ⪰0 or equivalently   P(k + 1) Ac(k) E (∗) P(k)−1 0 (∗) (∗) I   −1 γ2   0 CT DT   0 C D ⪰0   P(k + 1) Ac(k) E (∗) P(k)−1 −1 γ2CTC −1 γ2CTD (∗) (∗) −1 γ2DTD + I   ⪰0 or equivalently   P(k + 1) Ac(k) E (∗) P(k)−1 0 (∗) (∗) I   −1 γ2   0 CT DT   0 C D ⪰0 Using Schur complement, one obtains Using Schur complement, one obtains Using Schur complement, one obtains   P(k + 1) Ac(k) E 0 (∗) P(k)−1 0 CT (∗) (∗) I DT (∗) (∗) (∗) γ2I   ⪰0 (48) (48) Pre- and post-multiplication of (48) by Pre- and post-multiplication of (48) by Pre- and post-multiplication of (48) by   P(k + 1) A(k)G(k) + B(k)F(k) E 0 (∗) G(k)TP(k)−1G(k) 0 G(k)CT (∗) (∗) I DT (∗) (∗) (∗) γ2I   ⪰0 (49) (49) 17 Using Lemma 5, condition (49) holds if Using Lemma 5, condition (49) holds if Using Lemma 5, condition (49) holds if Using Lemma 5, condition (49) holds if   P(k + 1) A(k)G(k) + B(k)F(k) E 0 (∗) G(k) + G(k) −P(k) 0 G(k)CT (∗) (∗) I DT (∗) (∗) (∗) γ2I   ⪰0 This condition is (41). where Using (44), ∀x(0) such that x(0)TP(0)−1x(0) ≤α, one gets Using (44), ∀x(0) such that x(0)TP(0)−1x(0) ≤α, one gets Using (44), ∀x(0) such that x(0)TP(0)−1x(0) ≤α, one gets x(k)P(k)−1x(k) ≤x(0)TP(0)−1x(0) −1 γ2 k−1 P t=0 z(t)Tz(t) + k−1 P t=0 w(t)Tw(t) Consequently, Consequently, x(k)P(k)−1x(k) ≤x(0)TP(0)−1x(0) + k−1 X t=0 w(t)Tw(t) Hence x(k)P(k)−1x(k) ≤α + β x(k)P(k)−1x(k) ≤α + β (50) (50) Recall that −1 ≤v(k) = F(k)G(k)−1x(k) ≤1 Thus, using Lemma 5, one obtains Thus, using Lemma 5, one obtains 1 α + β −ejF(k)G(k)−1P(k)(G(k)−1)TF(k)TeT j ⪰0, ∀j = 1, m2m−1 Using Schur complement, this condition is equivalently rewritten as Using Schur complement, this condition is equivalently rewritten as   1 α+β ejF(k) F(k)TeT j G(k)TP(k)−1G(k)  ⪰0 Using Lemma 4, one gets Using Lemma 4, one gets Using Lemma 4, one gets   1 α+β ejF(k) F(k)TeT j G(k) + G(k)T −P(k)  ⪰0 The proof is complete. 2 The proof is complete. 2 The proof is complete. By using Lemma 2 to model the saturation non-linearity and by setting Fl = F, Gl = G in the conditions of Theorem 3, one recover Theorem 1 in [24]. Hence the result in [24] is a particular case of ours. 18 Condition (42) holds if and only if tion (42) holds if and only if Condition (42) holds if and only if   1 α+β ejFl ejF T l Gl + GT l −Pl  ⪰0, ∀l = 1, 2m, ∀j = 1, m2m−1 (51) (51) Define γs = γ2. Rewrite (41) as Define γs = γ2. Rewrite (41) as 2m X l1=1 2m X l2=1 λl1λl2Θr l1l2 ⪰0 (52) (52) with r = 1, 2m, and Θr l1l2 =   Pr (Al1Gl2 + Bl1Fl2) E 0 (∗) (Gl2 + Gl2 −Pl2) 0 Gl2CT (∗) (∗) I DT (∗) (∗) (∗) γsI   (53) (53) Combining (52) and Lemma 3 or Lemma 4, one obtains the following corollary. where Corollary 2: System (1), (3) is with a L2 gain less than γ = √γs, if for given scalars α > 0, β > 0, there exist Pl ⪰0, Gl, Fl, l = 1, 2m, and a scalar γs > 0, such that (51) hold and      Θr ll ⪰0, ∀r, ∀l = 1, 2m Θr l1l2 + Θr l2l1 ⪰0, ∀r, l1, l2 = 1, 2m, l1 < l2 (54) (54) or or      Θr ll ⪰0, ∀r, l = 1, 2m 2Θr l1l1 + Θr l1l2 + Θr l2l1 ⪰0, ∀r, l1, l2 = 1, 2m, l1 ̸= l2 (55) (55) Using Corollary 2, the problem of optimizing the L2 gain γ can be formulated as Using Corollary 2, the problem of optimizing the L2 gain γ can be formulated as as min Pl,Gl,Fl γ (56) min Pl,Gl,Fl γ subject to (51), (54) (56) (56) subject to (51), (54) (56) or or i or or or or min Pl,Gl,Fl γ subject to (51), (55) (57) min Pl,Gl,Fl γ (57) (57) subject to (51), (55) (57) subject to (51), (55) ( ) subject to (51), (55) ( ) For further use, we refer to the optimization problems (56), and (57), respec- tively, as algorithm 3 and algorithm 4. 19 Remark 3: In the unsaturated linear system case, it is well known [7] that the parameter β has no impact on the L2 gain γ. However, in the presence of the saturation non-linearity, this is no longer the case, i.e., γ is a function of β. Using (51), it is clear that this function is non-increasing, i.e., γ1 ≤γ2 if β1 ≥β2. For different initial conditions, Fig. 2 presents the state trajectories in the phase plane. 4 Examples Three examples are considered in this section. The CVX toolbox was used to solve SDP optimization problems. 4.1 Example 1 4.1 Example 1 4.1 Example 1 This example is taken from [4]. Consider the following system x(k + 1) =   1 1 0 1  x(k) +   0.5 1  sat(u(k)) (58) (58) The LQ controller with Q = I, and R = 0.1 is used, giving the state feedback gain The LQ controller with Q = I, and R = 0.1 is used, giving the state feedback gain K = [−0.6167 −1.2703] K = [−0.6167 −1.2703] For this example, results are almost identical for Algorithms 1 and 2. Hence only results of Algorithm 2 are presented. By solving the SDP problem (39), one obtains P1 =   66.5120 −26.0475 −26.0475 40.4144  , P2 =   103.7959 −29.9170 −29.9170 20.4263   Fig. 1 shows the intersection of two ellipsoids E(P1, 1) and E(P2, 1) (solid cyan and solid violet). For comparison, Fig. 1 also shows the intersection of two ellipsoids (dashed yellow and dashed red) obtained by using Theorem 1 in [4], and the ellipsoid obtained by using [11] (dash-dot green). We can see that the estimate of the invariant set obtained by using the nonlinear saturation- dependent auxiliary feedback gain is larger than that by the linear saturation independent ones. For different initial conditions, Fig. 2 presents the state trajectories in the phase plane. 20 -8 -6 -4 -2 0 2 4 6 8 x -4 -3 -2 -1 0 1 2 3 4 y Fig. 1. Invariant sets for our approach (solid cyan and solid violet), for [4] (dashed yellow and dashed red), and for [11] (dash-dot green) for example 1. -8 -6 -4 -2 0 2 4 6 8 x -4 -3 -2 -1 0 1 2 3 4 y Fig. 1. Invariant sets for our approach (solid cyan and solid violet), for [4] (dashed yellow and dashed red), and for [11] (dash-dot green) for example 1. -8 -6 -4 -2 0 2 4 6 8 x -4 -3 -2 -1 0 1 2 3 4 y -8 -6 -4 -2 0 2 4 6 8 x -4 -3 -2 -1 0 1 2 3 4 y Fig. 2. State trajectories for different initial conditions for example 1. Fig. 2. State trajectories for different initial conditions for example 1. 4.2 Example 2 Consider system (1), (3) with Consider system (1), (3) with Consider system (1), (3) with A =   0 1 −0.6 −0.62  , B =   0 1  , E =   0 1  , K = 0 −0.7 , C = 1 0 , D = 0 For this example, the L2 gain of the closed-loop system in the linear region is 4.7760. For α = 0, β = 15, by using algorithms 3 and 4, one gets, respectively, For this example, the L2 gain of the closed-loop system in the linear region is 4.7760. For α = 0, β = 15, by using algorithms 3 and 4, one gets, respectively, 21 γ = 13.4399, γ = 13.2680. The matrices obtained using algorithm 4 are γ = 13.4399, γ = 13.2680. The matrices obtained using algorithm 4 are P1 =   33.1911 −17.1499 −17.1499 17.0760  , P2 =   21.0983 −9.7412 −9.7412 19.6851  , G1 =   33.1911 −17.1499 −17.1499 17.0760  , G2 =   21.0983 −9.7412 −9.7412 19.6851  , F1 = 0.9861 −0.0336 , F2 = 1.1544 −0.7637 For different β ∈[0.5 20], Fig. 3 presents the L2 gain using algorithm 3 (dashed red), and algorithm 4 (solid blue). It can be observed that algorithm 4 slightly outperforms algorithm 3. For comparison, Fig. 3 also presents the L2 gain obtained by Theorem 2 in [6] (dashed violet), and by Theorem 1 in [24] (dash-dot yellow). Note that the dashed violet curve diverges to infinity as β approaches to 2.1. This implies that the maximal tolerable disturbance by [6] is 2.1. 0 2 4 6 8 10 12 14 16 18 20 6 8 10 12 14 16 18 20 22 Fang et al. Wada et al. Algorithm 3 Algorithm 4 Fig. 3. L2 gain as a function of β for Algorithm 3 (dashed red), for Algorithm 4 (solid blue), for Theorem 2 in [6] (dashed violet), and for Theorem 1 in [24] (dash-dot yellow). 0 2 4 6 8 10 12 14 16 18 20 6 8 10 12 14 16 18 20 22 Fang et al. Wada et al. Algorithm 3 Algorithm 4 Fig. 3. 4.2 Example 2 L2 gain as a function of β for Algorithm 3 (dashed red), for Algorithm 4 (solid blue), for Theorem 2 in [6] (dashed violet), and for Theorem 1 in [24] (dash-dot yellow). 4.3 Example 3 4.3 Example 3 To further illustrate the performance of our approach, we consider the follow- ing multi-input system To further illustrate the performance of our approach, we consider the follow- ing multi-input system x(k + 1) =   1.1 1 0 1.1  x(k) +   0.42 0.9 0.38 0.67  sat(u(k)) (59) (59) 22 Design the linear control law by the LQ approach with Q = I, R = 0.1I, giving the matrix gain   K =   0.0843 −0.6372 −0.8990 −0.8826   For this example, Algorithm 1 and Algorithm 2 give almost the same results. Hence only the results of Algorithm 2 is shown. By solving (39), one gets P1 =   36.3443 3.3024 3.3024 21.4723  , P2 =   36.3000 −9.2123 −9.2123 14.9668  , P3 =   36.2838 4.7992 4.7992 18.6164  , P4 =   36.6749 −7.5698 −7.5698 12.3944   Fig. 4 presents the intersection of four ellipsoids 4T l=1 E(Pl, 1) (solid lines). For comparison, Fig. 4 also shows the intersection of four ellipsoids (dashed lines) obtained by using Theorem 1 in [4], and the ellipsoid with [11] (dash-dot green). Finally, for different initial conditions, Fig. 5 presents the state trajec- -5 -4 -3 -2 -1 0 1 2 3 4 5 x1 -3 -2 -1 0 1 2 3 x2 Fig. 4. Invariant sets for our approach (solid lines), for [4] (dashed lines), and for [11] (dash-dot green) for example 3. -5 -4 -3 -2 -1 0 1 2 3 4 5 x1 -3 -2 -1 0 1 2 3 x2 Fig. 4. Invariant sets for our approach (solid lines), for [4] (dashed lines), and for [11] (dash-dot green) for example 3. tories in the phase plane. 5 Conclusion In this paper, a novel approach to the performance analysis of a saturated lin- ear system is proposed. The main contribution of the paper is a new nonlinear saturation-dependent auxiliary feedback law. Using the linear parameter vary- ing system modeling framework, sufficient conditions for the computation of 23 -5 -4 -3 -2 -1 0 1 2 3 4 5 x1 -3 -2 -1 0 1 2 3 x2 Fig. 5. State trajectories for different initial conditions for example 3. -5 -4 -3 -2 -1 0 1 2 3 4 5 x1 -3 -2 -1 0 1 2 3 x2 Fig. 5. State trajectories for different initial conditions for example 3. invariant set and for the L2 gain analysis are presented. The obtained con- ditions are expressed as a set of linear matrix inequalities constraints. It is shown that the proposed conditions are less conservative than the existing results in the literature. Three numerical examples with comparison to earlier solutions in the literature demonstrate the effectiveness of this new method. References [1] T Alamo, A Cepeda, and D Limon. Improved computation of ellipsoidal invariant sets for saturated control systems. In 44th IEEE Conference on Decision and Control, and European Control Conference, pages 6216–6221. IEEE, 2005. [2] Teodoro Alamo, A Cepeda, Daniel Lim´on, and Eduardo F Camacho. Estimation of the domain of attraction for saturated discrete-time systems. International journal of systems science, 37(8):575–583, 2006. [3] Stephen Boyd, Laurent El Ghaoui, Eric Feron, and Venkataramanan Balakrishnan. Linear matrix inequalities in system and control theory, volume 15. Siam, 1994. [4] Yong-Yan Cao and Zongli Lin. Stability analysis of discrete-time systems with actuator saturation by a saturation-dependent lyapunov function. Automatica, 39(7):1235–1241, 2003. [5] Maur´ıcio C De Oliveira, Jacques Bernussou, and Jos´e C Geromel. A new discrete-time robust stability condition. Systems & control letters, 37(4):261– 265, 1999. [6] Haijun Fang, Zongli Lin, and Tingshu Hu. Analysis of linear systems in the presence of actuator saturation and l2-disturbances. Automatica, 40(7):1229– 1238, 2004. 24 [7] Pascal Gahinet and Pierre Apkarian. A linear matrix inequality approach to hinf control. International journal of robust and nonlinear control, 4(4):421–448, 1994. [8] Michael Grant and Stephen Boyd. Cvx: Matlab software for disciplined convex programming, version 2.1, 2014. [9] Haitham Hindi and Stephen Boyd. Analysis of linear systems with saturation using convex optimization. In Proceedings of the 37th IEEE Conference on Decision and Control (Cat. No. 98CH36171), volume 1, pages 903–908. IEEE, 1998. [10] Tingshu Hu and Zongli Lin. Control systems with actuator saturation: analysis and design. Springer Science & Business Media, 2001. [11] Tingshu Hu, Zongli Lin, and Ben M Chen. Analysis and design for discrete- time linear systems subject to actuator saturation. Systems & Control Letters, 45(2):97–112, 2002. [12] Tingshu Hu, Zongli Lin, and Ben M Chen. An analysis and design method for linear systems subject to actuator saturation and disturbance. Automatica, 38(2):351–359, 2002. [13] Tingshu Hu, Andrew R Teel, and Luca Zaccarian. Stability and performance for saturated systems via quadratic and nonquadratic lyapunov functions. IEEE Transactions on Automatic Control, 51(11):1770–1786, 2006. [14] Mikael Johansson. Piecewise quadratic estimates of domains of attraction for linear systems with saturation. IFAC Proceedings Volumes, 35(1):187–192, 2002. [15] Vikram Kapila and Karolos Grigoriadis. Actuator saturation control. CRC Press, 2002. [16] Hassan K Khalil. Nonlinear systems third edition, volume 115. 2002. [17] Johan L¨ofberg. Yalmip: A toolbox for modeling and optimization in matlab. In Proceedings of the CACSD Conference, volume 3. Taipei, Taiwan, 2004. References [18] Yong-Mei Ma and Guang-Hong Yang. Performance analysis for linear discrete- time systems subject to actuator saturation. In 2008 American Control Conference, pages 3608–3613. IEEE, 2008. [19] Hoai-Nam Nguyen. Constrained control of uncertain, time-varying, discrete- time systems. Lecture Notes in Control and Infomration Sciences, 451:17, 2014. [20] Hoai-Nam Nguyen. l2-gain performance analysis for discrete-time systems with input saturation: an LPV approach. IFAC-PapersOnLine, 53(2):4588–4592, 2020. [21] C Paim, S Tarbouriech, JM Gomes da Silva, and EB Castelan. Control design for linear systems with saturating actuators and/spl lscr//sub 2/-bounded disturbances. In Proceedings of the 41st IEEE Conference on Decision and Control, 2002., volume 4, pages 4148–4153. IEEE, 2002. 25 [22] Sophie Tarbouriech, Germain Garcia, Jo˜ao Manoel Gomes da Silva Jr, and Isabelle Queinnec. Stability and stabilization of linear systems with saturating actuators. 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https://openalex.org/W4242106515	https://www.researchsquare.com/article/rs-72198/v1.pdf?c=1602868528000	English	null	Comparison of Irinotecan and Oxaliplatin as the First-Line Therapies for Metastatic Colorectal Cancer: A Meta-analysis	Research Square (Research Square)	2,020	cc-by	7,641	Research article Keywords: meta-analysis, metastatic colorectal cancer, chemotherapy Posted Date: October 16th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-72198/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on February 4th, 2021. See the published version at https://doi.org/10.1186/s12885-021-07823-7. Page 1/18 Page 1/18 Page 1/18 Methods This meta-analysis used data from the Cochrane Central Register of Controlled Trials, PubMed, and SCOPUS. The primary endpoint was OS, and the secondary endpoints were progression-free survival (PFS), objective response rate (ORR), and adverse events (AEs). Background Irinotecan (IRI) and oxaliplatin (Ox) are standard therapeutic agents of the first-line treatments for metastatic colorectal cancer (mCRC). Previous meta-analyses of randomized controlled trials (RCTs) showed that treatment with Ox-based compared with IRI-based regimens was associated with better overall survival (OS). However, these reports did not include trials of molecular targeting agents and did not take methods for the administration of concomitant drugs, such as bolus or continuous infusion of 5-fluorouracil, into account. A systematic literature review was performed to compare the efficacy and toxicity profiles between IRI- and Ox- based regimens as the first-line treatments for mCRC. Conclusion Although the safety profiles of IRI- and Ox-based regimens varied, their efficacy did not significantly differ. Therefore, both regimens could be used as the first-line treatments for mCRC with consideration of the patients’ condition or toxicity profiles. Results Nineteen trials involving 4,571 patients were included in the analysis. No statistically significant difference was observed between the two groups in terms of OS, PFS, and ORR. There was no significant heterogeneity. Regarding ≥ grade 3 AEs, IRI-based regimens were associated with a high incidence of leukopenia, febrile neutropenia, and diarrhea. Moreover, there was a high incidence of thrombocytopenia and peripheral sensory neuropathy in patients who received Ox-based regimens. In a subgroup analysis, IRI combined with bevacizumab was correlated with a better PFS (HR = 0.90, 95% CI = 0.82–0.98, P = 0.02), but not with OS. Background In clinical practice, physicians generally select the regimen based on adverse events (AEs) caused by the drugs or the patients’ condition. Recent reports showed that physicians preferred Ox-based regimen than IRI-based regimen. Field et al.8 conducted a survey on the use of chemotherapy for CRC. Results showed that 92.6% of medical oncologists in Australia select Ox-based regimen as the first-line treatment. They reported physicians commonly select this regimen as it has better efficacy and lower toxicity than IRI-based regimen. In addition, Marschner et al.9 conducted a prospective cohort study and revealed that Ox-based regimen was used as the first-line treatment in 430 out of 605 patients (71.0%) with mCRC. In clinical practice, physicians generally select the regimen based on adverse events (AEs) caused by the drugs or the patients’ condition. Recent reports showed that physicians preferred Ox-based regimen than IRI-based regimen. Field et al.8 conducted a survey on the use of chemotherapy for CRC. Results showed that 92.6% of medical oncologists in Australia select Ox-based regimen as the first-line treatment. They reported physicians commonly select this regimen as it has better efficacy and lower toxicity than IRI-based regimen. In addition, Marschner et al.9 conducted a prospective cohort study and revealed that Ox-based regimen was used as the first-line treatment in 430 out of 605 patients (71.0%) with mCRC. Previous meta-analyses10–12 of the first-line treatments for mCRC revealed that Ox-based regimen was correlated with a better OS. However, these studies included trials comparing continuous 5-FU infusion regimen with bolus 5-FU regimen, which is currently considered an inferior method. Moreover, these data were obtained from reports of regimens without molecular targeting agents. Therefore, they might not be applicable in the current clinical practice. Recently, a population-based observational study conducted by Teng et al.13 reported that IRI-based regimen might be more effective in improving OS than Ox-based regimen. Therefore, it remains unclear which regimen is associated with a better OS. Thus, a meta-analysis based on the current knowledge was performed to compare the efficacy and toxicity between IRI- and Ox-based regimens as the first-line treatments for mCRC. Background Currently, colorectal cancer (CRC) ranks fourth for incidence worldwide, with 550,000 deaths recorded annually.1 Patients with unresectable metastatic CRC (mCRC) have a poor prognosis, with a median overall survival (OS) of 6–8 months, without any therapy. Although chemotherapy is the standard treatment for these patients, the median OS is only 20–25 months.2 The combination of cytotoxic and molecular targeting agents is currently used as the first-line treatment for patients with unresectable mCRC. Irinotecan (IRI) or oxaliplatin (Ox) combined with fluoropyrimidines (FPs) is considered as the backbone of cytotoxic agents for the treatment of mCRC.3 In the meta-analysis conducted by Grothey et al.4, the use of all three active drugs (FPs, IRI, and Ox) for mCRC was associated with a long OS. Page 2/18 Page 2/18 First-line treatment is important for patients with mCRC, because the treatment period is the longest of the overall treatment time and significantly affects survival and quality of life (QOL). With regard to the use of cytotoxic agents, several trials investigated the best combination of regimens. Tournigand et al.5 and Colucci et al.6 conducted clinical trials to compare the efficacy between 5-fluorouracil (5-FU), folinic acid, and Ox (FOLFOX) regimen and 5-FU, folinic acid, and IRI (FOLFIRI) regimen. Results showed no significant differences between the two regimens. Moreover, Yamazaki et al.7 compared these regimens with bevacizumab (BEV), a molecular targeting agent. Results showed that the OS did not significantly differ between patients who received FOLFOX + BEV and FOLFIRI + BEV. Hence, both IRI- and Ox-based regimens could be used as the first- line treatments for mCRC. First-line treatment is important for patients with mCRC, because the treatment period is the longest of the overall treatment time and significantly affects survival and quality of life (QOL). With regard to the use of cytotoxic agents, several trials investigated the best combination of regimens. Tournigand et al.5 and Colucci et al.6 conducted clinical trials to compare the efficacy between 5-fluorouracil (5-FU), folinic acid, and Ox (FOLFOX) regimen and 5-FU, folinic acid, and IRI (FOLFIRI) regimen. Results showed no significant differences between the two regimens. Moreover, Yamazaki et al.7 compared these regimens with bevacizumab (BEV), a molecular targeting agent. Results showed that the OS did not significantly differ between patients who received FOLFOX + BEV and FOLFIRI + BEV. Hence, both IRI- and Ox-based regimens could be used as the first- line treatments for mCRC. Search Strategy and Selection Criteria We searched PubMed, SCOPUS, and the Central Registry of Controlled Trials of the Cochrane Library (CENTRAL) without language restrictions. The last search update was performed on December 17, 2018. The inclusion criteria were as follows: (1) randomized controlled trials comparing IRI- and Ox-based combination regimens as the first-line treatments for mCRC and (2) those using similar agents (IRI- or Ox-based combination regimens) or agents with comparable efficacy based on the results of previous randomized controlled trials. The efficacy of oral FPs was similar to that of continuous infusion of 5-FU. That is, capecitabine + Ox (CapOx) regimen and S-1 + Ox (SOX) regimen were considered as alternative to FOLFOX regimen.14–16 Similarly, capecitabine + IRI (CapIRI) and S-1 + IRI (IRIS) regimens were considered as alternative to FOLFIRI regimen.17, 18 (3) Abstracts or unpublished data were included if they had sufficient information on study design, characteristics of participants, interventions, and outcomes. We searched PubMed, SCOPUS, and the Central Registry of Controlled Trials of the Cochrane Library (CENTRAL) without language restrictions. The last search update was performed on December 17, 2018. The inclusion criteria were as follows: (1) randomized controlled trials comparing IRI- and Ox-based combination regimens as the first-line treatments for mCRC and (2) those using similar agents (IRI- or Ox-based combination regimens) or agents with comparable efficacy based on the results of previous randomized controlled trials. The efficacy of oral FPs was similar to that of continuous infusion of 5-FU. That is, capecitabine + Ox (CapOx) regimen and S-1 + Ox (SOX) regimen were considered as alternative to FOLFOX regimen.14–16 Similarly, capecitabine + IRI (CapIRI) and S-1 + IRI (IRIS) regimens were considered as alternative to FOLFIRI regimen.17, 18 (3) Abstracts or unpublished data were included if they had sufficient information on study design, characteristics of participants, interventions, and outcomes. Page 3/18 The search terms used were as follows: (1) terms suggestive of “colorectal” (i.e., “colorect,” “colon,” “colonic,” “bowel,” “recta,” or “rectum”), (2) “cancer” (i.e., “cancer,” “carcinoma,” “neoplas*,” or “tumor”), (3) “irinotecan” (i.e., “irinotecan,” “camptotecin-11,” or “topotecin”), (4) “oxaliplatin” (i.e., “L-OHP,” “eloxatine,” or “oxaliplatin”), and (5) “randomized trials” (i.e., “ramdomized controlled,”,“randomised,” or “randomly”). Data Extraction At least two of three review authors (SK, NT, and YH) independently scanned titles and abstracts to exclude studies that do not meet the criteria. The full text reports were then reviewed for further assessment. Disagreements were resolved via a consensus. Detailed data from eligible trials, such as year of publication, place where the study was conducted, study design, regimens, participants’ information, methodological evaluation, outcomes, and AEs, were extracted. If necessary, the authors were contacted for clarifications. Assessment of Risk of Bias At least two of three authors (SK, NT, and YH) independently assessed the risk of bias in the included studies in accordance with the Cochrane Handbook for Systematic Reviews of Interventions.19 The following domains were assessed: (1) sequence generation; (2) allocation concealment; (3) blinding of participants, personnel, and outcome assessors; (4) incomplete outcome data; (5) selective outcome reporting; and (6) other potential threats to validity. When inadequate details of methodological characteristics of trials were provided, the authors were contacted to obtain further information. Statistical Analysis The primary endpoint was OS. The secondary endpoints were progression-free survival (PFS), objective response rate (ORR), and ≥ grade 3 AEs according to the Common Terminology Criteria for Adverse Events version 4.0. Hazard ratios (HRs) and 95% confidence interval (CI), which were considered as relevant effect measures, were directly or indirectly assessed using the given data. When the Kaplan–Meier curve, but not the HR for PFS or OS, was included, we extracted the value using the Engauge Digitizer 10.8 and the method provided by Tierney et al.20 ORR were compared using odds ratio (OR). The risk difference was used to compare the risk of AEs. Statistical heterogeneity among the studies was assessed using the chi-square test and was expressed with the I2 index.19 The pooled effect was calculated with the random-effects model. Subgroup analyses were performed according to the combination of agents, which include regimens with or without molecular targeting agents, infusion of 5-FU or oral FPs, and anti-epidermal growth factor receptor (EGFR) or anti-ca (VEGF) antibodies. Since a previous report showed that CapOx plus anti-EGFR antibody might be less effective than FOLFOX plus anti-EGFR,21 we added the subgroup of anti-EGFR antibodies except CapOx + cetuximab. Statistical analyses were performed using the RevMan 5.3 software, which was provided by The Cochrane Collaboration. The corresponding funnel plots were used to examine the effect of publication bias visually. A two-sided P-value < 0.05 was considered statistically significant. Search Results and Characteristics of the Included Trials Page 4/18 A total of 4,451 potentially relevant studies were retrieved from the initial database search in PubMed, SCOPUS, and CENTRAL. Of these studies, 22 met all the inclusion criteria. However, one trial did not provide sufficient data, 22 and two were discontinued early due to poor accrual.23, 24 Finally, 19 RCTs5–7, 25–40 with 4,571 patients were included in the analysis. A flowchart of the search process is shown in Fig. 1. Table 1 shows the characteristics of RCTs included in the meta-analysis. Of these, 12 trials (n = 3,534) used 5- FU infusion regimens; 6 (n = 943), oral FPs; and 1, raltitrexed (n = 94). Nine RCTs (n = 2,154) used regimens with molecular targeting agents. Among them, five used BEV and four anti-EGFR antibodies (cetuximab or panitumumab). In total, 2 RCTs were conducted in North America, 13 in Europe, and 4 in Asia. The median age of the participants in all studies was 61–75 years. In one trial32, only elderly patient aged > 70 years was eligible. Page 5/18 Table 1 Characteristics of randomized controlled trials included in the meta-analysis. Study no. Search Results and Characteristics of the Included Trials Authors Region Year Trial phase Primary endpoint Interventions Patients Median OS (months) 1 Grothey et al.25 USA 2003 2 AEs CapOx CapIRI 82 79 NA NA 2 Tournigand et al.5 France 2004 3 PFS FOLFOX FOLFIRI 113 113 20.6 21.5 3 Kalofonos et al.26 Greece 2005 2 ORR Weekly Ox + LV + 5-FU Weekly IRI + LV + 5-FU 148 147 17.4 17.6 4 Comella et al.27 Italy 2005 3 ORR OXAFAFU IRIFAFU 140 133 18.9 15.6 5 Colucci et al.6 Italy 2005 3 ORR FOLFOX FOLFIRI 182 178 15 14 6 Feliu et al.28 Spain 2005 2 ORR Raltitrexed + Ox Raltitrexed + IRI 48 46 NA NA 7 Zheng et al.29 China 2006 2 ORR FOLFOX7 FOLFIRI 30 30 NA NA 8 Bajetta et al.30 Italy 2007 2 AEs TEGAFOX TEGAFIRI 73 68 19 20 9 Seymour et al.31 UK 2007 3 OS OxFU IrFU 357 356 15.4 16.7 10 Rosati et al.32 Italy 2010 2 ORR CapOx CapIRI 47 47 19.3 14 11 Ocvirk et al.33 Germany Austria 2010 2 PFS FOLFOX6 + Cmab FOLFIRI + Cmab 77 74 17.4 18.9 12 Moosmann et al.34 Germany 2011 2 ORR CapOx + Cmab CapIRI + Cmab 92 93 25.5 21.1 13 Schmiegel et al.35 Germany 2013 2 PFS CapOx + BEV CapIRI + BEV 127 128 24.4 25.5 14 Folprecht et al.36 Germany Austria 2014 2 ORR FOLFOX6 + Cmab FOLFIRI + Cmab 56 55 35.8 29 15 Yamazaki et al.7 Japan 2016 3 PFS FOLFOX6 + BEV FOLFIRI + BEV 200 202 30.4 31.4 16 Parikh et al 37 USA 2018 2 PFS FOLFOX6 + BEV FOLFIRI + BEV 188 188 23.9 27 5 Table 1 Characteristics of randomized controlled trials included in the met Page 6/18 17 Carrato et al.38 Spain 2017 2 ORR FOLFOX4 + Pmab FOLFIRI + Pmab 40 40 37 41 18 Yamada et al.39 Japan 2018 3 PFS FOLFOX(CapOx) + BEV S-1+IRI + BEV 244 243 33.6 34.8 19 Nakayama et al.40 Japan 2018 2 ORR CapOx + BEV CapIRI + BEV 54 53 26.7 28.7 OS, overall survival; AEs, adverse events; PFS, progression-free survival; ORR, objective response rate; NA, not available; CapOx, capecitabine + Ox; CapIRI, capecitabine + IRI; FOLFOX and OXAFAFU, 5-FU + folinic acid + Ox; FOLFIRI and IRIFAFU, 5-FU + folinic acid + IRI; TEGAFOX, tegafur + folinic acid + Ox; TEGAFIRI, tegafur + folinic acid + IRI; BEV, bevacizumab; Cmab, cetuximab; and Pmab, panitumumab OS, overall survival; AEs, adverse events; PFS, progression-free survival; ORR, objective response rate; NA, not available; CapOx, capecitabine + Ox; CapIRI, capecitabine + IRI; FOLFOX and OXAFAFU, 5-FU + folinic acid + Ox; FOLFIRI and IRIFAFU, 5-FU + folinic acid + IRI; TEGAFOX, tegafur + folinic acid + Ox; TEGAFIRI, tegafur + folinic acid + IRI; BEV, bevacizumab; Cmab, cetuximab; and Pmab, panitumumab Efficacy and Toxicity Sixteen trials included data on OS (Fig. 2A). Since seven RCTs did not describe the HR for OS, we estimated the value using the Kaplan–Meier curve. Results showed no significant difference between IRI- and Ox-based regimens (pooled HR = 0.96, 95% CI = 0.89–1.03, P = 0.28), and there was no significant heterogeneity among the trials (I2 = 2%). Thirteen trials included data on PFS (Fig. 2B). We estimated the HR for PFS in six trials using the Kaplan–Meier curve. No significant difference was observed between the two groups (pooled HR = 0.98, 95% CI = 0.94–1.04, P = 0.49), and there was no significant heterogeneity (I2 = 0%). All trials included data on ORR (Fig. 2C). There was no significant difference between the two groups. However, Ox-based regimen had a favorable outcome (pooled OR = 1.13, 95% CI = 1.00–1.27, P = 0.06). No significant heterogeneity was observed (I2 = 0%). We then performed a subgroup analysis according to the combination regimens. Overall, the OS did not significantly differ between IRI- and Ox-based regimens in all subgroups (Fig. 3A). The combined therapy of IRI and anti-VEGF was associated with a better PFS (pooled HR = 0.90, 95% CI = 0.82–0.98, P = 0.02), but not with OS (pooled HR = 0.91, 95% CI = 0.80–1.03, P = 0.15) (Fig. 3B and 3A). Ox-based regimens, especially those without molecular targeting agents, were associated with a better ORR (pooled OR = 1.22, 95% CI = 1.03–1.45, P = 0.02) (Fig. 3C). We then performed a subgroup analysis according to the combination regimens. Overall, the OS did not significantly differ between IRI- and Ox-based regimens in all subgroups (Fig. 3A). The combined therapy of IRI and anti-VEGF was associated with a better PFS (pooled HR = 0.90, 95% CI = 0.82–0.98, P = 0.02), but not with OS (pooled HR = 0.91, 95% CI = 0.80–1.03, P = 0.15) (Fig. 3B and 3A). Ox-based regimens, especially those without molecular targeting agents, were associated with a better ORR (pooled OR = 1.22, 95% CI = 1.03–1.45, P = 0.02) (Fig. 3C). The incidence of any ≥ grade 3 AEs did not significantly differ between patients who received the two regimens. Thrombocytopenia, peripheral sensory neuropathy, hand–foot syndrome, and allergic reaction were significantly correlated with Ox-based regimens. Meanwhile, leukopenia, diarrhea, and febrile neutropenia were commonly observed in patients who received IRI-based regimens (Fig. 4). Discussion Based on the current expert panels of guidelines3, 41, 42, there is no preferred regimen that can be used as the initial therapy for mCRC. Hence, IRI- or Ox-based regimen can be an alternative. In clinical practice, the regimen is selected based on efficacy of the regimens, physicians’ experience, and profiles of AEs. Our analysis showed that there was no significant difference in terms of OS between patients who received IRI- and Ox-based regimens as the first-line treatments for mCRC. This is the first meta-analysis that included treatments containing molecular targeting agents and clinically comparable combination of regimens. Therefore, the results of this study may be more applicable to current clinical practice than those of previous studies. The absence of significant heterogeneity between trials in terms of OS, PFS, and ORR indicated consistent results. Previous reports10–12 showed that Ox-based regimen was more preferred in terms of OS. This discrepancy in results may be attributed to the selection criteria of the trials. That is, previous studies included trials comparing FOLFOX with IFL (bolus 5-FU + IRI) regimen.43, 44 However, Delaunoit et al.45 showed that bolus 5-FU + IRI or Ox could be associated with severe gastrointestinal toxicity and high mortality rates. Thus, excluding these trials was appropriate for the evaluation of efficacy and toxicity between the two regimens. In the subgroup analysis of regimens without molecular targeting agents, Ox-based regimen was associated with a better ORR. However, the PFS and OS did not significantly differ. In colorectal cancer, the correlation between ORR and OS is not clearly elucidated.46 Hence, our results were not conflicting. In addition, in this subgroup, six of ten trials6, 26–29, 32 used ORR as the primary endpoint, and four6, 27, 29, 32 studies hypothesized that Ox-based regimen is associated with a better ORR. Thus, the evaluation of ORR might be biased in these trials. In the subgroup analysis of regimens without molecular targeting agents, Ox-based regimen was associated with a better ORR. However, the PFS and OS did not significantly differ. In colorectal cancer, the correlation between ORR and OS is not clearly elucidated.46 Hence, our results were not conflicting. In addition, in this subgroup, six of ten trials6, 26–29, 32 used ORR as the primary endpoint, and four6, 27, 29, 32 studies hypothesized that Ox-based regimen is associated with a better ORR. Thus, the evaluation of ORR might be biased in these trials. Assessment of Risk of Bias Risk of bias assessment is shown in Fig. 5. Overall, the quality of evidence in this study was moderate according to the risk of bias assessment (Fig. 5A). The funnel plot of each effect size in terms of OS, PFS, and ORR was symmetrical, with a similar number of studies on either side of the summary treatment effect. This result indicated a lack of major publication bias (Fig. 5B). Page 7/18 Page 7/18 Consent for publication Not applicable. Conclusions The efficacy of IRI- and Ox-based regimens when used as the first-line treatments for mCRC did not significantly differ. Hence, both regimens could be used in clinical practice with consideration of the patients’ condition or toxicity profiles. Ethics approval and consent to participate Not applicable. Ethical approval was not required for this study because this meta-analysis is literature based research and did not use individual data. Discussion In the subgroup analysis of regimens with molecular targeting agents, the combination of anti-VEGF antibody and IRI was more likely to be associated with better PFS and OS. However, the synergic effect of IRI and anti- VEFG antibody had not been reported. Although these data were mainly obtained from Japanese trials7, 39, 40, no data have shown that the combination was more beneficial for Japanese patients. Therefore, we speculated that this result was obtained by chance and was not clinically significant. With regard to the results of AEs, the incidence of ≥ grade 3 AEs did not significantly differ between patients who received the two regimens. Therefore, it may be reasonable for physicians to select each regimen according to AE profiles. However, Yamazaki et al.7 revealed that the FACT-C OQL score was more likely worse in patients treated with FOLFOX + BEV than in those treated with FOLFIRI + BEV. Moreover, a previous report indicated that peripheral sensory neuropathy lasts for a long period and affects QOL after treatment.47 Thus, physicians should consider QOL during not only the first-line treatment but also the overall survival time when selecting the regimens. This study had some limitations. That is, it is a meta-analysis. Moreover, there were no data on race, sex, primary site of the tumor, and status of RAS and BRAF genes. Thus, our data must be applied with caution to individual patients in clinical practice. Recently, some reports showed that the molecular subtypes or Page 8/18 Page 8/18 chromosomal variation of CRC might affect the efficacy of IRI- and Ox-based regimens.48, 49 Thus, in the future, suitable cytotoxic agents should be selected according to biomarker profile. chromosomal variation of CRC might affect the efficacy of IRI- and Ox-based regimens.48, 49 Thus, in the future, suitable cytotoxic agents should be selected according to biomarker profile. chromosomal variation of CRC might affect the efficacy of IRI- and Ox-based regimens.48, 49 Thus, in the future, suitable cytotoxic agents should be selected according to biomarker profile. Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors. Abbriviations IRI, irinotecan; Ox, oxaliplatin; mCRC, metastatic colorectal cancer; RCT, randomized controlled trial; OS, overall survival; PFS, progression free survival; ORR, objective response rate; AEs, adverse events; HR, hazard ratio; FPs, fluoropyrimidines; QOL, quality of life; CENTRAL, the Central Registry of Controlled Trials of the Cochrane Library; CI, confidence interval; EGFR, epidermal growth factor receptor; VEGF, vascular endothelial growth factor; CapOx, capecitabine + oxaliplatin; CapIRI, capecitabine + irinotecan; IFL, bolus 5-FU + irinotecan, 5-FU, 5- fluorouracil; BEV, bevacizumab; IRIS, S-1 + irinotecan, FOLFOX and OXAFAFU, 5-FU + folinic acid + oxaliplatin; FOLFIRI and IRIFAFU, 5-FU + folinic acid + irinotecan; TEGAFOX, tegafur + folinic acid + oxaliplatin; TEGAFIRI, tegafur + folinic acid + irinotecan; NA, not available; MTAs, molecular targeting agents Competing interests All authors declare no conflicts-of-interest related to this article. Acknowledgements We would like to show our greatest appreciation to Dr. Goro Nakayama and Dr. Gerardo Rosati for providing data of their clinical trials. Authors’ contributions Page 9/18 Page 9/18 Conceptualization and study design: SK, NT, YH, KY, AN, TK, KM, MY; Writing an original draft: SK; Project administration: YH, KY; Review and editing: SK, NT, YH, AN, MY, TK, KM, KY; Formal analysis: AN, TK, KM; Supervision: YH, KM, KY; Investigation of data: SK, NT, YH, MY; and All authors read and approved the final manuscript. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. References Irinotecan or oxaliplatin combined with 5-fluorouracil and leucovorin as first- linetherapy for advanced colorectal cancer: a meta-analysis. Chin Med J. 2010;123:3314–8. 11. Golfinopoulos V, Salanti G, Pavlidis N et al. 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JAMA Oncol. 2019;5:1574–81. doi:10.1001/jamaoncol.2019.2572 48. Aderka D, Stintzing S, Heinemann V. Explaining the unexplainable: discrepancies in results from the CALGB/SWOG 80405 and FIRE-3 studies. Lancet Oncol. 2019;20:e274–e283. doi:10.1016/S1470- 2045(19)30172-X 48. Aderka D, Stintzing S, Heinemann V. Explaining the unexplainable: discrepancies in results from the CALGB/SWOG 80405 and FIRE-3 studies. Lancet Oncol. 2019;20:e274–e283. doi:10.1016/S1470- 2045(19)30172-X 49. Fujita Y, Taguri M, Yamazaki K, et al. References aCGH analysis of predictive biomarkers for response to Bevacizumab plus Oxaliplatin- or irinotecan-Based chemotherapy in patients with metastatic colorectal cancer. Oncologist. 2019;24:327–37. doi:10.1634/theoncologist.2018-0119 49. Fujita Y, Taguri M, Yamazaki K, et al. aCGH analysis of predictive biomarkers for response to Bevacizumab plus Oxaliplatin- or irinotecan-Based chemotherapy in patients with metastatic colorectal cancer. Oncologist. 2019;24:327–37. doi:10.1634/theoncologist.2018-0119 49. Fujita Y, Taguri M, Yamazaki K, et al. aCGH analysis of predictive biomarkers for response to Bevacizumab plus Oxaliplatin- or irinotecan-Based chemotherapy in patients with metastatic colorectal cancer. Oncologist. 2019;24:327–37. doi:10.1634/theoncologist.2018-0119 Page 13/18 Page 13/18 Figure 1 Flowchart of the search process in randomized clinical trials (RCTs). Page 14/18 Figure 2 Pooled analyses of each endpoint. Black dot at the left side represents th using the Kaplan–Meier curve. (A) Pooled analysis of overall survival (OS free survival (PFS). (C) Pooled analysis of objective response rate (ORR). confidence interval Figure 2 Pooled analyses of each endpoint. Black dot at the left side represents the estimated hazard ratio obtained using the Kaplan–Meier curve. (A) Pooled analysis of overall survival (OS). (B) Pooled analysis of progression- free survival (PFS). (C) Pooled analysis of objective response rate (ORR). IRI, irinotecan; Ox, oxaliplatin; and CI, confidence interval Figure 2 Pooled analyses of each endpoint. Black dot at the left side represents the estimated hazard ratio obtained using the Kaplan–Meier curve. (A) Pooled analysis of overall survival (OS). (B) Pooled analysis of progression- free survival (PFS). (C) Pooled analysis of objective response rate (ORR). IRI, irinotecan; Ox, oxaliplatin; and CI, confidence interval Page 15/18 Figure 3 Subgroup analyses according to combination regimens. (A) Pooled analysis of progression-free survival (PFS). (C) Pooled analysis of o Ox, oxaliplatin; MTAs, molecular targeting agents; CapOx, capecitab 5-fluorouracil; FPs, fluoropyrimidines; EGFR, epidermal growth facto growth factor Figure 3 Subgroup analyses according to combination regimens. (A) Pooled analysis of overall survival (OS). (B) Pooled analysis of progression-free survival (PFS). (C) Pooled analysis of objective response rate (ORR). IRI, irinotecan; Ox, oxaliplatin; MTAs, molecular targeting agents; CapOx, capecitabine plus oxaliplatin; C-mab, cetuximab; 5-FU, 5-fluorouracil; FPs, fluoropyrimidines; EGFR, epidermal growth factor receptor; and VEGF, vascular endothelial growth factor Figure 3 Subgroup analyses according to combination regimens. (A) Pooled analysis of overall survival (OS). (B) Pooled analysis of progression-free survival (PFS). (C) Pooled analysis of objective response rate (ORR). IRI, irinotecan; Ox, oxaliplatin; MTAs, molecular targeting agents; CapOx, capecitabine plus oxaliplatin; C-mab, cetuximab; 5-FU, 5-fluorouracil; FPs, fluoropyrimidines; EGFR, epidermal growth factor receptor; and VEGF, vascular endothelial growth factor Page 16/18 Page 16/18 Page 16/18 Figure 4 Risk difference in the incidence of ≥ grade 3 AEs between patients who received IRI- and Ox-based regimens IRI, irinotecan; Ox, oxaliplatin Figure 5 Assessment of risk of bias. (A) Risk of bias assessment for each included RCT. The bar chart indicates the distribution of risk-of-bias judgments across the domains. (B) The funnel plot of each effect size in terms of overall survival (OS), progression-free survival (PFS), and objective response rate (ORR) SQ, sequence generation; AL, allocation concealment; BL, blinding; IN, incomplete outcome data; SL, selective outcome reporting; OT, other potential threats to validity; SE, standard error; HR, hazard ratio; OR, odds ratio Assessment of risk of bias. (A) Risk of bias assessment for each included RCT. The bar chart indicates the distribution of risk-of-bias judgments across the domains. (B) The funnel plot of each effect size in terms of overall survival (OS), progression-free survival (PFS), and objective response rate (ORR) SQ, sequence generation; AL, allocation concealment; BL, blinding; IN, incomplete outcome data; SL, selective outcome reporting; OT, other potential threats to validity; SE, standard error; HR, hazard ratio; OR, odds ratio PRISMAchecklistBMC.doc Figure 4 Risk difference in the incidence of ≥ grade 3 AEs between patients who received IRI- and Ox-based regimens IRI, irinotecan; Ox, oxaliplatin Risk difference in the incidence of ≥ grade 3 AEs between patients who received IRI- and Ox-based regimens IRI, irinotecan; Ox, oxaliplatin Page 17/18 Figure 5 Assessment of risk of bias. (A) Risk of bias assessment for each included RCT. The bar chart indicat distribution of risk-of-bias judgments across the domains. (B) The funnel plot of each effect size in t overall survival (OS), progression-free survival (PFS), and objective response rate (ORR) SQ, sequenc generation; AL, allocation concealment; BL, blinding; IN, incomplete outcome data; SL, selective outc reporting; OT, other potential threats to validity; SE, standard error; HR, hazard ratio; OR, odds ratio Figure 5 Assessment of risk of bias. (A) Risk of bias assessment for each included RCT. The bar chart indicates the distribution of risk-of-bias judgments across the domains. (B) The funnel plot of each effect size in terms of overall survival (OS), progression-free survival (PFS), and objective response rate (ORR) SQ, sequence generation; AL, allocation concealment; BL, blinding; IN, incomplete outcome data; SL, selective outcome reporting; OT, other potential threats to validity; SE, standard error; HR, hazard ratio; OR, odds ratio Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Page 18/18
https://openalex.org/W2473552772	https://inria.hal.science/hal-01369560/document	English	null	A Utility-Based Reputation Model for the Internet of Things	IFIP advances in information and communication technology	2,016	cc-by	6,668	A Utility-based Reputation Model for the Internet of Things Benjamin Aziz∗, Paul Fremantle∗, Rui Wei†, and Alvaro Arenas‡ ∗School of Computing University of Portsmouth Portsmouth, United Kingdom {benjamin.aziz,paul.fremantle}@port.ac.uk †Department of Computer and Information Technology Beijing Jiaotong University Beijing, China 12120463@bjtu.edu.cn ‡IE Business School IE University Madrid, Spain alvaro.arenas@ie.edu Abstract. The MQTT protocol has emerged over the past decade as a key protocol for a number of low power and lightweight communication scenarios including machine-to-machine and the Internet of Things. In this paper we develop a utility-based reputation model for MQTT, where we can assign a reputation score to participants in a network based on monitoring their behaviour. We mathematically deﬁne the reputation model using utility functions on participants based on the expected and perceived behaviour of MQTT clients and servers. We deﬁne an architec- ture for this model, and discuss how this architecture can be implemented using existing MQTT open source tools, and we demonstrate how exper- imental results obtained from simulating the architecture compare with the expected outcome of the theoretical reputation model. Keywords: Internet of Things, Utility Reputation, Trust Management 1 Introduction The Internet of Things (IoT) is an area where there is signiﬁcant growth: both in the number of devices deployed and the scenarios in which devices are being used. One of the challenges for the Internet of Things is supporting network protocols which utilise less energy, lower bandwidth, and support smaller footprint devices. One such protocol is the MQ Telemetry Transport (MQTT) protocol [15], which was originally designed to support remote monitoring and Supervisory Control And Data Acquisition (SCADA) scenarios but has become popular for the IoT. Another challenge with IoT networks is that small devices may not perform as well as needed due to a number of factors including: network outages or poor q ( ) p p Another challenge with IoT networks is that small devices may not perform as well as needed due to a number of factors including: network outages or poor network performance due to the use of 2G or other low bandwidth networks, A Utility-based Reputation Model for the Internet of Things 2 power outages for devices powered by batteries, deliberate vandalism or envi- ronmental damage for devices placed in public areas, and many other such chal- lenges. Therefore we identiﬁed that a reputation model for devices connecting by MQTT would be a useful construct to express consumers’ (applications’) trust in the behaviour and performance of these devices as well as measure the level of performance of the server aggregating data from such devices according to some predeﬁned Service Level Agreement (SLA). In addition, we implemented the reputation model to demonstrate that it could be used in real MQTT networks. Our model of reputation is based on the notion of a utility function, which formally expresses the consumer’s level of satisfaction related to various issues of interest against which the reputation of some entity is measured. In the case of MQTT networks, one notable such issue is the Quality of Service (QoS) with regards to the delivery of messages; whether messages are delivered exactly once, more than once or at most once to their consumers. The model, inspired by previous works [6, 19], is general enough to be capable of deﬁning the reputation of client devices and servers at various levels of abstraction based on their level of performance in relation to the delivery of messages issue of interest. The paper starts with an overview of the MQTT protocol (Section 2). 1 Introduction From this, we then mathematically deﬁne the reputation model for MQTT clients and server (Section 3), based on their ability to keep to the requirements of the protocol. We then outline a system architecture (Section 4) for monitoring the MQTT protocol and thereby being able to calculate the reputation by observing the behaviour of MQTT clients and server in a real network. We show how this system was implemented and we demonstrate the results of this implementation (Section 5). Finally we look at related work (Section 6) and conclude the paper outlining areas for further research (Section 7). 2 MQTT Overview MQTT [9] is described as a lightweight broker-based publish/subscribe messag- ing protocol that was designed to allow devices with small processing power and storage, such as those which the IoT is composed of, to communicate over low-bandwidth and unreliable networks. The publish/subscribe message pattern [10], on which MQTT is based, provides for one-to-many message distribution with three varieties of delivery semantics, based on the level of QoS expected from the protocol. In the “at most once” case, messages are delivered with the best eﬀort of the underlying communication infrastructure, which is usually IP- based, therefore there is no guarantee that the message will arrive. This protocol is termed the QoS = 0 protocol. In the second case of “at least once” semantics, certain mechanisms are incorporated to allow for message duplication. Despite the guarantee of delivering the message, there is no guarantee that duplicates will be suppressed. This protocol is also known as the QoS = 1 protocol. Fi- nally, for the last case of “exactly once” delivery semantics, also known as the QoS = 2 protocol, the published message is guaranteed to arrive only once at the subscribers. The protocol also deﬁnes message structures needed in commu- A Utility-based Reputation Model for the Internet of Things 3 3 nications between clients, i.e. end-devices responsible for generating data from their domain (the data source) and servers, which are the system components responsible for collating source data from clients/end-devices and distributing these data to interested subscribers. Servers are often also referred to as brokers, as they intermediate between the data publishers and subscribers. 3 A Reputation Model for MQTT We show in this section how the model of reputation deﬁned for business pro- cesses in [7, 8] can be adapted, with minimum changes, to the MQTT protocol to obtain the reputation of client devices and the server. 3.1 Monitoring Events Central to the model deﬁned by [7, 8] was the notion of an event, which is a signal produced by an independent monitor system, which is monitoring the interac- tions occurring between the diﬀerent entities in the monitored environment, in this case the client and server entities participating in the MQTT protocol. An event is deﬁned as follows: Event : TimeStamp × Ag × Msg × Id × N where TimeStamp is the timestamp of the event generated by the monitor system issuing it, Ag is the identity of the agent (client device or server) to whom the event is related, Msg is the speciﬁc message of interest (e.g. Publish and Pubrel messages), Id is an identity value of the protocol instance and ﬁnally, N is a natural number representing the number of times the message Msg has been monitored, i.e. was sent. For example, the following event, issued at monitor system’s local time: evex1 = (12:09:52, temp sensor, Publish, 1234, 2) denotes that the temp sensor device has been monitored, within the instance number 1234 of the protocol, to have sent twice the Publish message to the server responsible for collecting environment temperature data. On the other hand, the following event issued at local time 12:19:02: denotes that the temp sensor device has been monitored, within the instance number 1234 of the protocol, to have sent twice the Publish message to the server responsible for collecting environment temperature data. On the other hand, the following event issued at local time 12:19:02: evex2 = (12:19:02, temp server, Publish, 1234, 1) denotes that the server responsible for the environment temperature, temp server, has been monitored, within the same instance number 1234 of the protocol, to have published only once the speciﬁc message Publish to the subscribers of the temperature topic. In both these examples, the assumption is that the monitor system is capable of detecting that the protocol instance being monitored has terminated before it issues any events related to that instance. Although theo- retically this is impossible due to the halting problem, in practical terms, the denotes that the server responsible for the environment temperature, temp server, has been monitored, within the same instance number 1234 of the protocol, to have published only once the speciﬁc message Publish to the subscribers of the temperature topic. In both these examples, the assumption is that the monitor system is capable of detecting that the protocol instance being monitored has terminated before it issues any events related to that instance. Although theo- retically this is impossible due to the halting problem, in practical terms, the 4 A Utility-based Reputation Model for the Internet of Things monitor system can assume the protocol to have terminated after some reason- able amount of time has elapsed since the last protocol message. Event : TimeStamp × Ag × Msg × Id × N The monitor generates events in the above form, which are used by a repu- tation engine to determine the reputation values for client devices and servers in an MQTT-based environment. The reputation engine will then use a util- ity function pre-supplied to the engine by subscribers to determine the level of satisfaction of a subscriber with regards to the results reported within an event: utility : Event × SLA →[0, 1] ∀(t, a, m, i, n) ∈Event, sla ∈SLA • utility((t, a, m, i, n), sla) = r ∈R This utility function will consider a SLA, deﬁned as follows: SLA : Ag × Top × Iss →N0 Here the SLA considers an issue of interest to the subscriber, Iss, which will be in our case the QoS level value ﬁxed to one of 0, 1 or 2, expected from a particular agent Ag in relation to a speciﬁc topic Top. The outcome of the utility function is a real number r representing the satisfaction level of the subscriber in terms of both the SLA and the real values reported by events. For example, consider the following SLA instance sla = ((temp server, temperature, QoS), 2) then given the event evex2, the utility function could return the following value: utility(t, temp server, Publish, 1234, 1, ((temp server, temperature, QoS), 2)) = 1 then given the event evex2, the utility function could return the following value: utility(t, temp server, Publish, 1234, 1, ((temp server, temperature, QoS), 2)) = 1 This indicates that the subscriber’s requirements have been fulﬁlled, as indicated by their SLA (r = 1), with the results reported by the event evex2. On the other hand, considering the same SLA, the utility function might return: utility(t, temp server, Publish, 1234, 0, ((temp server, temperature, QoS), 2)) = 0 to show that the subscriber has a satisfaction value of 0 since the number of times the message was delivered to the subscriber is lower (i.e. 0) than what its QoS level is deﬁned in the SLA (i.e. 2), therefore breaching the exactly-once delivery semantics to the subscriber principle in MQTT. Since the number of times a message is delivered will either conﬁrm or not to the level of QoS expected by the subscriber, in all of the above cases, the score given will reﬂect either total satisfaction (i.e. 1) or total dissatisfaction (i.e. 0). 3.2 Reputation Models After introducing the main notions of an event and a utility function, we can now deﬁne models of reputation for the clients (e.g. sensor devices) and the MQTT A Utility-based Reputation Model for the Internet of Things 5 server (broker) that aggregates the messages from the clients before publishing them to the subscribers. The subscribers are assumed to be the business appli- cations or data consumers, and we do not include them in the reputation model. The MQTT standard does not prohibit a client from acting as both a device (i.e. source of data) and a subscriber (i.e. consumer of data). However, in our case, we only measure the reputation of the “source of data” clients. The Server Reputation Model The ﬁrst reputation model reﬂects the be- haviour of MQTT servers. Given a set of events, Event, captured by the monitor system and relevant to the server for whom the reputation is being calculated, then we can deﬁne the server’s reputation function computed at a particular point in time and parameterised by a speciﬁc SLA as follows: [Srv, SLA, TimeStamp] s rep sla : Srv × SLA × TimeStamp →[0, 1] ∀esets : ℘(Event) • s rep sla(s, sla, t) = P ev ∈esets.snd(ev) =fst(sla) = s ∧id top(ev, sla) ϕ(t,te)utility(ev,sla) #esets where #s denotes the cardinality of a set s and ϕ(t, te) is a time discount function that puts more importance (emphasis) on events registered closer in time to the moment of computing the reputation. One deﬁnition of ϕ(t, te) could be the time discount function deﬁned by [13], which we redeﬁne here as ϕ(t, te) = e−t−te λ , where t is the current time at which the reputation is calculated, te is the timestamp of the event being considered and λ is recency scaling factor used to adjust the value of the function to a scale required by the application. After this, the server reputation function, s rep sla, is deﬁned as the weighted average of the utilities obtained from all the generated events with respect to some SLA. The above deﬁnition aggregates the set of all relevant events, i.e. the events that ﬁrst have the same server name as that appearing in the SLA and second that are on an instance of the protocol related to the topic of the SLA. id top(12:09:52, temp server, Publish, 1234, 1, ((temp server, temperature, QoS), 2)) = True 3.2 Reputation Models The ﬁrst condition is checked using the two operators fst and snd, which will return the ﬁrst and second elements of a tuple, whereas the second condition is checked using the predicate id top(ev, sla), which returns a True outcome if and only if the identity number of an instance of a protocol captured by ev corresponds to the topic value mentioned in the SLA sla. Considering the example events of the previous section, we would have the following calculation of id top(ev, sla): id top(12:09:52, temp server, Publish, 1234, 1, ((temp server, temperature, QoS), 2)) = True The above deﬁnition calculates the sum of the time-discounted utility func- tion values, with respect to the given SLA and the events gathered, and average these over the total number of events gathered (#esets) in any one instance when this reputation value is calculated. 6 A Utility-based Reputation Model for the Internet of Things Based on the deﬁnition of s rep sla, we next aggregate the reputation of a server across every SLA that binds that server to its subscribers: [Srv, TimeStamp] s rep : Srv × TimeStamp →[0, 1] ∀slasets : ℘(SLA) • s rep(s, t) = P sla ∈slasets.fst(sla) = s s rep sla(s,sla,t) #slasets [Srv, TimeStamp] s rep : Srv × TimeStamp →[0, 1] ∀slasets : ℘(SLA) • s rep(s, t) = P sla ∈slasets.fst(sla) = s s rep sla(s,sla,t) #slasets Which provides a more general indication of how well a server s behaves in relegation to a number of subscribers. This reputation is again calculated in a particular point in time, t, however it is straightforward to further generalise this reputation function over some time range, between t and t′. Which provides a more general indication of how well a server s behaves in relegation to a number of subscribers. This reputation is again calculated in a particular point in time, t, however it is straightforward to further generalise this reputation function over some time range, between t and t′. The Client Device Reputation Model After introducing the reputation model of the server, we deﬁne here the client’s reputation model. Like the server, a client might also be implementing the QoS correctly, but it requires multiple reconnections, duplicate messages etc., while the server does not. 3.2 Reputation Models [Client, SLA, TimeStamp, KeepAlive] c rep sla ka : Client × SLA × TimeStamp × KeepAlive →[0, 1] ∀psets : ℘(Event) • c rep sla ka(c, sla, t, ka) = P ev∈kapingsets.snd(ev) =fst(sla) =c ∧id top(ev,sla) ϕ(t,te)utility(ev,sla) #kapingsets [Client, SLA, TimeStamp, KeepAlive] c rep sla ka : Client × SLA × TimeStamp × KeepAlive →[0, 1] ∀psets : ℘(Event) • c rep sla ka(c, sla, t, ka) = P ev∈kapingsets.snd(ev) =fst(sla) =c ∧id top(ev,sla) ϕ(t,te)utility(ev,sla) #kapingsets In this procedure, the client sends a Pingreq message within each KeepAlive time period, then the receiver answer with a Pingresp message when it receives a Pingreq message from the gateway to which it is connected. Clients should use KeepAlive timer to observe the liveliness of the gateway to check whether they are connected to broker. If a client does not receive a Pingresp from the gateway even after multiple retransmissions of the Pingresq message, it fails to connect with gateway during the Keep Alive period. Hence, for the above example, using id top to show a set of related events ev corresponds to the topic value mentioned in the SLA, sla, we would have that: id top(12 : 09 : 52, client, Pingreq, False, False, 1234, 1, ((client, temperature, QoS), 0)) = True id top(12 : 09 : 52, client, Pingreq, False, False, 1234, 1, ((client, temperature, QoS), 0)) = True The event evkaping = (12 : 09 : 52, client, Pingreq, False, False, 1234, 1) gen- erated by the monitor, could reﬂect a client device that has sent once the Pingreq message to connect to the gateway within the instance number 1234 of the protocol during the Keep Alive period. Then, given the SLA instance sla = ((client, temperature, QoS), 0), the client should deliver this Pingreq mes- sage in relation to a speciﬁc topic (in this case temperature) at most once within each KeepAlive time period, but there is no guarantee the message will arrive. 3.2 Reputation Models For instance, if the devices are not sending PINGs or responding to them, or this is delayed, it might indicate a problem is more likely to occur in the future. Similarly, if the device needs to send multiple duplicate messages or needs to be sent duplicate messages, it also might indicate possible failure in the future. Thus, the reputation model for a client may be based on either the “Keep Alive/PING” case or the “Client’s Retransmission Procedure” case. However, we start with the deﬁnition of an overall reputation model that generalises these two cases. Given a set of events, Event, captured by the monitor system relevant to some client, then we deﬁne the client’s reputation function computed at a particular point in time in a speciﬁc process (Keep Alive/PING procedure or retransmission procedure) and parameterised by a speciﬁc SLA as follows: [Client, SLA, TimeStamp, Procedure] c rep sla p : Client × SLA × TimeStamp × Procedure →[0, 1] ∀psets : ℘(Event) • c rep sla p(c, sla, t, p) = P ev∈psets.snd(ev) =fst(sla) = c ∧id top(ev,sla) ϕ(t,te)utility(ev,sla) #psets This deﬁnition gathers the set of all related events, i.e. the events that ﬁrst have the same client name as that appearing in the SLA and second that are on an instance of the protocol related to the topic of the SLA. The deﬁnition is parameterised by the client, an SLA, a timestamp and the speciﬁc procedure (e.g. Keep Alive/PING or retransmission). The SLA represents what the expectation is, from the server’s point of view, of the client’s behaviour in the context of the speciﬁc procedure. Similar to the case of s rep sla, a utility function is applied to measure the satisfaction of the server, in a time-discounted manner, in relation to the client’s behaviour and this is then averaged over the total number of events captured in a speciﬁc instance of time. 7 A Utility-based Reputation Model for the Internet of Things For example, consider the case of the Keep Alive/PING procedure, then c rep sla ka is deﬁned as the time-discounted average of the utilities obtained from all generated events with respect to the Keep Alive/PING procedure. 3.2 Reputation Models From the deﬁnition of c rep sla p, we generate a more general reputation for some client in a particular point in time t within a period, Period, as follows: [Client, SLA, TimeStamp] c rep sla : Client × SLA × TimeStamp →[0, 1] ∀periodsets : ℘(Period) • c repsla(c, sla, t) = P ev∈periodsets.snd(ev) =fst(sla) = c ∧id top(ev,sla) c rep sla p(c,sla,t,p) #periodsets [Client, SLA, TimeStamp] c rep sla : Client × SLA × TimeStamp →[0, 1] ∀periodsets : ℘(Period) • c repsla(c, sla, t) = P ev∈periodsets.snd(ev) =fst(sla) = c ∧id top(ev,sla) c rep sla p(c,sla,t,p) #periodsets [Client, SLA, TimeStamp] c rep sla : Client × SLA × TimeStamp →[0, 1] ∀periodsets : ℘(Period) • c repsla(c, sla, t) = P ev∈periodsets.snd(ev) =fst(sla) = c ∧id top(ev,sla) c rep sla p(c,sla,t,p) #periodsets Giving an example based on the Keep Alive/PING procedure, assume the KeepAliveTimer is set to 60, then calculating c rep sla(c, sla, t) will give us the reputation of the client device during the whole Keep Alive period of 60 seconds. In another example, based on the retransmission procedure, we assume that Nretry is set to 10. Aggregating over the c rep sla(c, sla, t) values yields reputation in relation to the client’s retransmissions within a 10 time-unit limit. 8 A Utility-based Reputation Model for the Internet of Things Finally, based on the deﬁnition of c rep sla, we can further generalise the reputation value over all relevant SLAs for a speciﬁc client, c, and in a particular point in time, t, as follows: [Client, SLA] c rep : Client × SLA →[0, 1] ∀slasets : ℘(SLA) • c rep(c, t) = P crep(c, t) = sla ∈slasets.fst(sla) = c c rep sla(s,sla,t) #slasets [Client, SLA] c rep : Client × SLA →[0, 1] ∀slasets : ℘(SLA) • c rep(c, t) = P crep(c, t) = sla ∈slasets.fst(sla) = c c rep sla(s,sla,t) #slasets This deﬁnition gives a more general indication of how well the client device generally behaves in relation to the SLAs it holds with the server (possibly on behalf of the subscribers dealing with the server). These could include scenarios where the clients might use the Keep Alive/PING procedure to observe the live- liness of the gateway to check whether they are connected to a broker. 3.2 Reputation Models A Utility based Reputation Model for the Internet of Things 9 A Utility-based Reputation Model for the Internet of Things 9 Similarly, another variation of the client’s reputation function may be based on the NRetry counter instead: [Client, SLA, TimeStamp, NRetry] c repnr sla nr : Client × SLA × TimeStamp × NRetry →[0, 1] ∀nretrysets : ℘(Event) • c reptr sla tr(c, sla, t, nr) = P ev ∈kapingsets.snd(ev) = fst(sla) = c ∧id top(ev, sla) ϕ(t,te)utility(ev,sla) #nretrysets Again, for the above deﬁnition, for sla = ((client, temperature, QoS), 2), and given the event evnretry = (12 : 09 : 52, client, Publish, False, False, 1234, 1), it could indicate that the client should not retransmit again in the retransmission period due to the fact that QoS is set to 2 (meaning the message is guaranteed to be delivered exactly-once to the subscribers). In this case, the DUP ﬂag must be set to False, in order to prevent a retransmission. 3.2 Reputation Models Moreover, in the case of messages that expect a response, if the reply is not received within a certain time period, the client will be expected to retransmit this message. The reputation model of a client in diﬀerent procedures might cause dif- ferent failures. Thus, as we demonstrated above the ﬁrst reputation model, c rep sla p, will lead to new models with slight variations capturing this variety of failures. For example, for the case of a client’s retransmission procedure, all messages that are “unicast” to the gateway and for which a gateway’s response is expected are supervised by a retry timer Tretry and a retry counter Nretry. The retry timer Tretry is started by the client when the message is sent and stopped when the expected gateway’s reply is received. If the client does not receive the expected gateway’s reply during the Tretry period, it will retransmit the message. In addition, the client should terminate this procedure after Nretry number of retransmissions and should assume that it is disconnected from the gateway. The client should then try to connect to another gateway only if it fails to re-connect again to the previous gateway. One such client reputation, is deﬁned based on a speciﬁc TRetry timer: [Client, SLA, TimeStamp, TRetry] c reptr sla tr : Client × SLA × TimeStamp × TRetry →[0, 1] ∀tretrysets : ℘(Event) • c reptr sla tr(c, sla, t, tr) = P ev∈kapingsets.snd(ev) = fst(sla) = c ∧id top(ev, sla) ϕ(t,te)utility(ev,sla) #tretrysets [Client, SLA, TimeStamp, TRetry] c reptr sla tr : Client × SLA × TimeStamp × TRetry →[0, 1] ∀tretrysets : ℘(Event) • c reptr sla tr(c, sla, t, tr) = P ev∈kapingsets.snd(ev) = fst(sla) = c ∧id top(ev, sla) ϕ(t,te)utility(ev,sla) #tretrysets For example, consider the following sla = ((client, temperature, QoS), 1), then given the event evtretry = (12 : 09 : 52, client, Publish, False, True, 1234, 2), it could reﬂect an event in the retransmission procedure. If the client does not receive a Puback message with QoS level 1 within a time period deﬁned by the TRetry value, the client may resend the Publish message with the DUP ﬂag set. When the server receives a duplicate message from the client, it re-publishes the message to the subscribers, and sends another Puback message. 5 Simulation of the Model We implemented the architecture, described in the previous section, by running a number of oﬀ-the-shelf open source tools. First, we used the Mosquitto tool [1] as the broker (server). Mosquitto is an open source MQTT broker written in the C language that implements the MQTT protocol version 3.1. The Mosquitto project is highly portable and runs on a number of systems, which made it an eﬀective choice for our experiments. In order to simulate the client, we used the source code for the Eclipse Paho MQTT Python client library [2], which comes with Java MQTT client API and implements versions 3.1 and 3.1.1 of the MQTT protocol. This code provides a client class, which enables applications to connect to an MQTT broker to publish messages, receive published messages and to subscribe to topics. It also provides some helper functions to make publishing one oﬀmessages to an MQTT server very straightforward. Using this library we created a set of programs that would publish and subscribe to the Mosquitto broker. Finally, to implement the monitoring function we needed to capture all the traﬃc between the client and the server. For this we extended the Paho MQTT test proxy [2], which acts as a “reverse proxy”, impersonating an MQTT server and sending all the traﬃc on to a real broker after capturing the messages. The proxy represents a mediator between clients and the broker. By extending this proxy we were able to trace all the packets being sent and received and send monitoring information to our reputation engine in order to calculate the reputation of the client and broker. 4 A Reputation System Architecture for MQTT Our architecture for a reputation system for an MQTT network is composed of a reputation monitor and a reputation engine, as shown in Figure 1. Fig. 1. The Reputation System Architecture. Fig. 1. The Reputation System Architecture. The architecture deﬁnes the capabilities of the various components in an MQTT network. The reputation monitor (also sometimes referred to as the proxy) will monitor the MQTT interactions that take place among the MQTT network components, namely the client devices, server and subscribers. Monitor- ing implies that the reputation monitor will issue events to the reputation engine A Utility-based Reputation Model for the Internet of Things 10 whenever these are required after each time it has captured an MQTT commu- nication relevant to the utility functions predeﬁned by the consumers (possibly the subscribers). These events could represent aggregations/abstractions of data collected from such communications, in order to minimise the additional network traﬃc created by this process. Once an event has arrived at the reputation engine, it is either stored for applying further aggregations/abstractions or it is used immediately to compute new updates for the various reputation values for the clients and the server. The calculations are based on the reputation models deﬁned in the previous sections, and the updates to these reputation values are then stored in a local reputation database. In our architecture, we only consider the monitoring problem, however, it is easy to extend this architecture in the future to include a control step, where the reputation values for diﬀerent participants are then used to impact/feed back into the MQTT network communications. 5.2 Reputation Results To compute the reputation value of clients and servers, we collected events re- lated to the QoS level agreed between the client and the server throughout the 5-minute measurement window, and used the server and client reputation model proposed in Section 3.2 to calculate their reputation values. The QoS level mon- itoring is important as it is directly related to the issue of message suppression when messages are communicated over the unreliable network. In the presence of such abnormal behaviour, the reputation values of the clients and the server are shown in Figure 2 versus the rate of successful message delivery (0.5 to 1). From this ﬁgure, we note that despite starting at low reputation levels in line with the low delivery rate of messages, these reputation values will increase reaching the optimal value of 1 when the rate of delivery of messages is 1. This optimal case represents the case of normal behaviour when every message is delivered successfully to its destination. 5.1 Results To begin with, we assume that the network will misbehave with regards to the messages that are exchanged among the various entities in the system. This mis- behaviour is modelled as the network dropping some messages according to a A Utility-based Reputation Model for the Internet of Things 11 predeﬁned rate (e.g. 0–100%). There could be other sources of network misbe- haviour, such as the insertion of new messages and the repetition or modiﬁcation of transmitted messages, however, for simplicity, we consider only the suppres- sion of messages as our example of how the network could misbehave and how such misbehaviour would aﬀect the reputation of MQTT clients and servers. In our case, we chose the rate of successful message delivery to be in the range of 50% to 100%, where 50% means that one message in every two is dropped by the network, and 100% means that every message is delivered suc- cessfully to its destination. This latter case is equivalent to the normal behaviour discussed above. There are a number of tools that can drop network packets se- lectively. However, we created a new tool based on the above-mentioned proxy that speciﬁcally targets disrupting MQTT ﬂows by dropping MQTT packets. The tool allowed us to target a percentage of dropped packets and therefore calculate the reputation under a given percentage of packet loss. Since our aim is to demonstrate, in general terms, how reputation-based trust can be obtained in an IoT system such as an MQTT network, and for simplicity, we opted to consider only one source of misbehaviour, namely message suppression, without considering the other sources. Despite the fact that such sources are also interesting, they do not aﬀect the generality of our approach. 6 Related Work Reputation is a general concept widely used in all aspects of knowledge rang- ing from humanities, arts and social sciences to digital sciences. In computing systems, reputation is considered as a measure of how trustworthy a system is. There are two approaches to trust in computer networks: the ﬁrst involves a “black and white” approach based on security certiﬁcates, policies, etc. For example, SPINS [17], develops a trusted network. The second approach is prob- abilistic in nature, where trust is based on reputation, which is deﬁned as a A Utility-based Reputation Model for the Internet of Things 12 Fig. 2. Reputation Values for the Clients (c rep) and Servers (s rep) vs. the rate of Successful Message Delivery. Fig. 2. Reputation Values for the Clients (c rep) and Servers (s rep) vs. the rate of Successful Message Delivery. probability that an agent is trustworthy. In fact, reputation is often seen as one measure by which trust or distrust can be built based on good or bad past experiences and observations (direct trust) [14] or based on collected referral information (indirect trust) [5]. In recent years, the concept of reputation has shown itself to be useful in many areas of research in computer science, particularly in the context of distributed and collaborative systems, where interesting issues of trust and security manifest themselves. Therefore, one encounters several deﬁnitions, models and systems of reputation in distributed computing research (e.g. [12, 14, 20]). There is considerable work into reputation and trust for wireless sensor net- works, much of which is directly relevant to IoT trust and reputation. The Her- mes [22] and E-Hermes [23] systems utilise Bayesian statistical methods to cal- culate reputation based on how eﬀectively nodes in a mesh network propagate messages including the reputation messages. Similarly TRM-IoT [11] evaluates reputation based on the packet-forwarding trustworthiness of nodes, in this case using fuzzy logic to provide the evaluation framework. Another similar work is CORE [16] which again looks at the packet forwarding reputation of nodes. Our approach diﬀers from the existing research in two regards: ﬁrstly, the existing reputation models for IoT utilise the ability of nodes to operate in consort as the basis of reputation. 7 Conclusion To conclude, we deﬁned in this paper a model of reputation for IoT systems, in particular, for MQTT networks, which is based on the notion of utility functions. The model can express the reputation of client and server entities in an MQTT system at various levels, and in relation to a speciﬁc issue of interest, in our case the QoS level of the delivery of messages in the presence of a lossy network. We demonstrated that it is possible, using oﬀ-the-shelf open source MQTT tools, to implement an architecture of the reputation system that monitors the MQTT components, and we showed that the experimental results obtained from running such a system validate the theoretical model. Future work will focus on adapting the reputation model and its architecture and implementation to other IoT standards, e.g. the Advanced Message Queu- ing Protocol (AMQP) [21], the Extensible Messaging and Presence Protocol (XMPP) [4], the Constrained Application Protocol (CoAP) [18] and the Sim- ple/Streaming Text Oriented Messaging Protocol (STOMP) [3]. We also plan to consider other issues of interest when calculating reputation where satisfaction is not necessarily a binary decision, for example, the quality of data generated by client devices and the quality of any ﬁltering, aggregation or analysis functions the server may apply to such data in order to generate new information to be delivered to the consumers. Further, we intend to apply Bayesian statistics to the results to improve the probabilistic calculation of the reputation values. Some other interesting, though more advanced areas of research, include the strengthening of the model to be able to cope with malicious forms of client and server behaviour, for example, collusion across such entities in order to produce fake reputation values for a targeted victim, and a study on the welfare of IoT ecosystems based on the diﬀerent rates of the presence of ill-behaved and well- behaved entities in the ecosystem, and how variations in the presence ratio of such entities would lead to a notion of reputation reﬂecting the wider ecosystem. 6 Related Work While this is important in wireless sensor networks, there are many IoT applications that do not utilise mesh network topologies and therefore there is a need for a reputation model that supports client-server IoT protocols such as MQTT. Secondly, the work we have done evaluates the reputation of a reliable messaging system based on the number A Utility-based Reputation Model for the Internet of Things 13 of retries needed to successfully transmit a message. Although many reputation models have been based on rates of packet forwarding, the analysis of a reliable messaging system (like MQTT with QoS > 1) is diﬀerent as messages are always delivered except in catastrophic circumstances. Therefore we looked at the eﬀort and retries required to ensure reliable delivery instead. We have not seen any similar approach to this and consider this the major contribution of the paper. 1. Mosquitto: An open source mqtt v3.1/v3.1.1 broker. http://mosquitto.org/, ac- cessed: 2016-03-11 2. Paho. http://www.eclipse.org/paho/, accessed: 2016-03-11 3. STOMP: The Simple Text Oriented Messaging Protocol. https://stomp.github. io, accessed: 2016-03-11 4. XMPP Standards Foundation. http://xmpp.org, accessed: 2016-03-11 4. XMPP Standards Foundation. http://xmpp.org, accessed: 2016-03-11 References A Utility-based Reputation Model for the Internet of Things 14 5. Abdul-Rahman, A., Hailes, S.: Supporting trust in virtual communities. In: HICSS ’00: Proceedings of the 33rd Hawaii International Conference on System Sciences- Volume 6. IEEE Computer Society, Washington, DC, USA (2000) ( ) 6. Arenas, A.E., Aziz, B., Silaghi, G.C.: Reputation management in collaborative computing systems. Security and Communication Networks 3(6), 546–564 (2010) ( ) ( ) 7. Aziz, B., Hamilton, G.: Reputation-controlled business process workﬂows. In: Pro- ceedings of the 8th International Conference on Availability, Reliability and Secu- rity. pp. 42–51. IEEE CPS (2013) 8. Aziz, B., Hamilton, G.: Enforcing reputation constraints on business process work- ﬂows. Journal of Wireless Mobile Networks, Ubiquitous Computing, and Depend- able Applications (JoWUA) 5(1), 101–121 (Mar 2014) 9. 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Locke, D.: MQ Telemetry Transport (MQTT) V3 16. Michiardi, P., Molva, R.: Core: a collaborative reputation mechanism to enforce node cooperation in mobile ad hoc networks. In: Advanced Communications and Multimedia Security, pp. 107–121. Springer (2002) ( ) 17. Perrig, A., Szewczyk, R., Tygar, J., Wen, V., Culler, D.E.: Spins: Security protocols for sensor networks. Wireless networks 8(5), 521–534 (2002) 18. Shelby, Z., Hartke, K., Bormann, C.: Constrained Application Pro- tocol (CoAP) draft-ietf-core-coap-18. https://tools.ietf.org/html/ draft-ietf-core-coap-18, accessed: 2016-03-11 19. Silaghi, G.C., Arenas, A., Silva, L.: Reputation-based trust management sys- tems and their applicability to grids. Tech. Rep. TR-0064, Institutes on Knowl- edge and Data Management & System Architecture, CoreGRID - Network of Excellence (February 2007), http://www.coregrid.net/mambo/images/stories/ TechnicalReports/tr-0064.pdf 20. 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https://openalex.org/W3210107689	https://www.e3s-conferences.org/10.1051/e3sconf/202131207006/pdf	English	null	Development of a 0D multi-zone model for fast and accurate prediction of homogeneous charge compression ignition (HCCI) engine	E3S web of conferences	2,021	cc-by	8,242	E3S Web of Conferences 312, 07006 (2021) 76° Italian National Congress ATI E3S Web of Conferences 312, 07006 (2021) 76° Italian National Congress ATI https://doi.org/10.1051/e3sconf/202131207006 Development of a 0D multi-zone model for fast and accurate prediction of homogeneous charge compression ignition (HCCI) engine Giovanni Fasulo1, Fabio Bozza1, Enrica Malfi1, and Luigi Teodosio1 1University of Naples, Federico II, Napoli, Italia Abstract. Homogeneous Charge Compression Ignition (HCCI) is a promising advanced combustion mode, featured by both high thermal efficiency and low emissions. In this context, a 0D multi-zone model has been developed, where the thermal stratification in the combustion chamber has been taken into account. The model is based on a control mass Lagrangian multi-zone approach. In addition, a procedure based on a tabulated approach (Tabulated Kinetic of Ignition - TKI) has been developed, to perform an accurate and fast prediction of the air/fuel mixture auto-ignition. This methodology allows combining the accuracy of detailed chemistry with a negligible computational effort. The tabulated procedure has been preliminarily verified through the comparison with the results of a commercial software (GT-Power™). In this assessment, single zone simulations have been performed comparing the TKI strategy to a conventional chemical kinetics one, in four different cases at varying the intake temperature and the equivalence ratio. Then, the proposed 0D multi- zone model has been validated against experimental data available in the literature. The analyses are carried out with reference to an HCCI engine fuelled with pure hydrogen and working in a single operating point, namely 1500 rpm, 2.2 bar IMEP and with a fuel/air equivalence ratio of 0.24. Three different temperatures, i.e., 373, 383, and 393 K, have been considered for the intake air. The experimental/numerical comparisons of pressure cycles and burn rates proved that the proposed numerical approach can reproduce the experiments with good accuracy, without the need for case-by-case tuning. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). 1 Introduction In the present-day scenario, the automotive industry is facing a challenging path consisting in the design of low emission and low fuel-consumption vehicles, through the development of innovative Internal Combustion Engine (ICE) architectures. To this aim, an increasing interest is addressed to innovative combustion modes. Among these options, the Homogeneous Charge Compression Ignition (HCCI), as a low-temperature combustion (LTC) concept proposed by Onishi et al. [1], represents a reliable technique, able to produce ultra-low NOx levels and to provide greater fuel conversion efficiencies, compared to those of conventional ICEs [2-4]. https://doi.org/10.1051/e3sconf/202131207006 E3S Web of Conferences 312, 07006 (2021) 76° Italian National Congress ATI This combustion mode merges the benefits of the two conventional engine combustion processes. Indeed, the presence of homogeneous air/fuel charge preparation, like SI combustion, removes the fuel-rich region, resulting in low smoke emission. The mixture burns in a controlled auto-ignition, like DI combustion, allowing the adoption of a lean air/fuel mixture and higher compression ratios with relevant improvements for fuel consumption. p However, HCCI combustion presents some drawbacks which should be overcome [4-8]. They include the lack of any direct control method for combustion phasing; the high production of HC and CO emissions; limitations in achieving high engine loads (knock issue); misfiring; and cold start problems. To overcome the above-listed issues, the HCCI concept is still under investigation and development with the aim to become a feasible and reliable alternative to conventional combustion engines. In this context, both 1D and 3D-CFD numerical approaches represent valuable tools to support the study and the improvement of HCCI. The engine modelling and simulation studies are applied to enhance the engine design, improve engine calibration, developing the optimal engine control strategies. The combustion process of conventional SI engines in 1D codes is successfully modelled employing a phenomenological model based on a two-zone schematization of the combustion chamber, namely the burned and unburned zone separated by a turbulent propagating flame [9,10]. Instead, since the HCCI engines are characterized by the absence of the flame front propagation, the combustion phenomenon is described through the evolution of the mixture composition in the cylinder via a detailed chemical kinetic mechanism [11-13]. For this reason, the most common HCCI modelling is based on a single zone approach [14- 16]. 1 Introduction However, this schematization is not able to accurately predict the combustion duration, the heat release rate, and the pressure rise rate since a unique auto-ignition event of all mixture instantaneously occurs. In this case, an over-prediction of the pressure and temperature peaks is obtained [17-23]. To overcome these limits, phenomenological multi-zone models have been developed, where the combustion chamber is subdivided into smaller volumes. The number of zones and their configurations are determined through a balance between the accuracy and the computational effort. Either two zone, the core and the boundary layer [24]) or many zones (up to thirty [25]) can be specified. The multi-zone schematization improves the prediction capabilities since it allows to compute a temperature gradient between the zones, ensuring a progressive ignition of the charge [26-39,49]. It is known that the thermal stratification before ignition is primarily developed by heat loss to the cylinder walls, with little contribution from mixing with internal residuals [35]. However, the multi-zone approach has got some issues: one of them is the computational time, due to the resolution of chemical kinetics in each zone. In order to reduce the computational effort in the HCCI combustion modelling, two approaches can be followed. The first one consists of simplifying the kinetic mechanism and finally use a scheme with few species. Because of the complex nature of hydrocarbon fuels, those simplifications typically produce misleading results. The second uncommon approach is the so-called Tabulated Kinetic of Ignition (TKI). It is based on the tabulation and interpolation methods, which record the essential parameters of an adiabatic reactor simulation in a database to be employed at runtime for the engine simulation [36]. [ ] In this framework, this study takes place focusing on the development of a multi-zone model, based on the TKI approach, to reproduce the combustion in an HCCI engine. 2 E3S Web of Conferences 312, 07006 (2021) 76° Italian National Congress ATI E3S Web of Conferences 312, 07006 (2021) 76° Italian National Congress ATI https://doi.org/10.1051/e3sconf/202131207006 The paper is arranged as follows. First, the balance equations for the multi-zone model have been described, to derive the temporal evolution of the thermodynamic properties during the engine cycle. Then, the adopted tabulated procedure is verified in a single-zone schematization, through the comparison with results obtained by a commercial software employing a chemical kinetic simulator, under various operating conditions. 1 Introduction Once validated, the model is utilized in its multi-zone version, and a sensitivity analysis is performed on the tuning parameters of the temperature stratification in the combustion chamber. Finally, the whole procedure is applied to reproduce the experimental data related to an HCCI engine fuelled with hydrogen. 2 Methodology In the study of HCCI engines, 0D multi-zone modelling presents many advantages over the 0D single-zone approach and the 3D CFD one. It must be noted that the 3D CFD model for HCCI combustion treats any cell as a homogeneous reactor and therefore it does not account for turbulence-chemistry interactions (TCI) [37-39]. Since the HCCI combustion is primarily controlled by chemistry, the 0D multi-zone model is an excellent option when a broad variety of parameters have to be modified, thanks to the low computational cost. There are two macro-categories of quasi-dimensional multi-zone models: Eulerian control- volume and Lagrangian control-mass approaches. Both assume that the pressure in the combustion chamber is uniform, and both require a correlation for the overall heat transfer with the combustion chamber wall, such as the Hohenberg correlation. In the Eulerian control-volume model [29-32], the knowledge of the relative zonal size and position is assumed. Since the pressure is uniform, the mass that crosses the zonal boundaries is calculated using the ideal gas law. Instead, the Lagrangian model [33-39] is developed on a control mass basis (each zone has a specified constant mass) and no information is given on zone’s position inside the combustion chamber. Here, the ideal gas law allows the computation of the zonal volume. The model proposed in this paper is based on the latter framework. It allows allocating a mass fraction quantity in each zones, based, for example, on a normal distribution, as reported in Fig. 1. This distribution assumes an almost constant level for very large values of the standard deviation, 𝜎𝜎. Fig. 1: The effect of the standard deviation of the Gaussian distribution on the zonal mass fraction distribution, in the case of a 21-zone schematization. Fig. 1: The effect of the standard deviation of the Gaussian distribution on the zonal mass fraction distribution, in the case of a 21-zone schematization. 3 https://doi.org/10.1051/e3sconf/202131207006 E3S Web of Conferences 312, 07006 (2021) 76° Italian National Congress ATI Further, in the Eulerian model the heat transfer to the walls only occurs in the thermal boundary layers (outer zone) and the heat is transferred between the inner zones through interzonal heat transfers. Since there are multiple mechanisms involved in this model, such as the diffusion and convective exchanges, several empirical correlations must be applied. This tuning condition severely limits the implementation and predictability of Eulerian models, although this model appears to be closer to reality. 2 Methodology The Lagrangian approach has been conceived to avoid the specification of interzonal mass and heat transfer. However, it still requires tuning for the assigning a predefined zone-by- zone heat transfer fraction. Indeed, the thermal stratification is achieved by setting different fractions of the total cylinder's heat transfer to each zone, following the rule that the charge closer to the boundaries (typically outer regions and boundary layer) loses more heat than the charge further away (typically inner regions). A fully adiabatic inner zone could be also specified. The above heat transfer fraction distribution can be derived by experiments or 3D-CFD outcomes and was verified to be generalized to a broad variety of operating conditions [35,36]. Fig. 2a depicts such a distribution of heat transfer fraction per unit mass versus the cumulative mass fraction (CMF). With this curve, each infinitesimal mass layer, starting from the core of the combustion chamber (CMF = 0) to its walls (CMF = 1), exchanges a certain amount of the overall heat to create the desired thermal stratification in the combustion chamber. For consistency, the integral of this curve must be equal to 1. Fig. 2: a) The distribution of heat transfer fraction per unit mass versus the cumulative mass fraction (CMF), in the figure are indicated the positions of the 1𝑠𝑠𝑠𝑠,10𝑡𝑡ℎ,𝑎𝑎𝑎𝑎𝑎𝑎 20𝑡𝑡ℎ zones; b) Zonal Heat Transfer Fraction, resulting from the above distribution and constant zonal mass fraction distribution. The continuous curve versus the CMF in Fig. 2a cannot be directly used during the simulation, since the model needs a discrete distribution based on the number of zones and zonal mass distribution. Once the number of zones and the zonal mass fraction distribution are known, the heat transfer fraction for a specific zone, called 𝑥𝑥𝑤𝑤,𝑧𝑧, can be easily derived by integrating the curve in Fig. 2a in the appropriate interval. Fig. 2b depicts the integration results for each zone. Although no spatial information is required, the first zone is presumably located in the core of the combustion chamber, while the last one is adjacent to the cylinder wall, as the schematization of the boundary layer. Fig. 2: a) The distribution of heat transfer fraction per unit mass versus the cumulative mass fraction (CMF), in the figure are indicated the positions of the 1𝑠𝑠𝑠𝑠,10𝑡𝑡ℎ,𝑎𝑎𝑎𝑎𝑎𝑎 20𝑡𝑡ℎ zones; b) Zonal Heat Transfer Fraction, resulting from the above distribution and constant zonal mass fraction distribution. Fig. 2 Methodology 2: a) The distribution of heat transfer fraction per unit mass versus the cumulative mass fraction (CMF), in the figure are indicated the positions of the 1𝑠𝑠𝑠𝑠,10𝑡𝑡ℎ,𝑎𝑎𝑎𝑎𝑎𝑎 20𝑡𝑡ℎ zones; b) Zonal Heat Transfer Fraction, resulting from the above distribution and constant zonal mass fraction distribution. The continuous curve versus the CMF in Fig. 2a cannot be directly used during the simulation, since the model needs a discrete distribution based on the number of zones and zonal mass distribution. 𝑥 The continuous curve versus the CMF in Fig. 2a cannot be directly used during the simulation, since the model needs a discrete distribution based on the number of zones and zonal mass distribution. 𝑥 Once the number of zones and the zonal mass fraction distribution are known, the heat transfer fraction for a specific zone, called 𝑥𝑥𝑤𝑤,𝑧𝑧, can be easily derived by integrating the curve in Fig. 2a in the appropriate interval. Fig. 2b depicts the integration results for each zone. Although no spatial information is required, the first zone is presumably located in the core of the combustion chamber, while the last one is adjacent to the cylinder wall, as the schematization of the boundary layer. 4 4 https://doi.org/10.1051/e3sconf/202131207006 E3S Web of Conferences 312, 07006 (2021) 76° Italian National Congress ATI Table 1 summarizes a comparison between the described approaches. Table 1. Comparison between Eulerian and Lagrangian multi-zone models [36]. Eulerian model (Control volume, “onion skin type”) Lagrangian model (Control mass, “balloon” type”) Knowledge of the zonal position Required Not Required Interzonal mass/heat transfer correlations Required Not Required Balance the interzonal pressure gradient Mass transfer from the higher to lower pressure zones The higher pressure zones expand and the lower pressure contract Heat Transfer Considered for the outer zone. This model also requires a distinct correlation for the interzonal heat transfer Different zones have different heat transfer fraction, which reflects different potential positions in the cylinder Table 1 summarizes a comparison between the described approaches. Table 1. Comparison between Eulerian and Lagrangian multi-zone models [36]. 2.1 Lagrangian Multi-zone model: balance equations The proposed HCCI model is implemented in the commercial software GT-Power through user coding, and it is utilized during the closed valve phase for engine simulations. The multi-zone model switch to single-zone model during the open-valve period, coupled to the 1D resolution of the intake/exhaust pipes. The calculation of the initial conditions at Inlet Valve Closure (IVC) is provided by the 1D flow model at each engine cycle. Starting from the initial conditions at IVC, the average properties and the composition of each zone (pressure, temperature, and gas composition) can be initialized. Furthermore, an initial temperature stratification among the zones can be also defined, maintaining the mean energy content of the cylinder. gy y For the considered model, several assumptions are used: gy y For the considered model, several assumptions are used: gy y For the considered model, several assumptions are used: 1) each specie in the combustion chamber is treated as an ideal gas, taking into account the variability of gas properties; 2) since no interzonal mass transfer is allowed in the Lagrangian approach, the mass fraction of each zone remains constant during the simulation; 3) blow-by and crevices effects are neglected. 3) blow-by and crevices effects are neglected. The zonal energy evolves during time due to liquid species evaporation, combustion, heat transfer to the wall, and interzonal work exchange (i.e., work done by one zone on another). The zonal composition, on the other hand, only varies due to the auto-ignition process. Eq. (1) depicts the first law of thermodynamics for each zone. 𝑑𝐸𝑧𝑝𝑑𝑉𝑧𝛿𝑄𝑊𝑧𝛿𝑄𝑉𝑉𝑉𝑧 The zonal energy evolves during time due to liquid species evaporation, combustion, heat transfer to the wall, and interzonal work exchange (i.e., work done by one zone on another). The zonal composition, on the other hand, only varies due to the auto-ignition process. Eq. (1) depicts the first law of thermodynamics for each zone. 𝑑𝐸𝑧𝑝𝑑𝑉𝑧𝛿𝑄𝑊𝑧𝛿𝑄𝑉𝑉𝑉𝑧 𝑑𝑑𝐸𝐸𝑧𝑧 𝑑𝑑𝑑𝑑= −𝑝𝑝 𝑑𝑑𝑉𝑉𝑧𝑧 𝑑𝑑𝑑𝑑− 𝛿𝛿𝑄𝑄𝑊𝑊,𝑧𝑧 𝑑𝑑𝑑𝑑 − 𝛿𝛿𝑄𝑄𝑉𝑉𝑉𝑉𝑉𝑉,𝑧𝑧 𝑑𝑑𝑑𝑑 (1) (1) where 𝐸𝐸𝑧𝑧= 𝑚𝑚𝑧𝑧𝑒𝑒𝑧𝑧= 𝑚𝑚𝑧𝑧∑ 𝑥𝑥𝑖𝑖,𝑧𝑧𝑒𝑒𝑖𝑖,𝑧𝑧(𝑇𝑇𝑧𝑧) 𝑁𝑁𝑠𝑠𝑠𝑠𝑠𝑠 𝑖𝑖 , with 𝑒𝑒𝑖𝑖,𝑧𝑧 and 𝑥𝑥𝑖𝑖,𝑧𝑧 are the specific internal energy and the mass fraction of species i and zone z. ( 𝛿𝛿𝑄𝑄𝑊𝑊,𝑧𝑧 𝑑𝑑𝑑𝑑) is the heat transfer to the combustion chamber walls and ( 𝛿𝛿𝑄𝑄𝑉𝑉𝑉𝑉𝑉𝑉,𝑧𝑧 𝑑𝑑𝑑𝑑 ) is the heat loss due to the evaporation, in the case of liquid fuel injection. j . (1) can be rearranged to get temperature evaluation of each zone: 𝑑𝑇𝑧𝑑𝑑𝑥𝑤𝑧𝛿𝑄𝑊𝛿𝑄𝑉𝑉𝑉𝑧𝑑𝑑𝑖𝑧𝑖𝑧𝑇𝑧𝑁𝑠𝑠𝑠𝑖 j Eq. 2.1 Lagrangian Multi-zone model: balance equations (1) can be rearranged to get temperature evaluation of each zone: 𝑑𝑇𝑧𝑑𝑑𝜌𝑐𝑑𝑑𝑑𝑑𝑚𝑐𝑥𝑤𝑧𝛿𝑄𝑊𝑑𝑑𝛿𝑄𝑉𝑉𝑉𝑧𝑑𝑑𝑐𝑑𝑁𝑠𝑠𝑠𝑖 𝑑𝑑𝑇𝑇𝑧𝑧 𝑑𝑑𝑑𝑑= − 1 𝜌𝜌𝑧𝑧𝑐𝑐𝑝𝑝,𝑧𝑧 𝑑𝑑𝑑𝑑 𝑑𝑑𝑑𝑑 − 1 𝑚𝑚𝑧𝑧𝑐𝑐𝑝𝑝,𝑧𝑧 (𝑥𝑥𝑤𝑤,𝑧𝑧 𝛿𝛿𝑄𝑄𝑊𝑊 𝑑𝑑𝑑𝑑+ 𝛿𝛿𝑄𝑄𝑉𝑉𝑉𝑉𝑉𝑉,𝑧𝑧 𝑑𝑑𝑑𝑑 ) − 1 𝑐𝑐𝑝𝑝,𝑧𝑧 (∑ 𝑑𝑑𝑑𝑑𝑖𝑖,𝑧𝑧 𝑑𝑑𝑑𝑑ℎ𝑖𝑖,𝑧𝑧(𝑇𝑇𝑧𝑧) 𝑁𝑁𝑠𝑠𝑠𝑠𝑠𝑠 𝑖𝑖 ) (2) 5 https://doi.org/10.1051/e3sconf/202131207006 E3S Web of Conferences 312, 07006 (2021) 76° Italian National Congress ATI The calculation of the pressure derivative ( 𝑑𝑑𝑑𝑑 𝑑𝑑𝑑𝑑) can be obtained by applying the equation of state (𝑝𝑝𝑝𝑝= ∑ 𝑚𝑚𝑧𝑧𝑅𝑅𝑧𝑧𝑇𝑇𝑧𝑧 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑧𝑧 ), furnishing: 𝑑𝑑𝑝𝑑𝑑𝑑𝑑𝜌𝑧𝑐𝑚𝑑𝑑𝑖𝑧𝑑𝑑𝑇𝑁𝑠𝑠𝑠𝑥𝛿𝑄𝑊𝑑𝑑𝛿𝑄𝑉𝑉𝑉𝑧𝑑𝑑𝑁𝑁𝑁𝑁𝑁 𝑑𝑑𝑑𝑑 𝑑𝑑𝑑𝑑= −𝑝𝑝𝑑𝑑𝑑𝑑 𝑑𝑑𝑑𝑑−∑ [ 𝜌𝜌𝑧𝑧 𝑐𝑐𝑝𝑝,𝑧𝑧(𝑚𝑚𝑧𝑧∑ 𝑑𝑑𝑑𝑑𝑖𝑖,𝑧𝑧 𝑑𝑑𝑑𝑑ℎ𝑖𝑖,𝑧𝑧(𝑇𝑇𝑢𝑢) 𝑁𝑁𝑠𝑠𝑠𝑠𝑠𝑠 𝑖𝑖 + 𝑥𝑥𝑤𝑤,𝑧𝑧𝛿𝛿𝑄𝑄𝑊𝑊 𝑑𝑑𝑑𝑑+ 𝛿𝛿𝑄𝑄𝑉𝑉𝑉𝑉𝑉𝑉,𝑧𝑧 𝑑𝑑𝑑𝑑 )] 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑧𝑧 𝑉𝑉−∑ ( 𝜌𝜌𝑧𝑧 𝑐𝑐𝑝𝑝,𝑧𝑧𝑉𝑉𝑧𝑧) 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑧𝑧 (3) 𝛿𝑄𝑊𝑑𝑑 (3) The term 𝛿𝛿𝑄𝑄𝑊𝑊 𝑑𝑑𝑑𝑑 is the overall heat transfer and is calculated using the Hohenberg correlation [40], used with the average cylinder temperature. Then, it is subdivided into zones through the coefficient 𝑥𝑥𝑤𝑤,𝑧𝑧, computed as specified in Fig. 2b. Moreover, if the engine is fuelled with gaseous fuel, the evaporation heat is zero. The last term in Eq. (2) takes into account the energy released by the variation in the composition of the species within the zone related to the auto-ignition event. composition of the species within the zone related to the auto-ignition event. For any specified condition, the following equation must be solved for each chemical specie: 𝑑𝑑𝑖𝑧𝑑𝑑𝑀𝑖𝜌𝜔 p p g For any specified condition, the following equation must be solved for each chemical specie: 𝑑𝑑𝑖𝑧𝑑𝑑𝑀𝑖𝜌𝜔 𝑑𝑑𝑑𝑑𝑖𝑖,𝑧𝑧 𝑑𝑑𝑑𝑑= 𝑀𝑀𝑖𝑖 𝜌𝜌𝑧𝑧 𝜔𝜔̇ 𝑖𝑖,𝑧𝑧 (4) 𝜌𝑧𝜔𝑖𝑧 (4) 𝑀𝑀𝑖𝑖 being the molar mass of species i, 𝜌𝜌𝑧𝑧 being the density of the zone z, and 𝜔𝜔̇ 𝑖𝑖,𝑧𝑧 being the molar consumption/production rate of specie i per unit volume [𝑚𝑚𝑚𝑚𝑚𝑚 𝑚𝑚3𝑠𝑠] during combustion. Typically, this term is provided by the solution of a chemical kinetic scheme at each time step and for each zone. 𝑀𝑀𝑖𝑖 being the molar mass of species i, 𝜌𝜌𝑧𝑧 being the density of the zone z, and 𝜔𝜔̇ 𝑖𝑖,𝑧𝑧 being the molar consumption/production rate of specie i per unit volume [𝑚𝑚𝑚𝑚𝑚𝑚 𝑚𝑚3𝑠𝑠] during combustion. Typically, this term is provided by the solution of a chemical kinetic scheme at each time step and for each zone. As known, the complex chemical processes occurring inside an HCCI engine would require a large-scale mechanism, able to take into account both low and high temperature reactions. 2.1 Lagrangian Multi-zone model: balance equations For a typical hydrocarbon fuel, such as gasoline, more than hundred species are usually required and, in this case, the computational time becomes too long for a practical application. Thus, the multi-zone chemical kinetics models offer no substantial advantages over the CFD ones. Furthermore, the detailed knowledge of species concentrations in a single zone is not mandatory; indeed, only the development of the most important species (fuel, O2, N2, CO2, H2O) is required for the computation of the zonal heat release. For these reasons, a methodology, known as Tabulated Kinetic of Ignition - TKI [41-47], is designed to perform an accurate and fast prediction of the air/fuel mixture auto-ignition. The TKI technique will be briefly described and tested in the next section. 3 Description and validation of TKI As mentioned, the TKI approach aims to describe the heat release due to the auto-ignition process based on the results obtained from a priori simpler chemistry calculations. The burn rate prediction in engine simulation can be achieved through this approach by exploiting a phenomenon called Burn Rate Equality [36]. According to this property, the burn rate of an auto-ignition combustion engine, at each time step, equals the burn rate of a homogeneous adiabatic constant-volume (CV) or constant-pressure (CP) reactor with the appropriate initial pressure, temperature, composition, and the same combustion progress variable [36]. One of the challenges in the TKI approach is the definition of the combustion progress variable, which defines the location of the engine chemical state between the initial pre- 6 6 6 E3S Web of Conferences 312, 07006 (2021) 76° Italian National Congress ATI https://doi.org/10.1051/e3sconf/202131207006 76° Italian National Congress ATI reaction state and the final equilibrium one. It is used to find a similar and unique state between the engine simulation and the adiabatic reactor one. reaction state and the final equilibrium one. It is used to find a similar and unique state between the engine simulation and the adiabatic reactor one. reaction state and the final equilibrium one. It is used to find a similar and unique state between the engine simulation and the adiabatic reactor one. Indeed, the progress variable definition has been widely explored in literature works [41- between the engine simulation and the adiabatic reactor one. Indeed, the progress variable definition has been widely explored in literature works [41- 47]. Typically, it is based on the time evolution of species concentration (CO2, CO, O2, etc.), formation enthalpy or temperature. I hi k h d fi i i f h i bl d b L h i i i l [45] h Indeed, the progress variable definition has been widely explored in literature works [41- 47]. Typically, it is based on the time evolution of species concentration (CO2, CO, O2, etc.), formation enthalpy or temperature. In this work, the definition of the progress variable proposed by Lehtiniemi et al. [45] has been adopted. It defines the c variable as a non-dimensional heat released by combustion, calculated as: 𝑐𝑓𝑡𝑓𝑒𝑒𝑒 𝑐𝑐= ℎ𝑓𝑓(𝑡𝑡) −ℎ𝑓𝑓(0) ℎ𝑓𝑓(𝑒𝑒𝑒𝑒𝑒𝑒) −ℎ𝑓𝑓(0) (5) (5) ℎ𝑓𝑓 being the reactor enthalpy of formation at reference temperature (298 K), ℎ298. 3 Description and validation of TKI The ℎ𝑓𝑓(0) and ℎ𝑓𝑓(𝑒𝑒𝑒𝑒𝑒𝑒) represent the initial and the final values of reactor enthalpy of formation, respectively. Each point in the thermochemical state space is uniquely characterize by the definition of ℎ𝑓𝑓: 𝑡𝑥𝑡𝑁𝑁𝑁𝑁 ℎ𝑓𝑓(𝑡𝑡) = ∑ℎ298,𝑖𝑖𝑥𝑥𝑖𝑖(𝑡𝑡) 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑖𝑖=1 (6) (6) Preliminary, a database for engine simulations is built from the solution of the chemistry in the constant-volume or constant-pressure reactors with specified initial conditions. At the end of each reactor calculation, progress variable reaction rates (𝑐𝑐̇) are stored as a function of the discrete values of the normalized progress variable c. The initial conditions in the reactor simulation are selected to replicate all the possible in- cylinder conditions. They are defined based on two parameters describing the thermodynamic state (initial pressure, 𝑝𝑝0, and fresh gas temperature, 𝑇𝑇0) and two variables defining the mixture composition (fuel/air equivalence ratio, 𝜙𝜙 , and EGR fraction, 𝑥𝑥𝐸𝐸𝐸𝐸𝐸𝐸). For each parameter, a variation range for the initial conditions is selected to properly match the local sensitivity of chemistry to that parameter. Table 2 shows the number of tabulated values, and the maximum and minimum levels for each parameter in the look-up tables. The initial conditions in the reactor simulation are selected to replicate all the possible in- cylinder conditions. They are defined based on two parameters describing the thermodynamic state (initial pressure, 𝑝𝑝0, and fresh gas temperature, 𝑇𝑇0) and two variables defining the mixture composition (fuel/air equivalence ratio, 𝜙𝜙 , and EGR fraction, 𝑥𝑥𝐸𝐸𝐸𝐸𝐸𝐸). thermodynamic state (initial pressure, 𝑝𝑝0, and fresh gas temperature, 𝑇𝑇0) and two variables defining the mixture composition (fuel/air equivalence ratio, 𝜙𝜙 , and EGR fraction, 𝑥𝑥𝐸𝐸𝐸𝐸𝐸𝐸). For each parameter, a variation range for the initial conditions is selected to properly match the local sensitivity of chemistry to that parameter. Table 2 shows the number of tabulated values, and the maximum and minimum levels for each parameter in the look-up tables. For each parameter, a variation range for the initial conditions is selected to properly match the local sensitivity of chemistry to that parameter. Table 2 shows the number of tabulated values, and the maximum and minimum levels for each parameter in the look-up tables. Table 2. 3 Description and validation of TKI Extreme levels and number of division points of the auto-ignition look-up table 𝒑 Minimum Maximum Number of points 𝒑𝒑𝟎𝟎, bar 5 165 15 𝑻𝑻𝟎𝟎, K 600 1210 21 𝝓𝝓, − 0.2 2 13 𝒙𝒙𝑬𝑬𝑬𝑬𝑬𝑬, − 0 0.6 11 𝑝𝑇𝜙𝑥 At each time step of the simulation, the solver, based on thermodynamic engine state and the table used (CP or CV), identifies the appropriate initial properties (𝑝𝑝0, 𝑇𝑇0, 𝜙𝜙 and 𝑥𝑥𝐸𝐸𝐸𝐸𝐸𝐸), and inquires a specific row of the table. Here, the value of tabulated c closest to the engine simulation one is identified and the corresponding 𝑐𝑐 ̇ value is retrieved from the table, to integrate the progress variable in each zone. This datum is also used in the energy balance equation to calculate the amount of heat released by the on-going auto-ignition (AI) process, as a consequence of the species composition variation. During a time step, a fraction of fuel in the specified zone, calculated by 𝑑𝑑𝑑𝑑𝑖𝑖,𝑧𝑧 𝑑𝑑𝑑𝑑∝𝜔𝜔̇ 𝑖𝑖,𝑧𝑧∝ 𝑐𝑐̇𝑖𝑖,𝑧𝑧, is assumed to react with the air, leading to the formation of chemical equilibrium species in the in-cylinder mixture. 7 7 7 https://doi.org/10.1051/e3sconf/202131207006 E3S Web of Conferences 312, 07006 (2021) 76° Italian National Congress ATI To assess the reliability of the c integration and the TKI method, Fig. 3 reports the pressure trend of a single-zone model, related to four different cases at varying operating conditions (i.e., intake temperature and fuel/air equivalence ratio). To assess the reliability of the c integration and the TKI method, Fig. 3 reports the pressure trend of a single-zone model, related to four different cases at varying operating conditions (i.e., intake temperature and fuel/air equivalence ratio). These single zone simulations are performed to compare the online chemical kinetics, realized using a conventional chemical kinetics software, and the tabulated kinetics (TKI approach), either performed in a homogeneous adiabatic reactor at constant pressure or constant volume. The simulations are carried out considering an engine fuelled with hydrogen, modelled with Hong’s kinetic scheme [48], with 10 species and 20 reactions. Fig. 3: Comparison of the pressure results between the online chemical kinetics approach (the red line, “CHEMKIN”), the TKI approach (the blue and green lines, based, respectively, on the CP- reactor and CV-reactor), on different intake temperatures and relative fuel/air equivalence ratios. Fig. 3: Comparison of the pressure results between the online chemical kinetics approach (the red line, “CHEMKIN”), the TKI approach (the blue and green lines, based, respectively, on the CP- reactor and CV-reactor), on different intake temperatures and relative fuel/air equivalence ratios. In Fig. 3 a perfect agreement between the results of online chemical kinetics and TKI can be observed. These results highlight that the TKI-based HCCI model enables fast computations and accuracy comparable to one of the conventional chemical kinetics models. In Fig. 3 a perfect agreement between the results of online chemical kinetics and TKI can be observed. These results highlight that the TKI-based HCCI model enables fast computations and accuracy comparable to one of the conventional chemical kinetics models. Furthermore, the adopted tabulated approach, although characterized by a scheme of just 10 species and 20 reactions, is three times faster than the conventional chemical kinetics. This disparity would be much greater if a more detailed kinetic scheme is utilized, as, for example, occurring for a hydrocarbon fuel. p g y Although both CV and CP approaches can be successfully used with an appropriate interpolation, CP simulations are chosen for the remainder of the paper. 7 Conversely to traditional chemical kinetics methods, TKI model is characterized by three different benefits. First, the numerical method is non-stiff, which makes the simulation much faster. Second, the simulation time for the pre-generated data is much faster than solving the chemical kinetics in the engine simulation. Third, a more accurate and detailed chemical kinetics mechanisms can be used to generate the relative tables, without impacting the computational effort, even if the table build-up will be more time demanding. 8 8 E3S Web of Conferences 312, 07006 (2021) 76° Italian National Congress ATI https://doi.org/10.1051/e3sconf/202131207006 4 Sensitivity analysis on the model tuning parameters In this paragraph, a sensitivity analysis is performed to evaluate and quantify the impact of tuning parameters on the multi-zone HCCI model. These results will be employed during the model calibration to reproduce the experimental data, as deeply treated in the following paragraph. The tuning parameters of the proposed multi-zone model are the number of zones; the zonal mass fraction distribution; the profile for the heat transfer fraction distribution (Fig. 2a); and initial temperature distribution between the various zones. The analyses are performed at a fixed engine speed of 1500 rpm, an intake pressure of 1 bar, an intake temperature of 383 K, and a fuel/air equivalence ratio of 0.3. Except otherwise specified, the set of tuning parameters is selected as listed in the second column of Table 3, while the third column describes the values used in the sensitivity analyses. Table 3. The set of tuning parameters in the sensitivity analyses. Tuning Parameters Value Sensitivity value Number of zones 21 1 – 5 – 10 – 25 – 50 Mass Fraction Distribution Constant (𝜎𝜎= 10) 1 – 1.5 – 2.5 – 5 – 10 Heat Transfer Distribution Blue curve in Fig. 5 Six different profiles Temperature Stratification Absent 0 – 3 – 8 – 15 – 20 K between the hottest and coldest zone Table 3. The set of tuning parameters in the sensitivity analyses. The outcomes of the sensitivity study on the number of zones and the mass fraction distribution within the zones are reported in Fig. 4. In this figure, the pressure has been plotted shifted of 10 bar on the y-axis for each simulation to ensure a better representation. Fig. 4: The outcomes of the sensitivity study on the number of zones (a) and the zonal mass fraction distribution (b). The shifted pressure is used for a better graphic representation of the results. Fig. 4: The outcomes of the sensitivity study on the number of zones (a) and the zonal mass fraction distribution (b). The shifted pressure is used for a better graphic representation of the results. 9 9 E3S Web of Conferences 312, 07006 (2021) 76° Italian National Congress ATI https://doi.org/10.1051/e3sconf/202131207006 Fig. 4a displays the impact of a different number of zones. 4 Sensitivity analysis on the model tuning parameters Here, the single-zone model (red line) burns with a great delay compared to the multi-zone model (even with 5 zones), since the single-zone model does not take into account the presence of the adiabatic core of the combustion chamber. Furthermore, in the multi-zone model, the pressure rises generated by the instantaneous auto-ignition of the first zone leads to the auto-ignition of the other zones. This mechanism allows to take into account the combustion duration. A smoother pressure rise can be obtained by increasing the number of zones, even though a higher computational effort is required. The pressure traces between the 25- and 50-zones simulations are essentially indistinguishable. The impact of the zonal mass fraction distribution are plotted in Fig. 4b. In these simulations, the standard deviation of the reference Gaussian distribution for the mass fraction between zones has been varied as listed in Table 3, following the trends reported in Fig. 1. 𝜎𝑝 An increased mass fraction in the intermediate zones (small 𝜎𝜎 value) implies a corresponding higher ∆𝑝𝑝. Similar behaviour can be noted in the evolution of the progress variable, although the overall results are very close to each other. These simulations are carried out with just 5 zones, since the differences are even lower by increasing the number of zones. A further sensitivity analysis is performed on the distribution of heat transfer between the different zones and on the initial temperature stratification at IVC. Concerning the heat transfer parameters, six different profiles have been tested, some of which derived from the literature papers (namely, the profile 1 and 3 [35,36] in Fig. 5). The functional form of the heat transfer distribution mainly depends on engine geometry than on operating conditions. For the engine analysed in this work, no experimental or numerical data are available to derive a thermal stratification profile. Basing on the above consideration, the profiles 1 and 3 were parameterized to create additional trends plotted in Fig. 5, which will be used in the calibration with the experimental data. Fig. 5: Six different trends for the heat transfer fraction per unit mass versus the cumulative mass fraction. They derive from the parametrization of two trends obtained from the paper [35, 36]. Fig. 5: Six different trends for the heat transfer fraction per unit mass versus the cumulative mass fraction. They derive from the parametrization of two trends obtained from the paper [35, 36]. Fig. 4 Sensitivity analysis on the model tuning parameters 5: Six different trends for the heat transfer fraction per unit mass versus the cumulative mass fraction. They derive from the parametrization of two trends obtained from the paper [35, 36]. 10 E3S Web of Conferences 312, 07006 (2021) 76° Italian National Congress ATI E3S Web of Conferences 312, 07006 (2021) https://doi.org/10.1051/e3sconf/202131207006 76° Italian National Congress ATI Fig. 6: The outcomes of the sensitivity study on the heat transfer fraction distributions (a) and the initial zonal temperature gradient (b). Fig. 6: The outcomes of the sensitivity study on the heat transfer fraction distributions (a) and the initial zonal temperature gradient (b). Fig. 6a reports the results of the application of the above defined profiles. With the yellow trend (#1), most of the heat transfer is located at the cylinder periphery and a quasi- adiabatic inner zone arises with an early auto-ignition. On the contrary, the magenta trend (#6) corresponds, as expected, to a single-zone model, since all zones evolve with the same temperature. p In Fig. 6b, a linear temperature distribution is imposed at IVC between the first and the last zone. The results of this analysis are similar to the ones of the heat transfer distribution. Once the initial temperature stratification is increased, the combustion start is advanced and its duration increases, as a consequence of the wider thermal stratification existing in the combustion chamber. In Fig. 6b, a linear temperature distribution is imposed at IVC between the first and the last zone. The results of this analysis are similar to the ones of the heat transfer distribution. Once the initial temperature stratification is increased, the combustion start is advanced and its duration increases, as a consequence of the wider thermal stratification existing in the combustion chamber. After understanding the effects of these parameters and selecting the actual degrees of freedom of the model, a comparison of the outcomes of the multi-zone model and the experimental data can be attempted, as reported in the following section. 5 Model Validation Here, the validation of the proposed model through the comparison with literature-derived experimental outcomes is presented. In particular, the experimental results reported by Ibrahim et al. [50] are considered. The adopted engine is a single-cylinder, four-stroke, water-cooled, naturally aspirated unit fuelled with hydrogen through a direct injector (Engine manufacturer: Kirloskar, Model: AV1). Main engine characteristics are reported in Table 4. Table 4. Engine characteristics used in the experimental data. Compression Ratio 16 Bore and Stroke 80 mm – 110 mm Displacement Volume 533 𝑐𝑐𝑐𝑐3 Table 4. Engine characteristics used in the experimental data. Compression Ratio 16 Bore and Stroke 80 mm – 110 mm Displacement Volume 533 𝑐𝑐𝑐𝑐3 Table 4. Engine characteristics used in the experimental data. 11 E3S Web of Conferences 312, 07006 (2021) 76° Italian National Congress ATI https://doi.org/10.1051/e3sconf/202131207006 Model validation is proposed in terms of the numerical/experimental assessment for the in- cylinder pressure traces and the burn rate profiles. The latter are derived by the authors’ reverse procedure processing the measured pressure cycle. The analyses are carried out considering a single operating point, namely 1500 rpm, 2.2 bar IMEP, and with a fuel/air equivalence ratio of 0.24. Three different temperature levels at intake valve closing (IVC) were considered: 373 K, 383 K, and 393 K. The validation results are reported in Fig. 7, showing the numerical/experimental comparisons of in-cylinder pressure traces and burn rate profiles as a function of the engine crank angle by varying the intake temperature. g g y y g p To reproduce the experimental data, the model was set with 40 zones, a constant mass fraction distribution, the yellow curve in Fig. 5 for the heat transfer distribution, and an initial temperature stratification at IVC of 2 K. Fig. 7 shows a quite satisfactory agreement between the model outcomes and experimental data. Fig. 7: Numerical/experimental comparison of in-cylinder pressure traces and burn rate profiles at varying the intake temperature. Fig. 7: Numerical/experimental comparison of in-cylinder pressure traces and burn rate profiles at varying the intake temperature. Fig. 7: Numerical/experimental comparison of in-cylinder pressure traces and burn rate profiles at varying the intake temperature. The proposed multi-zone approach, considering temperature stratification in the combustion chamber, accurately predicts the engine characteristics in terms of combustion phasing and burn rate. 5 Model Validation 9: The zonal and bulk temperature (top) and the zonal and bulk combustion variable progress (bottom) for all considered cases in the experimental comparison Fig. 9: The zonal and bulk temperature (top) and the zonal and bulk combustion variable progress (bottom) for all considered cases in the experimental comparison From these figures, it can be noted that the pressure gradient, generated by the zone auto- ignition, is the only connection between the various zones. In particular, referring to the highest intake temperature of 393 K, Fig. 9a shows that the combustion occurs almost completely before the firing TDC, i.e., during the compression phase, and all zones burn close to each other. The close auto-ignition of the zones is due to the increase in pressure, and consequently in temperature, generated by the auto-ignitions of the previous zones. p Instead, at lower intake temperature (third case of 373 K), Fig. 9c reports a much longer overall oxidation since it takes place entirely in the expansion phase. Here, the pressure increase produced by the early burning zones is mitigated by the expansion; thus, the colder near-wall zones are not able to reach the auto-ignition conditions. 5 Model Validation Moreover, it provides reasonable estimations of the maximum pressure rise rate; this parameter allows the calculation of the Ringing Intensity, which is used to properly forecast the load limit of HCCI engines. A greater error occurs for the lower intake temperature of 373 K (green line in Fig. 7). This case is close to the auto-ignition threshold and, in this condition, the model is extremely sensitive to the mixture temperature. Indeed, a slight temperature difference causes a relevant variation in the auto-ignition timing. A greater error occurs for the lower intake temperature of 373 K (green line in Fig. 7). This case is close to the auto-ignition threshold and, in this condition, the model is extremely sensitive to the mixture temperature. Indeed, a slight temperature difference causes a relevant variation in the auto-ignition timing. The Fig. 8 shows the evolution of the overall combustion progress variable for the three considered cases. In each case, some unburned mixture is still present at EVO. None of the three trends reaches the unity, and, particularly, for the case with the intake temperature at 373 K around 20% of gases remains unburned. Thus, this model is able to predict the unburned hydrocarbons from unreacted unburned zones, although a more refined HC simulation also requires the characterization of the crevices flows and the post-oxidation. 12 https://doi.org/10.1051/e3sconf/202131207006 E3S Web of Conferences 312, 07006 (2021) 76° Italian National Congress ATI Fig. 8: Evolution of Combustion Progress Variable versus the engine crank angle at varying the intake temperature. Fig. 8: Evolution of Combustion Progress Variable versus the engine crank angle at varying the intake temperature. The observed incomplete combustion can be better clarified through the analysis of the temperature and zonal progress variable trends, reported in Fig. 9a-c. Depending on the actual zone temperature, different auto-ignition events occur zone-by- zone during time. However, in each operating condition, the very low temperature of the outer zones determines the absence or partial auto-ignition reactions, leaving some unburned hydrocarbons inside the combustion chamber. 13 https://doi.org/10.1051/e3sconf/202131207006 E3S Web of Conferences 312, 07006 (2021) 76° Italian National Congress ATI 76° Italian National Congress ATI Fig. 9: The zonal and bulk temperature (top) and the zonal and bulk combustion variable progress (bottom) for all considered cases in the experimental comparison Fig. 3. R. H. Stanglmaier, E. C. Roberts, SAE trans. (1999): 2138-2145. 6 Conclusion Th d l d t t d t bilit t d th b h i f HCCI Finally, the reliability of the HCCI multi-zone model has been proved through the comparisons with the experimental data in the case of an engine fuelled with hydrogen. It is worth underling that the comparison has been made by keeping constant the model tuning parameters at varying operating conditions. p y g p g The model demonstrates an adequate capability to reproduce the behaviour of an HCCI engine, also perceiving the effect of the intake temperature. It can be usefully employed to help the development of new engines featured by HCCI combustion. y g g The model demonstrates an adequate capability to reproduce the behaviour of an HCCI engine, also perceiving the effect of the intake temperature. It can be usefully employed to help the development of new engines featured by HCCI combustion. p p g y As the next developments of this model, the research should be conducted in these four areas: p p g y As the next developments of this model, the research should be conducted in these four areas: 1) carrying out more experiments in various operating conditions to extend the model validation. This will allow showing the ability of the model in predicting operations in a wide range of fuel/air and EGR ratios; 2) developing a more accurate model for the heat transfer distribution. Currently, the tuning parameters are not correlated with any physical phenomenon in the combustion chamber. Thanks to 3D CFD calculations or experimental data, these values could be correlated to some variables, including turbulence values, walls’ temperatures, the amount of EGR, etc; 3) calculating the NO emission and considering the blow-by and crevices effects for a better prediction of the HC and CO emissions; 4) extending the use of this model to other combustion strategies (SACI, RCCI), including the integration of this model with a conventional flame front propagation model. Definitions/Abbreviations ( ) 2. P. Duret, et al., SAE trans. (2004): 987-1001. Definitions/Abbreviations aTDC after Top Dead Centre BDC Bottom Dead Centre CAD Crank Angle Degree CFD Computational Fluid Dynamics CMF Cumulative Mass Fraction CP Constant-Pressure CV Constant-Volume DI Direct Injection EVO Exhaust Valve Opening HC Hydrocarbon unburned HCCI Homogeneous Charge Compression Ignition ICE Internal Combustion Engine IMEP Indicated Mean Effective Pressure IVC Intake Valve Closing PFI Port Fuel Injection TCI Turbulence-Chemistry Interactions TDC Top Dead Centre TKI Tabulated Kinetic of Ignition 6 Conclusion This work proposes and validates a phenomenological 0D multi-zone model, implemented as an “user routine” in the commercial software GT-POWER, for a fast and accurate prediction of HCCI combustion. The model is based on the control-mass Lagrangian approach and on the TKI method. The TKI approach gives some significant benefits, such as the possibility to operate with a good accuracy and a larger number of zones, preserving the computational effort. Primarily, the TKI methodology has been validated by comparing the results of single-zone simulation to the ones of the conventional chemical kinetic model. Then, a parametric analysis on the tuning parameters of the multi-zone model has been performed. The model is based on the control-mass Lagrangian approach and on the TKI method. The TKI approach gives some significant benefits, such as the possibility to operate with a good accuracy and a larger number of zones, preserving the computational effort. e TKI approach gives some significant benefits, such as the possibility to operate with a od accuracy and a larger number of zones, preserving the computational effort. Primarily, the TKI methodology has been validated by comparing the results of single-zone simulation to the ones of the conventional chemical kinetic model. Then, a parametric analysis on the tuning parameters of the multi-zone model has been performed. Here, the results show that it is required a larger number of zones for a smoother pressure trajectory, while the zonal mass distribution does not play a significant role. Furthermore, it 14 E3S Web of Conferences 312, 07006 (2021) 76° Italian National Congress ATI https://doi.org/10.1051/e3sconf/202131207006 is fundamental to describe the zonal heat transfer distribution and initial ∆𝑇𝑇𝐼𝐼𝐼𝐼𝐼𝐼, since the thermal stratification is crucial to forecast the burn rate profile of HCCI combustion. is fundamental to describe the zonal heat transfer distribution and initial ∆𝑇𝑇𝐼𝐼𝐼𝐼𝐼𝐼, since the thermal stratification is crucial to forecast the burn rate profile of HCCI combustion. is fundamental to describe the zonal heat transfer distribution and initial ∆𝑇𝑇𝐼𝐼𝐼𝐼𝐼𝐼, since the thermal stratification is crucial to forecast the burn rate profile of HCCI combustion. 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https://openalex.org/W4255151951	https://strathprints.strath.ac.uk/74694/1/Shiramizu_etal_POne_2020_Sexual_orientation_predicts_mens_preferences.pdf	English	null	Sexual orientation predicts men’s preferences for sexually dimorphic face-shape characteristics: a replication study.	null	2,020	cc-by	3,635	RESEARCH ARTICLE Sexual orientation predicts men’s preferences for sexually dimorphic face-shape characteristics: A replication study Victor ShiramizuID, Ciaran Docherty, Lisa M. DeBruine, Benedict C. Jones Institute of Neuroscience & Psychology, University of Glasgow, United Kingdom Victor ShiramizuID, Ciaran Docherty, Lisa M. DeBruine, Benedict C. Jones Institute of Neuroscience & Psychology, University of Glasgow, United Kingdom a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Editor: Tapio Mappes, University of Jyva¨skyla¨, FINLAND Together, these findings indicate the sexual orientation contributes to individual differences in men’s face preferences. Received: March 2, 2020 Accepted: October 29, 2020 Published: November 13, 2020 Published: November 13, 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.pone.0242262 OPEN ACCESS Citation: Shiramizu V, Docherty C, DeBruine LM, Jones BC (2020) Sexual orientation predicts men’s preferences for sexually dimorphic face-shape characteristics: A replication study. PLoS ONE 15(11): e0242262. https://doi.org/10.1371/journal. pone.0242262 Editor: Tapio Mappes, University of Jyva¨skyla¨, FINLAND Editor: Tapio Mappes, University of Jyva¨skyla¨, FINLAND Editor: Tapio Mappes, University of Jyva¨skyla¨, FINLAND PLOS ONE PLOS ONE RESEARCH ARTICLE Sexual orientation predicts men’s preferences for sexually dimorphic face-shape characteristics: A replication study RESEARCH ARTICLE Abstract Many researchers have proposed that straight men prefer women’s faces displaying femi- nine shape characteristics at least partly because mating with such women will produce healthier offspring. Although a prediction of this adaptation-for-mate-choice hypothesis is that straight men will show stronger preferences for feminized versus masculinized versions of women’s faces than will gay men, only one previous study has directly tested this predic- tion. Here we directly replicated that study by comparing 623 gay and 3163 straight men’s preferences for feminized versus masculinized versions of faces. Consistent with the adap- tation-for-mate-choice hypothesis of straight men’s femininity preferences, we found that straight men showed significantly stronger preferences for feminized female faces than did gay men. Consistent with previous research suggesting that gay men place a premium on masculinity in potential romantic partners, we also found that gay men showed significantly stronger preferences for masculinized versions of male faces than did straight men. Together, these findings indicate the sexual orientation contributes to individual differences in men’s face preferences Introduction Many studies of straight men’s face preferences have reported that straight men show strong preferences for female faces with pronounced feminine shape characteristics [1, 2]. These pref- erences for feminine female faces are widely assumed to at least partly reflect adaptations for mate choice [1, 3]. Specifically, straight men are thought to show strong preferences for femi- nized versions of women’s faces because mating with such women would produce healthier or more viable offspring [1, 3]. However, evidence that women displaying more feminine facial characteristics possess traits that would cause them to produce healthier, more viable offspring is mixed. For example, while some studies have reported that women with more feminine faces report better general health, such as less frequent colds and other illnesses [e.g. 4, 5], other studies have not found significant correlations between facial femininity and measures of women’s health [e.g. 6, 7]. Although these mixed results suggest the proposed link between women’s actual health and facial femininity, women with more feminine facial characteristics are perceived to be healthier and as likely to be better parents [2]. Copyright: © 2020 Shiramizu 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: The data used in this study are available at https://osf.io/cuqdz. Funding: This work was funded by a European Funding: This work was funded by a European Research Council (ERC) grant (KINSHIP - 647910) PLOS ONE \| https://doi.org/10.1371/journal.pone.0242262 November 13, 2020 1 / 6 PLOS ONE Sexual orientation and men’s face preferences awarded to LMD. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. If straight men show strong preferences for feminized versions of women’s faces because mating with such women would produce healthier or more viable offspring, one might reason- ably predict that gay men’s preferences for feminized versions of women’s faces would be weaker than those of straight men. Such results would complement those showing opposite- sex biases in straight participants’ face preferences, which have been widely interpreted as evi- dence that face preferences at least partly reflect adaptations for mate choice [1, 3]. Participants Participants for the online study, which was run at faceresearch.org, were 623 men (mean age = 26.4 years, SD = 7.41 years) who reported that their preferred sex of partner was male and 3163 straight men (mean age = 26.65 years, SD = 7.52 years) who reported that their pre- ferred sex of partner was female. No other exclusion or inclusion criteria were applied. 60% of participants reported their ethnicity as White, 20% opted not to report their ethnicity, 6% reported their ethnicity as West Asian, 5% reported their ethnicity as East Asian, and 2% reported their ethnicity as African. All other ethnicities accounted for <1% of our sample. All participants provided informed consent and all procedures were approved by the Psychology Ethics Committee (University of Glasgow). Introduction However, and perhaps surprisingly, only one study has directly tested this hypothesis. In a study of 311 gay men and 215 straight men, Glassenberg et al. [8] found that straight men did indeed show stronger preferences for feminized versus masculinized versions of women’s faces than did gay men. Interestingly, Glassenberg et al. [8] also found that gay men showed stronger preferences for masculinized versions of men’s faces than did straight men, consistent with the proposal that gay men place a premium on masculinity in romantic partners. Competing interests: The authors have declared that no competing interests exist. Many previously reported findings for individual differences in preferences for sexually dimorphic face shapes have not replicated well in recent large-scale studies [see, e.g., 9]. Consequently, we attempted a direct replication of Glassenberg et al. [8]. We compared 623 gay and 3163 straight men’s preferences for feminized versus masculinized versions of female and male faces. While Glassenberg et al. tested both men and women, our replication study focused on men’s face preferences. Following Glassenberg et al., we predicted that straight men would show stronger preferences for femininity in women’s faces than gay men did and that gay men would show stronger preferences for masculinity in men’s faces than straight men did. awarded to LMD. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. PLOS ONE \| https://doi.org/10.1371/journal.pone.0242262 November 13, 2020 Results The main effect of sexual orientation was sig- nificant and positive (estimate = 0.39, SE = 0.03, df = 88.8, t = 11.72, p<0.001), indicating that straight men generally showed stronger preferences for feminized versions of faces than did gay men (see Fig 1). The main effect of sex of face was significant and negative (estimate = -0.90, SE = 0.12, df = 39.2, t = -7.07, p<0.001), indicating that men generally showed stronger preferences for feminized versions of faces when judging the attractiveness of female than male faces (see Fig 1). The effect of participant age was not significant (estimate = 0.01, SE = 0.01, df = 226.6, t = 1.14, p = 0.252). The interaction between sex of face and sexual orien- tation was significant (estimate = 0.23, SE = 0.06, df = 68.1, t = 3.70 p<0.001) and is illustrated in Fig 1. Repeating this analysis without participant age as a covariate showed the same pattern of results (see robustness analysis reported at https://osf.io/cuqdz/). SE = 0.01, df = 226.6, t = 1.14, p = 0.252). The interaction between sex of face and sexual orien- tation was significant (estimate = 0.23, SE = 0.06, df = 68.1, t = 3.70 p<0.001) and is illustrated in Fig 1. Repeating this analysis without participant age as a covariate showed the same pattern of results (see robustness analysis reported at https://osf.io/cuqdz/). Next, we repeated the analysis described above separately for male and female faces and with sex of face removed from the model. Our analysis of female faces showed a significant effect of sexual orientation (estimate = 0.28, SE = 0.03, df = 53.9, t = 7.26, p<0.001), indicating that straight men showed stronger preferences for feminized versions of women’s faces than did gay men (see Fig 1). The effect of participant age was not significant (estimate = -0.01, SE = 0.01, df = 115.4, t = -0.58, p = 0.560). Our analysis of male faces showed a significant effect of sexual orientation (estimate = 0.51, SE = 0.05, df = 37.0, t = 9.89, p<0.001), indicating that gay men showed stronger preferences for masculinized versions of men’s faces than did straight men (see Fig 1). Stimuli Following previous studies of individual differences in women’s preferences for masculine faces [e.g., 9, we used prototype-based image transformations to objectively manipulate sexual dimorphism of 2D shape in face images. First, male and female prototype (i.e. average) faces were manufactured using established computer graphic methods that have been widely used in studies of face perception [10]. These prototypes were manufactured using face images of 20 young White male adults and 20 young White female adults, respectively. Next, 50% of the lin- ear differences in 2D shape between symmetrized versions of the male and female prototypes were added to or subtracted from face images of 20 young White male adults and 20 young White female adults. This process created masculinized and feminized versions of the individ- ual face images that differ in sexual dimorphism of 2D shape and that are matched in other regards. Stimuli are publicly available [11]. 2 / 6 PLOS ONE \| https://doi.org/10.1371/journal.pone.0242262 November 13, 2020 PLOS ONE Sexual orientation and men’s face preferences Procedure Participants were shown the 40 pairs of face images and were asked to choose the face in each pair that was more attractive. As in Glassenberg et al. [8], the specific instruction was “Which face do you consider more attractive?”. Participants also indicated the strength of these prefer- ences by choosing from the options ‘slightly more attractive’, ‘somewhat more attractive, ‘more attractive’, and ‘much more attractive’. The order in which pairs of faces were shown was fully randomized and the side of the screen on which any particular image was shown was also fully randomized. Responses were coded using a 0 (masculinized face judged as much more attractive than feminized face) to 7 (feminized face judged as much more attractive than masculinized face). These preference scores were centered on chance before being used in our analyses. Results Analyses were conducted using R v3.6.0 [12]. Data, analysis code, and full results output are publicly available at https://osf.io/cuqdz/. First, we analyzed preference scores using a mixed effect model using lmer and lmerTest [13, 14] with the factors sexual orientation (effect coded so that gay = -0.5 and straight = 0.5) and sex of face (effect coded so that female = -0.5 and male = 0.5), and the covariate participant age (centered and scaled on mean of sample). We included participant age as a covariate to control for possible effects of age on face preferences [15]. Random intercepts were included for participant and stimulus, with random slopes speci- fied maximally [16, 17]. We did not include the interaction between sexual orientation and participant age in our model. Results of this initial analysis are summarized in Fig 1. The intercept was significant and positive (estimate = 0.29, SE = 0.06, df = 39.8, t = 4.54, p<0.001), indicating that participants generally preferred feminized versions of faces. The main effect of sexual orientation was sig- nificant and positive (estimate = 0.39, SE = 0.03, df = 88.8, t = 11.72, p<0.001), indicating that straight men generally showed stronger preferences for feminized versions of faces than did gay men (see Fig 1). The main effect of sex of face was significant and negative (estimate = -0.90, SE = 0.12, df = 39.2, t = -7.07, p<0.001), indicating that men generally showed stronger preferences for feminized versions of faces when judging the attractiveness of female than male faces (see Fig 1). The effect of participant age was not significant (estimate = 0.01, SE = 0.01, df = 226.6, t = 1.14, p = 0.252). The interaction between sex of face and sexual orien- tation was significant (estimate = 0.23, SE = 0.06, df = 68.1, t = 3.70 p<0.001) and is illustrated in Fig 1. Repeating this analysis without participant age as a covariate showed the same pattern of results (see robustness analysis reported at https://osf.io/cuqdz/). Results of this initial analysis are summarized in Fig 1. The intercept was significant and positive (estimate = 0.29, SE = 0.06, df = 39.8, t = 4.54, p<0.001), indicating that participants generally preferred feminized versions of faces. PLOS ONE \| https://doi.org/10.1371/journal.pone.0242262 November 13, 2020 Results The effect of participant age was significant (estimate = 0.03, SE = 0.01, df = 107.0, t = 2.36, p = 0.019) and indicated that older men showed stronger prefer- ences for feminized male faces. SE = 0.01, df = 107.0, t = 2.36, p = 0.019) and indicated that older men showed stronger prefer- ences for feminized male faces. Preferences for femininity in women’s faces were significantly greater than chance when gay and straight men judged the attractiveness of women’s faces (both ps < .001). Preferences 3 / 6 PLOS ONE \| https://doi.org/10.1371/journal.pone.0242262 November 13, 2020 PLOS ONE Sexual orientation and men’s face preferences Fig 1. The interaction between sexual orientation and sex of face on femininity preference in our study. The box plots and distributions represent the average femininity score for each individual face. The box plots are showing the median, first and third quartile, and the minimum and maximum femininity score for gay (purple) and straight (yellow) participants. On the y-axis, zero equals chance. Fig 1. The interaction between sexual orientation and sex of face on femininity preference in our study. The box plots and distributions represent the average femininity score for each individual face. The box plots are showing the median, first and third quartile, and the minimum and maximum femininity score for gay (purple) and straight (yellow) participants. On the y-axis, zero equals chance. https://doi.org/10.1371/journal.pone.0242262.g001 for femininity in men’s faces were significantly lower than chance when gay men judged the attractiveness of men’s faces (p = .002), but did not differ significantly from chance when straight men judged the attractiveness of men’s faces (p = .28). for femininity in men’s faces were significantly lower than chance when gay men judged the attractiveness of men’s faces (p = .002), but did not differ significantly from chance when straight men judged the attractiveness of men’s faces (p = .28). PLOS ONE \| https://doi.org/10.1371/journal.pone.0242262 November 13, 2020 Discussion Our analyses showed that straight men demonstrated stronger preferences for feminized ver- sions of women’s faces than did gay men. This pattern of results replicates those reported by Glassenberg et al. [8]. This pattern of results is also consistent with the proposal that straight men’s strong preferences for women’s faces with pronounced feminine characteristics at least partly reflect mating-related motivations [1, 3]. Since women’s faces displaying feminine char- acteristics are generally ascribed prosocial traits [2], that both gay and straight men showed preferences for feminine version of women’s faces that were significantly greater than chance suggests that men’s preferences for feminine female faces might also partly reflect general pref- erences for prosocial associates. Our analyses also showed that gay men demonstrated stronger preferences for masculine men that did straight men. This pattern of results also replicates those reported by Glassenberg et al. [8]. This pattern of results is also consistent with other previous research suggesting that PLOS ONE \| https://doi.org/10.1371/journal.pone.0242262 November 13, 2020 4 / 6 PLOS ONE Sexual orientation and men’s face preferences gay men show stronger preferences for men described in vignettes as possessing masculine traits [18]. It is currently unclear why gay men place this premium on masculinity. A potential limitation of our study was the use of a forced-choice paradigm for assessing men’s preferences for sexually dimorphic face shapes. We used this paradigm in our study because it was the same as that used in the study by Glassenberg et al. [8] that we were attempt- ing to (and successfully did) replicate. However, some recent research suggests that forced- choice paradigms can produce qualitatively different patterns of results than other methods for assessing preferences for sexually dimorphism face-shape characteristics [19]. Establishing the extent to which the effects of sexual orientation on face preferences that we observed in the current study and that were also observed by Glassenberg et al. [8] generalize to other methods for assessing face preferences would be an important direction for future research. Since we only tested male participants, it is also not known whether Glassenberg et al’s findings for women’s face preferences would replicate. In conclusion, we found that straight men showed stronger preferences for feminized ver- sions of women’s faces than did gay men, consistent with an adaptation-for-mate-choice explanation of straight men’s preferences for feminine women. Discussion We also found that gay men showed stronger preferences for masculine men that did straight men. Together these results suggest that sexual orientation influences men’s face preferences. Author Contributions Conceptualization: Victor Shiramizu, Lisa M. DeBruine, Benedict C. Jones. onceptualization: Victor Shiramizu, Lisa M. DeBruine, Benedict C. Jones. Formal analysis: Victor Shiramizu, Ciaran Docherty, Lisa M. DeBruine. Formal analysis: Victor Shiramizu, Ciaran Docherty, Lisa M. DeBruine. Methodology: Victor Shiramizu, Lisa M. DeBruine, Benedict C. Jones. Resources: Lisa M. DeBruine, Benedict C. Jones. Supervision: Lisa M. DeBruine, Benedict C. Jones. Supervision: Lisa M. DeBruine, Benedict C. Jones. Writing – original draft: Victor Shiramizu, Ciaran Docherty, Lisa M. DeBruine, Benedict C. Jones. Writing – review & editing: Victor Shiramizu, Ciaran Docherty, Lisa M. DeBruine, Benedict C. Jones. PLOS ONE \| https://doi.org/10.1371/journal.pone.0242262 November 13, 2020 References 1. Little A. C., Jones B. C., & DeBruine L. M. (2011). Facial attractiveness: evolutionary based research. Philosophical Transactions of the Royal Society B: Biological Sciences, 366(1571), 1638–1659. https:// doi.org/10.1098/rstb.2010.0404 PMID: 21536551 2. Perrett D. I., Lee K. J., Penton-Voak I., Rowland D., Yoshikawa S., Burt D. M., et al. (1998). Effects of sexual dimorphism on facial attractiveness. Nature, 394(6696), 884–887. https://doi.org/10.1038/ 29772 PMID: 9732869 3. Thornhill R., & Gangestad S. W. (1999). Facial attractiveness. Trends in cognitive sciences, 3(12), 452–460. https://doi.org/10.1016/s1364-6613(99)01403-5 PMID: 10562724 4. Thornhill R., & Gangestad S. W. (2006). Facial sexual dimorphism, developmental stability, and suscep- tibility to disease in men and women. Evolution and Human Behavior, 27(2), 131–144. 5. Gray A. W., & Boothroyd L. G. (2012). Female facial appearance and health. Evolutionary Psychology, 10(1), 147470491201000108. PMID: 22833849 6. Cai Z., Hahn A. C., Zhang W., Holzleitner I. J., Lee A. J., DeBruine L. M., et al. (2019). No evidence that facial attractiveness, femininity, averageness, or coloration are cues to susceptibility to infectious ill- nesses in a university sample of young adult women. Evolution and Human Behavior, 40(2), 156–159. PLOS ONE \| https://doi.org/10.1371/journal.pone.0242262 November 13, 2020 5 / 6 PLOS ONE Sexual orientation and men’s face preferences 7. Rhodes G., Chan J., Zebrowitz L. A., & Simmons L. W. (2003). Does sexual dimorphism in human faces signal health?. Proceedings of the Royal Society of London. Series B: Biological Sciences, 270 (suppl_1), S93–S95. 8. Glassenberg A. N., Feinberg D. R., Jones B. C., Little A. C., & DeBruine L. M. (2010). Sex-dimorphic face shape preference in heterosexual and homosexual men and women. Archives of Sexual Behavior, 39(6), 1289–1296. https://doi.org/10.1007/s10508-009-9559-6 PMID: 19830539 9. Jones B. C., Hahn A. C., Fisher C. I., Wang H., Kandrik M., Han C., et al. (2018). No compelling evi- dence that preferences for facial masculinity track changes in women’s hormonal status. Psychological science, 29(6), 996–1005. 10. Tiddeman B., Burt M., & Perrett D. (2001). Prototyping and transforming facial textures for perception research. IEEE computer graphics and applications, 21(5), 42–50. 11. DeBruine L. M. & Jones B. C. (2017). Young Adult White Faces with Manipulated Versions. https://doi. org/10.6084/m9.figshare.4220517.v1 12. R Core Team. (2018). R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing. Retrieved from https://www.R-project.org/ 13. Bates D., Ma¨chler M., Bolker B., & Walker S. (2015). Fitting linear mixed-effects models using lme4. Journal of Statistical Software, 67(1). 14. PLOS ONE \| https://doi.org/10.1371/journal.pone.0242262 November 13, 2020 References Kuznetsova A., Brockhoff P. B., & Christensen R. H. B. (2017). lmerTest package: tests in linear mixed effects models. Journal of Statistical Software, 82(13). 15. Marcinkowska U. M., Dixson B. J., Kozlov M. V., Prasai K., & Rantala M. J. (2017). Men’s preferences for female facial femininity decline with age. The Journals of Gerontology: Series B, 72(1), 180–186. 16. Barr D. J. (2013). Random effects structure for testing interactions in linear mixed-effects models. Fron- tiers in Psychology, 4, 328. https://doi.org/10.3389/fpsyg.2013.00328 PMID: 23761778 17. Barr D. J., Levy R., Scheepers C., & Tily H. J. (2013). Random effects structure for confirmatory hypoth- esis testing: Keep it maximal. Journal of Memory and Language, 68(3), 255 278. https://doi.org/10. 1016/j.jml.2012.11.001 PMID: 24403724 18. Bailey J. M., Kim P. Y., Hills A., & Linsenmeier J. A. (1997). Butch, femme, or straight acting? Partner preferences of gay men and lesbians. Journal of personality and social psychology, 73(5), 960. 19. Jones A. L., & Jaeger B. (2019). Biological bases of beauty revisited: The effect of symmetry, average- ness, and sexual dimorphism on female facial attractiveness. Symmetry, 11(2), 279. 6 / 6
https://openalex.org/W2398092487	https://europepmc.org/articles/pmc5122388?pdf=render	English	null	The BRG1 chromatin remodeling enzyme links cancer cell metabolism and proliferation	Oncotarget	2,016	cc-by	8,552	The BRG1 chromatin remodeling enzyme links cancer cell metabolism and proliferation Qiong Wu1, Pasil Madany1, Jason R. Dobson1, Jake M. Schnabl1, Soni Sharma2, Tara C. Smith1 Andre J. van Wijnen3, Janet L. Stein4, Jane B. Lian4, Gary S. Stein4, Rohini Muthuswami2, Anthony N. Imbalzano1, Jeffrey A. Nickerson1 1Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, MA, USA 2School of Life Sciences, Jawaharlal Nehru University, New Delhi, Delhi, India 3Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA 4Department of Biochemistry and Vermont Cancer Center for Basic and Translational Research, University of Vermont College of Medicine, Burlington, WA, USA Correspondence to: Anthony N. Imbalzano, email: anthony.imbalzano@umassmed.edu Jeffrey A. Nickerson, email: jeffrey.nickerson@umassmed.edu Correspondence to: Anthony N. Imbalzano, email: anthony.imbalzano@umassmed.edu Jeffrey A. Nickerson, email: jeffrey.nickerson@umassmed.edu Keywords: breast cancer, metabolism, lipogenesis, gene regulation, BRG1 Keywords: breast cancer, metabolism, lipogenesis, gene regulation, BRG1 Received: January 07, 2016 Accepted: May 01, 2016 Published: May 20, 2016 Published: May 20, 2016 ABSTRACT Cancer cells reprogram cellular metabolism to meet the demands of growth. Identification of the regulatory machinery that regulates cancer-specific metabolic changes may open new avenues for anti-cancer therapeutics. The epigenetic regulator BRG1 is a catalytic ATPase for some mammalian SWI/SNF chromatin remodeling enzymes. BRG1 is a well-characterized tumor suppressor in some human cancers, but is frequently overexpressed without mutation in other cancers, including breast cancer. Here we demonstrate that BRG1 upregulates de novo lipogenesis and that this is crucial for cancer cell proliferation. Knockdown of BRG1 attenuates lipid synthesis by impairing the transcription of enzymes catalyzing fatty acid and lipid synthesis. Remarkably, exogenous addition of palmitate, the key intermediate in fatty acid synthesis, rescued the cancer cell proliferation defect caused by BRG1 knockdown. Our work suggests that targeting BRG1 to reduce lipid metabolism and, thereby, to reduce proliferation, has promise for epigenetic therapy in triple negative breast cancer. www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No. 25 The BRG1 chromatin remodeling enzyme links cancer cell metabolism and proliferation Qiong Wu1, Pasil Madany1, Jason R. Dobson1, Jake M. Schnabl1, Soni Sharma2, Tara C. Smith1 Andre J. van Wijnen3, Janet L. Stein4, Jane B. Lian4, Gary S. Stein4, Rohini Muthuswami2, Anthony N. Imbalzano1, Jeffrey A. Nickerson1 1Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, MA, USA 2School of Life Sciences, Jawaharlal Nehru University, New Delhi, Delhi, India 3Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA 4Department of Biochemistry and Vermont Cancer Center for Basic and Translational Research, University of Vermont Research Paper www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No. 25 The BRG1 chromatin remodeling enzyme links cancer cell metabolism and proliferation Qiong Wu1, Pasil Madany1, Jason R. Dobson1, Jake M. Schnabl1, Soni Sharma2, Tara C. Smith1 Andre J. van Wijnen3, Janet L. Stein4, Jane B. Lian4, Gary S. Stein4, Rohini Muthuswami2, Anthony N. Imbalzano1, Jeffrey A. Nickerson1 1Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, MA, USA 2School of Life Sciences, Jawaharlal Nehru University, New Delhi, Delhi, India 3Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA 4Department of Biochemistry and Vermont Cancer Center for Basic and Translational Research, University of Vermont Research Paper www.impactjournals.com/oncotarget/ INTRODUCTION Re-introducing BRG1 largely restored FASN and ACC expression, de novo lipid synthesis and cell proliferation. Supplementing the cell media with exogenous palmitate completely restored cell proliferation in BRG1 knockdown cells, thereby demonstrating a causal link between lipid synthesis and cancer cell proliferation and identifying a novel mechanism by which lipogenic signaling is crucial for cancer cell growth. BRG1 upregulates lipogenic gene expression in triple negative breast cancer cells The reduction in de novo lipid synthesis in BRG1 knockdown breast cancer cells was directly caused by reduction of lipogenic gene expression. De novo fatty- acid synthesis involves two key enzymes, ACC and FASN. ACC carboxylates acetyl-CoA to form malonyl-CoA, and the malonyl-CoA product is subsequently converted by FASN to palmitate, a precursor for longer-chain fatty acids [31]. As shown in Figure 2A, mRNA levels of ACC and FASN were down-regulated after BRG1 knockdown in breast cancer cells but not in non-tumorigenic MCF-10A mammary epithelial cells. The protein levels of these enzymes were also reduced by BRG1 knockdown in breast cancer cells, but not in non-tumorigenic MCF-10A mammary epithelial cells (Figure 2B). y p ( g ) The expression of other genes involved in the synthesis or metabolism of lipids was also regulated by BRG1 (Figure 2C). Elevated glucose catabolism produces an excess of the glycolytic end-product, pyruvate. Most excess pyruvate is converted to lactate, but some is converted to acetyl-CoA by ATP citrate lyase (ACLY) and can be used in de novo fatty-acid synthesis. Therefore, ACLY is the link between the metabolism of carbohydrates and the production of fatty acids and represents an important step in fatty acid biosynthesis [32]. Long- chain acyl CoA synthetase (ACSL) catalyzes the first step in fatty acid activation for intracellular metabolism by converting long-chain fatty acids into acyl-CoA thioesters [33, 34]. ACSL1 is the best-studied and the major ACSL isoform, is highly expressed in major energy-metabolizing tissues, and plays a key role in lipid biosynthesis and fatty acid degradation [35]. Lipin-1 (LPIN1) is a magnesium- dependent phosphatidic acid phosphohydrolase that catalyzes the penultimate step in triglyceride synthesis including the dephosphorylation of phosphatidic acid to yield diacylglycerol [36]. The expression of all these genes was decreased by BRG1 knockdown (Figure 2C). INTRODUCTION for fatty acid biosynthesis, are correlated with poor prognosis in breast cancer patients [1, 7]. Increases in both FASN expression and activity are observed early in oncogenesis and correlate with cancer progression, with FASN-overexpressing tumors exhibiting more aggressive phenotypes [1]. Chemical or RNAi-mediated inhibition of key enzymes involved in fatty acid synthesis, including FASN, ACC and ACLY, reduces cell proliferation, induces apoptosis of cancer cells and retards the growth of human tumors in mouse xenograft models [1, 9–13]. Upregulation of lipogenic genes and overall lipogenesis are hallmarks of cancer [1]. Depending on the tumor type, tumor cells synthesize up to 95% of saturated and mono-unsaturated fatty acids (FA) de novo despite sufficient exogenous supply [2]. Lipogenic enzymes such as fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC), and ATP citrate lyase (ACLY) that are involved in fatty acid biosynthesis and sterol regulatory element binding protein 1 (SREBP1), the master regulator of lipogenic gene expression, are overexpressed in a number of cancers including breast, prostate, ovarian, lung, and colon [3–6]. Several lines of evidence suggest that activation of the de novo fatty acid synthesis pathway is required for carcinogenesis [1, 7, 8]. For example, elevated levels of FASN, the major enzyme responsible Whereas various tumor types display increased endogenous fatty acid biosynthesis irrespective of extracellular lipid availability, most normal cells, even those with comparatively high proliferation rates, preferentially use dietary/exogenous lipids for synthesis of new structural lipids [1, 12]. We sought to investigate how lipogenic pathways are re-wired in cancer. Mammalian www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 38270 and HDQ-P1) that were treated with a previously validated pool of siRNAs targeting BRG1 [22, 27] (Figure 1G–1H). ADAADi (Active DNA-dependent ATPase A Domain inhibitor), a minor product generated by the bacterial APH (3′)-III enzyme that encodes for aminoglycoside resistance, inhibits the ATPase activity of the SWI2/SNF2 family of ATPases [28, 29] and increases the chemosensitivity of triple negative breast cancer cells to clinically relevant therapeutic drugs [30]. Pharmacological inhibition of the BRG1 ATPase domain by ADAADi in MDA-MB- 231cells also decreased de novo lipid synthesis (Figure 1I). Collectively, the data show a role for BRG1 in promoting de novo lipid synthesis in triple negative breast cancer cells. SWI/SNF complexes are evolutionarily conserved, multisubunit enzymes that mobilize nucleosomes and remodel chromatin using the energy of ATP hydrolysis [14–16]. INTRODUCTION These enzymes are important in DNA replication and repair, cell growth control, maintenance of pluripotency, and promotion of cell lineage differentiation. Increasing evidence supports an important role for human SWI/SNF enzyme subunits in cancer development [17, 18]. Meta-analyses of cancer genome-sequencing data estimates that nearly 20% of human cancers harbor mutations in one or more SWI/SNF genes [17–20]. We and others reported that knockdown of BRG1 reduces cell proliferation in both breast epithelial and cancer cells in vitro [21–23] and attenuates tumor growth in a xenograft model [21, 22]. However, the underlying mechanisms remained unknown. Here we report that BRG1 directly regulates triple negative breast cancer cell proliferation via regulation of lipogenic pathways. Knockdown of BRG1 decreased de novo lipid synthesis in breast cancer cells, but not in breast epithelial cells, with concomitant reduction in cell proliferation. BRG1 knockdown significantly reduced lipogenic gene expression. Chromatin immunoprecipitation analysis revealed that BRG1 was bound to sequences at lipogenic genes. Re-introducing BRG1 largely restored FASN and ACC expression, de novo lipid synthesis and cell proliferation. Supplementing the cell media with exogenous palmitate completely restored cell proliferation in BRG1 knockdown cells, thereby demonstrating a causal link between lipid synthesis and cancer cell proliferation and identifying a novel mechanism by which lipogenic signaling is crucial for cancer cell growth. SWI/SNF complexes are evolutionarily conserved, multisubunit enzymes that mobilize nucleosomes and remodel chromatin using the energy of ATP hydrolysis [14–16]. These enzymes are important in DNA replication and repair, cell growth control, maintenance of pluripotency, and promotion of cell lineage differentiation. Increasing evidence supports an important role for human SWI/SNF enzyme subunits in cancer development [17, 18]. Meta-analyses of cancer genome-sequencing data estimates that nearly 20% of human cancers harbor mutations in one or more SWI/SNF genes [17–20]. We and others reported that knockdown of BRG1 reduces cell proliferation in both breast epithelial and cancer cells in vitro [21–23] and attenuates tumor growth in a xenograft model [21, 22]. However, the underlying mechanisms remained unknown. Here we report that BRG1 directly regulates triple negative breast cancer cell proliferation via regulation of lipogenic pathways. Knockdown of BRG1 decreased de novo lipid synthesis in breast cancer cells, but not in breast epithelial cells, with concomitant reduction in cell proliferation. BRG1 knockdown significantly reduced lipogenic gene expression. Chromatin immunoprecipitation analysis revealed that BRG1 was bound to sequences at lipogenic genes. Reduction of BRG1 in cancer cells attenuated de novo lipid synthesis One of the most conserved features of all cancers is the reprogramming of cellular metabolism in favor of biosynthetic processes that support high proliferation rates and survival in the tumor microenvironment [24]. To support unlimited growth, cancer cells exhibit higher rates of glucose metabolism, protein synthesis and de novo lipid synthesis [25, 26]. We surveyed these pathways by metabolic labeling in MDA-MB-231 triple negative breast cancer cells in the presence of a scrambled sequence shRNA or shRNA targeting BRG1 [21, 22, 27]. Glucose uptake and protein synthesis were not affected in MDA- MB-231 BRG1 knockdown cells (Figure 1A–1C). Interestingly, de novo lipid synthesis was reduced by 40% in the MDA-MB-231 BRG1 knockdown cells (Figure 1D) but not in MCF-10A breast epithelial cells expressing the same shRNA against BRG1 (Figure 1E). Western blot analysis confirmed the knockdown of BRG1 in both cell lines (Figure 1F). This observation was reproduced in other triple negative breast cancer lines (MDA-MB-468 We next showed that BRG1 directly bound to these genes having BRG1-dependent expression. We predicted BRG1 binding sites based on BRG1 ChIP-seq www.impactjournals.com/oncotarget Oncotarget 38271 re 1: BRG1 knockdown reduced de novo lipid synthesis in triple negative breast cancer cells but not in MCF-10A mmary epithelial cells. (A) MDA-MB-231 cells expressing either a scrambled sequence shRNA or an shRNA targeting BRG incubated with 14C-glucose and incorporation of radioactivity into cells was measured to determine glucose uptake. There was n ficant change. (B) BRG1 knockdown cells also had no change in the rate of protein synthesis compared to control cells as measure -leucine incorporation. (C) Phosphorimage of the blot of 35S-labeled protein extracted from control and BRG1 knockdown cells (left Coomassie brilliant blue staining of the blot (right). (D) BRG1 knockdown cells had a decrease in de novo lipid synthesis as measure C-acetate incorporation in total extracted lipids. (E) MCF-10A cells expressing a scrambled sequence shRNA or shRNA targetin 1 were incubated with 14C-acetate and incorporation of radioactivity into extracted total lipids was measured. BRG1 knockdown di ause a significant decrease in lipid synthesis. (F) Western blot analysis verified the shRNA-mediated knockdown of BRG1 in MDA 231 and MCF-10A cells. (G) Western blot analysis verified the siRNA-mediated knockdown of BRG1 in MDA-MB-468 and HDQ-P (H) Three triple negative breast cancer cell lines were treated with scrambled sequence siRNA or a cocktail of siRNAs targetin 1 and 14C-acetate incorporation into extracted total lipids was measured. Reduction of BRG1 in cancer cells attenuated de novo lipid synthesis (I) A small molecule inhibitor (ADAADi) of BRG1 inhibite vo lipid synthesis in MDA-MB-231 cells. Each data point represents the mean of 3 independent experiments performed in triplicate b t d d d i ti *P 0 001 Figure 1: BRG1 knockdown reduced de novo lipid synthesis in triple negative breast cancer knockdown reduced de novo lipid synthesis in triple negative breast cancer cells but not in MC Figure 1: BRG1 knockdown reduced de novo lipid synthesis in triple negative breast cancer cells but not in MCF-10A mammary epithelial cells. (A) MDA-MB-231 cells expressing either a scrambled sequence shRNA or an shRNA targeting BRG1 were incubated with 14C-glucose and incorporation of radioactivity into cells was measured to determine glucose uptake. There was no significant change. (B) BRG1 knockdown cells also had no change in the rate of protein synthesis compared to control cells as measured by 3H-leucine incorporation. (C) Phosphorimage of the blot of 35S-labeled protein extracted from control and BRG1 knockdown cells (left) and Coomassie brilliant blue staining of the blot (right). (D) BRG1 knockdown cells had a decrease in de novo lipid synthesis as measured by 14C-acetate incorporation in total extracted lipids. (E) MCF-10A cells expressing a scrambled sequence shRNA or shRNA targeting BRG1 were incubated with 14C-acetate and incorporation of radioactivity into extracted total lipids was measured. BRG1 knockdown did not cause a significant decrease in lipid synthesis. (F) Western blot analysis verified the shRNA-mediated knockdown of BRG1 in MDA- MB-231 and MCF-10A cells. (G) Western blot analysis verified the siRNA-mediated knockdown of BRG1 in MDA-MB-468 and HDQ-P1 cells. (H) Three triple negative breast cancer cell lines were treated with scrambled sequence siRNA or a cocktail of siRNAs targeting BRG1 and 14C-acetate incorporation into extracted total lipids was measured. (I) A small molecule inhibitor (ADAADi) of BRG1 inhibited de novo lipid synthesis in MDA-MB-231 cells. Each data point represents the mean of 3 independent experiments performed in triplicate. Error bars are standard deviations. P < 0.001. Figure 1: BRG1 knockdown reduced de novo lipid synthesis in triple negative breast cancer cells but not in MCF-10A mammary epithelial cells. (A) MDA-MB-231 cells expressing either a scrambled sequence shRNA or an shRNA targeting BRG1 were incubated with 14C-glucose and incorporation of radioactivity into cells was measured to determine glucose uptake. There was no significant change. Reduction of BRG1 in cancer cells attenuated de novo lipid synthesis (B) BRG1 knockdown cells also had no change in the rate of protein synthesis compared to control cells as measured by 3H-leucine incorporation. (C) Phosphorimage of the blot of 35S-labeled protein extracted from control and BRG1 knockdown cells (left) and Coomassie brilliant blue staining of the blot (right). (D) BRG1 knockdown cells had a decrease in de novo lipid synthesis as measured by 14C-acetate incorporation in total extracted lipids. (E) MCF-10A cells expressing a scrambled sequence shRNA or shRNA targeting BRG1 were incubated with 14C-acetate and incorporation of radioactivity into extracted total lipids was measured. BRG1 knockdown did not cause a significant decrease in lipid synthesis. (F) Western blot analysis verified the shRNA-mediated knockdown of BRG1 in MDA- MB-231 and MCF-10A cells. (G) Western blot analysis verified the siRNA-mediated knockdown of BRG1 in MDA-MB-468 and HDQ-P1 cells. (H) Three triple negative breast cancer cell lines were treated with scrambled sequence siRNA or a cocktail of siRNAs targeting BRG1 and 14C-acetate incorporation into extracted total lipids was measured. (I) A small molecule inhibitor (ADAADi) of BRG1 inhibited de novo lipid synthesis in MDA-MB-231 cells. Each data point represents the mean of 3 independent experiments performed in triplicate. Error bars are standard deviations. P < 0.001. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 38272 data from K562 and HeLa cells and global H3K27Ac and DNase I hypersensitivity analyses [37] and used these for PCR primer design within the region 1.5 Kb upstream of the TSS of the ACC, FASN, ACLY and ACSL1 genes (Supplementary Figure 1). There were no active transcriptional marks in sequences upstream of the LPIN1 TSS, however, there were multiple active marks in the first intronic region of this gene, allowing the design of PCR primers in this region (Supplementary Figure 1). Chromatin immunoprecipitation (ChIP) analysis detected BRG1 binding at these lipogenic genes in control cells that was markedly decreased in BRG1 knockdown cells (Figure 2D). Additional ChIP controls are shown in Supplementary Figure 2. These data suggest that BRG1 directly and transcriptionally regulates lipid biosynthesis and metabolism pathways in these breast cancer cells. the knockdown cells. Re-expression or over-expression of wildtype or catalytically inactive BRG1 is negligible in some cell types if Brahma (BRM), the closely related ATPase that can act as the catalytic subunit in mammalian SWI/SNF complexes in a manner that is mutually exclusive with BRG1, is expressed [38–40]. Reduction of BRG1 in cancer cells attenuated de novo lipid synthesis In addition, we have previously shown that knockdown of either BRG1 or BRM in triple negative breast cancer cell lines results in increased expression of the remaining ATPase [22] and that both BRG1 and BRM contribute to triple negative cell proliferation [30]. Therefore we re-established BRG1 expression in MDA-MB-231 cells after expressing shRNA against both BRG1 and BRM [22] by transient transfection with plasmid vectors. Re-expression of BRG1 in this double knockdown background resulted in increasing levels of FASN and in a dose-dependent manner (Figure 3A), as well as a concomitant increase in de novo lipid synthesis (Figure 3B). As we previously reported [22], re-expression of BRG1 in double knockdown cells caused only a partial rescue in cell proliferation (Figure 3C). This is expected since we have shown that BRM also contributes to cell proliferation [30]. These data show that BRG1 regulates both de novo lipid synthesis and cell proliferation. Reduction in de novo lipid synthesis impaired cancer cell proliferation To further confirm that BRG1 is the direct cause for the reduction in lipid biosynthesis, a complementary experiment was performed to restore BRG1 expression in Figure 2: BRG1 was required for the expression of genes involved in fatty acid and lipid synthesis. (A) mRNA levels of ACC and FASN in MDA-MB-231 and MCF-10A cells expressing either a scrambled sequence shRNA or an shRNA targeting BRG1 were measured by qPCR. (B) Western blot analysis measuring protein levels of ACC and FASN in MDA-MB-231 and MCF-10A cells expressing a scrambled sequence shRNA or shRNA targeting BRG1. (C) mRNA levels of ACLY, ACSL1 and LPIN1 in MDA-MB-231 cells expressing either a scrambled sequence shRNA or an shRNA targeting BRG1 were determined by qPCR. (D) ChIP experiments with MDA-MB-231 cells expressing either a scrambled sequence shRNA or an shRNA targeting BRG1 demonstrated that BRG1 binds to sequences upstream of each gene. Negative control sequences 1 and 2 are within the coding sequences of the ACC and FASN genes, respectively. Each dataset represents the mean of 3 independent experiments performed in triplicate. Error bars are standard deviations. P < 0.01, *P < 0.001. Figure 2: BRG1 was required for the expression of genes involved in fatty acid and lipid synthesis. (A) mRNA levels of ACC and FASN in MDA-MB-231 and MCF-10A cells expressing either a scrambled sequence shRNA or an shRNA targeting BRG1 were measured by qPCR. (B) Western blot analysis measuring protein levels of ACC and FASN in MDA-MB-231 and MCF-10A cells expressing a scrambled sequence shRNA or shRNA targeting BRG1. (C) mRNA levels of ACLY, ACSL1 and LPIN1 in MDA-MB-231 cells expressing either a scrambled sequence shRNA or an shRNA targeting BRG1 were determined by qPCR. (D) ChIP experiments with MDA-MB-231 cells expressing either a scrambled sequence shRNA or an shRNA targeting BRG1 demonstrated that BRG1 binds to sequences upstream of each gene. Negative control sequences 1 and 2 are within the coding sequences of the ACC and FASN genes, respectively. Each dataset represents the mean of 3 independent experiments performed in triplicate. Error bars are standard deviations. P < 0.01, **P < 0.001. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 38273 We reasoned that, as an upstream regulator of key lipogenic enzymes, depletion of BRG1 should also increase the sensitivity of cells to fatty acid synthesis inhibitors. 5-tetradecyloxy-2-furoic acid (TOFA) is an inhibitor of ACC [41]. Reduction in de novo lipid synthesis impaired cancer cell proliferation When TOFA was added to cells, the decrease in viable cell number was larger after BRG1 knockdown than in control cells (Figure 4A). The FASN inhibitor c75 [42] decreased cell viability in control cells, underscoring the need for de novo lipid biogenesis in highly proliferative tumor cells. As shown in Figure 4B, c75 potency was significantly enhanced in BRG1 knockdown cells compared to control cells. ACC and FASN have been reported to be essential to cancer cell survival, and knocking down either ACC or FASN dramatically decreases cancer cell proliferation [9, 43, 44]. After reducing BRG1 levels, breast cancer cells showed increased sensitivity to both inhibitors. cancer cells with BRG1 knockdown [22]. Following BRG1 knockdown, the cell culture medium was replaced with medium containing increasing doses of nonfat BSA-conjugated palmitic acid, and cell proliferation was measured. As shown in Figure 4C, addition of exogenous palmitic acid in the culture medium completely rescued the cell proliferation defect in BRG1 knockdown cells but had no effect on control cells. This result shows that BRG1- dependent contributions to de novo lipid synthesis in turn regulate the rate of the breast cancer cell proliferation. Addition of palmitate did not affect ACC or FASN expression in either control of BRG1 knockdown cells (Figure 4D). Thus BRG1 function is upstream of ACC and FASN expression, which is upstream of palmitate production and de novo lipid synthesis, which are upstream of cell proliferation. The data do not exclude possible feedback signals whereby the altered proliferation rate also affects de novo lipid synthesis. Regardless, the results demonstrate that decreased cell proliferation in BRG1 knockdown cells can be attributed to the decrease in expression of the key metabolic enzymes for fatty acid synthesis. Since the end product of FASN and common intermediate in all de novo fatty acid synthesis is palmitate, we asked if the decrease in palmitate production was responsible for the impaired cell proliferation in Figure 3: Restoration of BRG1 expression in cells depleted for BRG1 partially rescued the decrease in de novo ipid synthesis and cell proliferation. (A) Western blot analysis showing that re-expression of BRG1 partially restored ACC and FASN protein levels. (B) Re-expression of BRG1 partially reversed the inhibition of de novo lipid synthesis caused by loss of BRG1. C) Re-expression of BRG1 partially reversed the inhibition of cell growth caused by loss of BRG1. DISCUSSION its promise as a target for anti-cancer therapeutics [1, 12, 50, 51], the mechanism by which FASN is dysregulated in cancer is unknown. Similarly, two enzymes catalyzing rate limiting steps upstream of FASN in de novo fatty acid synthesis, ACC and ACLY, are often dysregulated in cancer and have been proposed as breast cancer therapeutic targets [52–54]. One of the hallmarks of cancer is elevated de novo fatty acid synthesis [45]. Clinical and basic scientific investigation has shown that human cancers synthesize fatty acids via the de novo fatty acid synthesis pathway even when exogenous fatty acids are abundant, seemingly independent of the regulatory signals that control fatty- acid synthesis in normal cells. The role of FASN in normal human biology includes energy storage from excess carbohydrates to fat in liver and adipose tissue and specialized functions that facilitate lactation in the breast and reproduction in endometrium and decidua [46–48]. FASN expression during these processes is strictly regulated by nutrition and hormonal levels [49]. In contrast, FASN is highly expressed in many cancers and precancerous lesions. In this context, the expression of FASN is independent of nutrition, and in many cases, it appears independent of hormonal regulation [45]. Despite BRG1 is a known epigenetic regulator of chromatin structure and gene expression that may also play an architectural role in gene organization [55]. We show here that loss of BRG1 attenuated FASN, ACC, and ACLY expression and impaired de novo lipid synthesis in breast cancer cells with a concomitant decrease in cell proliferation. Importantly, this phenomenon was only seen in cancer cells and not in non-tumorigenic mammary epithelial cells. Re-introducing BRG1 to BRG1- and BRM-depleted cancer cells increased FASN and ACC expression, increased de novo lipid synthesis, and partially restored cell proliferation. When the culture medium was Figure 4: Fatty acid levels regulated breast cancer cell proliferation. (A–B) BRG1 knockdown rendered cells more sensitive to growth inhibition by the ACC inhibitor TOFA or the FASN inhibitor c75. (C) Addition of palmitic acid to the cell culture media completely reversed the growth inhibition caused by BRG1 knockdown. (D) Addition of palmitic acid did not affect FASN or ACC expression in control or in BRG1 knockdown cells. Each dataset represents the mean of 3 independent experiments performed in triplicate. Error bars are standard deviations. P < 0.05, **P < 0.001, n.s. not significant. Figure 4: Fatty acid levels regulated breast cancer cell proliferation. Reduction in de novo lipid synthesis impaired cancer cell proliferation Each dataset represents the mean of 3 independent experiments performed in triplicate. Error bars are standard deviations. P < 0.05, P < 0.01, P < 0.001. Figure 3: Restoration of BRG1 expression in cells depleted for BRG1 partially rescued the decrease in de novo lipid synthesis and cell proliferation. (A) Western blot analysis showing that re-expression of BRG1 partially restored ACC and FASN protein levels. (B) Re-expression of BRG1 partially reversed the inhibition of de novo lipid synthesis caused by loss of BRG1. (C) Re-expression of BRG1 partially reversed the inhibition of cell growth caused by loss of BRG1. Each dataset represents the mean of 3 independent experiments performed in triplicate. Error bars are standard deviations. P < 0.05, P < 0.01, P < 0.001. www.impactjournals.com/oncotarget Oncotarget 38274 MATERIALS AND METHODS supplemented with palmitic acid, the end product of FASN and the key intermediate in the synthesis of longer chain and desaturated fatty acids, cell proliferation in BRG1 knockdown cells was completely rescued. The complete rescue in cells where only BRG1 was knocked down and BRM was present suggests that BRG1 is the SWI/ SNF ATPase predominantly responsible for regulation of these metabolic pathways. Thus our results provide the first evidence of a direct relationship between BRG1, lipogenic enzyme transcriptional control, de novo fatty acid synthesis, and cell proliferation. Strategically targeting BRG1 for cancer therapy BRG1 function in cancer appears to be context dependent. It is mutated in lung and other cancers [56, 57], and cancers featuring loss of the SNF5/INI1 subunit may require BRG1 [18], thereby suggesting the potential of targeting BRG1 to treat such tumors [58, 59]. In addition, BRG1 is upregulated with little evidence of mutation in primary breast and prostate tumors, in melanoma and neuroblastoma, and in pancreatic, gastric, and colorectal carcinomas [22, 23, 60–67]. Therefore, developing small molecule inhibitors that interfere with BRG1 function or that fine-tune the expression of BRG1 back to physiological levels might provide therapeutic benefits. Indeed, we have recently demonstrated that knockdown of BRG1 or use of a BRG1 inhibitor sensitizes triple negative breast cancer cells to commonly used chemotherapeutic agents, perhaps via modulation of ABC transporter expression [30]. DISCUSSION (A–B) BRG1 knockdown rendered cells more sensitive to growth inhibition by the ACC inhibitor TOFA or the FASN inhibitor c75. (C) Addition of palmitic acid to the cell culture media completely reversed the growth inhibition caused by BRG1 knockdown. (D) Addition of palmitic acid did not affect FASN or ACC expression in control or in BRG1 knockdown cells. Each dataset represents the mean of 3 independent experiments performed in triplicate. Error bars are standard deviations. P < 0.05, ***P < 0.001, n.s. not significant. www.impactjournals.com/oncotarget Oncotarget 38275 Reagents Doxycycline, palmitic acid, TOFA, c75, MTT (3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide), and anti-GAPDH antibody were purchased from Sigma (Sigma-Aldrich, St. Louis, MO). FASN (C20G5) and acetyl- CoA carboxylase (C83B10) antibodies were purchased from Cell Signaling (Cell Signaling Technology, Inc., Danvers, MA, USA). Anti-rabbit and anti-mouse IgG, and HRP-linked antibodies were from GE (GE Healthcare Life Science, Pittsburgh, PA, USA). BRG1 antiserum [70] was used in western blot and ChIP experiments. The BRM antibody (ab15597) used in western blots was purchased from Abcam (Cambridge, MA, USA). D-14C-Glucose, 3H-Leucine, 35S-Methionine and [2–14C] acetic acid were purchased from PerkinElmer (PerkinElmer Life Sciences, Waltham, MA, USA). ADAADi was prepared as described [28]. The data presented here show that BRG1 is specifically required for fatty acid and lipid synthesis in triple negative breast cancer cells but not in mammary epithelial cells. This specificity increases the promise for BRG1- based therapies in triple negative breast cancer compared to treatment approaches that non-specifically target fatty acid and lipid synthesis. We conclude that targeting BRG1 and its cooperating factors are a promising and novel strategy for attacking both cancer cell proliferation and cancer metabolism. Cell culture MCF-10A cells from the Karmanos Cancer Institute (Detroit, MI) were maintained in monolayer as described [68]. MDA-MB-231 cells were obtained from T. Guise [69]. MCF-10A cells inducibly expressing shRNA targeting BRG1 or a control shRNA were previously described [21]. MDA-MB-231 cells inducibly expressing shRNA targeting BRG1, BRG1 + BRM, or a control shRNA were previously described [22]. MDA-MB-468 cells were obtained from Professor Hong Zhang (UMass Medical School). HDQ-P1 cells were purchased from the Leibniz Institute DSMZ- German Collection of Microorganisms and Cell Culture (Braunschweig, Germany). MDA-MB-231, MDA-MB-468 and HDQ-P1 cells were maintained in DMEM containing 10% FBS and Penicillin/Streptomycin. Doxycycline- inducible knockdown was performed as described [21, 22, 27] using 500 ng/ml doxycycline for MCF-10A cells and 100 ng/ml doxycycline for MDA-MB-231 cells. siRNA-mediated knockdown of BRG1 was performed using the validated pool of siRNAs and methods described previously [22, 27]. We and others have demonstrated that BRG1 levels are elevated in primary breast cancer [22, 23]. Our results here show that the elevated levels of lipid synthesis found in breast cancer are dependent on BRG1 acting to upregulate the transcription of FASN, ACC, ACLY and other genes involved in fatty acid synthesis. BRG1 is therefore an epigenetic link between breast cancer cell proliferation and fatty acid synthesis, but it is likely to be only the first factor identified in a novel regulatory circuit up-regulating lipogenic enzyme expression in cancer with downstream consequences on cell proliferation. Further investigation of BRG1 and the BRG1-interacting factors of this circuit will elucidate mechanisms by which cancer cells rewire signaling pathways controlling de novo lipid biosynthesis. The identities of the breast cancer cell lines were authenticated by Short Tandem Repeat profiling at the Genetic Resources Core Facility, Johns Hopkins School of Medicine, Institute of Genetic Medicine. MCF-10A cells were similarly authenticated at the University of Vermont Cancer Center. Proliferation assays Five thousand cells were seeded in each well of 96-well plates, and treated as indicated in each figure legend the following day. MTT solution at a final concentration of 5 ug/mL was added to each well and samples were incubated for 4 hours. The media was www.impactjournals.com/oncotarget Oncotarget 38276 RT-qPCR Protein synthesis rate was determined by labeling cells with 1 μCi 3H-Leucine for 4 h. After three washes in ice cold PBS, cells were lysed with PBS containing 2% SDS before scintillation counting. All readings were normalized by cell number determined from parallel plates. For other protein synthesis assays, cells were labeled with 10 μCi 35S-Methionine overnight, washed 3 times with ice cold PBS, and then lysed with PBS containing 1% NP-40. Radiolabeled proteins (2 µg) were run on SDS-PAGE 4–20% gradient gels before transfer to a PVDF membrane. Radioactivity was measured by Phosphoimager and checked for equal protein loading by staining the membrane with Coomassie Brilliant Blue. One million cells were used for extraction of total RNA using RNeasy Plus following manufacturer’s instructions (Qiagen Inc., Valencia, CA, USA). cDNA synthesis was accomplished using a SuperScript III kit (Invitrogen, San Diego, CA, USA). Gene expression was measured by real time qPCR using following primers. ACC alpha (ACACA) forward 5′-ATGCG GTCTATCCGTAGG- 3′, reverse 5′-GGTGTGACCATGA CAAC-3′; FASN, forward 5′-GTTTGATGCCTCCTTCTTC-3′, reverse 5′-CGGAG TG AATCTGGGTTG-3′; ACLY forward 5′- CCCAAGTCCAAGATCCCTGC-3′, reverse 5′- TCGTCTCGG GAGCAGACATA-3′; ACSL1 forward 5′- GGAAGAGCCAACAGA CGGAA-3′, reverse 5′- CATTGCTCCTTTGGGGTTGC-3′; LPIN1 forward 5′- GAGGCAGAC AGCACCATACA-3′, reverse 5′- GGCTAACTGCCCCACGTAAT-3′. Western blotting subsequently removed and the plates were air-dried. One hundred microliters of DMSO were added to each well and incubated for 30 minutes at room temperature with gentle shaking. Absorbance was measured at OD540 in a Synergy H4 Hybrid microplate reader (Bio Tek, Winooski, VT). Cells were washed, trypsinized, and counted and whole cell lysates from one million cultured cells were prepared by lysis in 200 μL of 1× Laemmli sample buffer and boiled for 5 min. 10 μL of lysate was separated on SDS-polyacrylamide gels (4–20% gradient) and transferred to PVDF membrane. Membranes were blocked in 5% non-fat dry milk in PBS, incubated with primary antibodies overnight at 4oC. Following repeated washing in 5% milk/PBS, membranes were incubated with secondary antibody conjugated to HRP for 1 hour at room temperature, washed repeatedly with 5%milk/PBS, and developed using Amersham ECL Western Blotting Detection Reagents and Amersham Hyperfilm ECL (GE LifeScience). De novo lipid synthesis assay In experiments with ADAADi, cells were pre- treated with 2 μM ADAADi for 48 h prior to addition of radiolabeled acetate. Breast cancer cells were labeled with 1 μCi /mL 14C-acetate for 1 h. MCF-10A cells were labeled with 5 μCi /mL 14C acetate for 1 h. After removal of the labeling medium, cells were washed 3 times with PBS and then cultured in fresh medium for 24 h. At the end of the incubation, cells were trypsinized and counted. One million cells were used for lipid extraction. Total lipids were extracted as described [71], transferred to a scintillation vial and counted in a scintillation counter (Beckman Coulter LS6500). Glucose uptake assay Cells were pulse labeled with 1 μCi 14C-glucose for 5 minutes, and then washed 3 times with PBS. Cells were trypsinized and counted. One million cells were lysed by adding 200μL 0.2N NaOH for 15 min. The cell lysate were transferred to a scintillation vial, and radioactive signal was measured in a scintillation counter (Beckman Coulter LS6500). Chromatin immunoprecipitation (ChIP) ChIP was performed as described previously [38] with modifications. Cells were cooled to room temperature before crosslinking which was done by replacing the media with ice-cold growth medium containing 3.7% formaldehyde for 40 min at 4ºC. Each ChIP reaction used 5 μg of BRG1 antiserum [70] or control IgG (Millipore, Billerica, MA) and 50 μg of chromatin extract. Primers used for measuring BRG1 binding at those promoters measured by qPCR are listed below: ACC alpha (ACACA), forward 5′- GTCCCACCCCGTAAGGATTT -3′ (-537 ~ -556 bp to TSS), reverse 5′- GGCGCTAGCTCCAAACTAAC -3′ (-708~-727 bp to TSS); c75, TOFA and palmitic acid treatment MDA-MB-231 SCRAM and shBRG1 cells were plated in 96-well plates at a density of 3,000 cells/well and treated with 0.1 μg/ml doxycycline or with vehicle for 48 h. Reagents were added to the cell culture medium at 0, 15, or 20 nM for 24 h (palmitic acid) or at 10, 25, or 50 μM for 72 h (c75, TOFA). To assess cell proliferation, 10 µL of MTT reagent was added to each well and incubated for 4 h prior to an MTT assay. ( p ) FASN, forward 5′- CTCCCGAGTGATTCCTCGAA -3′(-1086 ~ -1105 bp to TSS), reverse 5′- CTCAAAGT AGGACACGCAGC -3′ (-1233 ~ -1252 bp to TSS); ACLY, forward 5′- GTAAGCAAGTGGGGCTAGG AG -3′ (-570 ~ -590 bp to TSS), reverse 5′CTTCGCT GGAATCTCGCATTG -3′ (-665~ -684 bp to TSS); FASN, forward 5′- CTCCCGAGTGATTCCTCGAA -3′(-1086 ~ -1105 bp to TSS), reverse 5′- CTCAAAGT AGGACACGCAGC -3′ (-1233 ~ -1252 bp to TSS); ACLY, forward 5′- GTAAGCAAGTGGGGCTAGG AG -3′ (-570 ~ -590 bp to TSS), reverse 5′CTTCGCT GGAATCTCGCATTG -3′ (-665~ -684 bp to TSS); FASN, forward 5′- CTCCCGAGTGATTCCTCGAA -3′(-1086 ~ -1105 bp to TSS), reverse 5′- CTCAAAGT AGGACACGCAGC -3′ (-1233 ~ -1252 bp to TSS); ACLY, forward 5′- GTAAGCAAGTGGGGCTAGG AG -3′ (-570 ~ -590 bp to TSS), reverse 5′CTTCGCT GGAATCTCGCATTG -3′ (-665~ -684 bp to TSS); ACLY, forward 5′- GTAAGCAAGTGGGGCTAGG AG -3′ (-570 ~ -590 bp to TSS), reverse 5′CTTCGCT GGAATCTCGCATTG -3′ (-665~ -684 bp to TSS); www.impactjournals.com/oncotarget Oncotarget 38277 2. Vazquez-Martin A, Colomer R, Brunet J, Lupu R, Menendez JA. Overexpression of fatty acid synthase gene activates HER1/HER2 tyrosine kinase receptors in human breast epithelial cells. Cell Prolif. 2008; 41:59–85. ACSL1, forward 5′- CCAGACTGCCTCGGA TTTCATA -3′ (-130 ~ -151 bp to TSS), reverse 5′- GGCGGTCCAATGTACCCTT -3′ (-172 ~ -191 bp to TSS); LIPIN1, forward 5′-TGCAGCCCATTTCCTGG ATT-3′ (+66,769 bp ~ +66,788 bp to TSS), reverse 5′-GAGGAAGGAGGGGCTGAGTA-3′ (+66,842 bp ~ (+66,861 bp to TSS). ACSL1, forward 5′- CCAGACTGCCTCGGA TTTCATA -3′ (-130 ~ -151 bp to TSS), reverse 5′- GGCGGTCCAATGTACCCTT LIPIN1, forward 5′-TGCAGCCCATTTCCTGG ATT-3′ (+66,769 bp ~ +66,788 bp to TSS), reverse 5′-GAGGAAGGAGGGGCTGAGTA-3′ (+66,842 bp ~ (+66,861 bp to TSS). LIPIN1, forward 5′-TGCAGCCCATTTCCTGG ATT-3′ (+66,769 bp ~ +66,788 bp to TSS), reverse 5′-GAGGAAGGAGGGGCTGAGTA-3′ (+66,842 bp ~ (+66,861 bp to TSS). 3. Martel PM, Bingham CM, McGraw CJ, Baker CL, Morganelli PM, Meng ML, Armstrong JM, Moncur JT, Kinlaw WB. GRANT SUPPORT 12. Mashima T, Seimiya H, Tsuruo T. De novo fatty-acid synthesis and related pathways as molecular targets for cancer therapy. Br J Cancer. 2009; 100:1369–1372. This work was supported by NIH grants P01 CA82834, R01 EB014869, and R21 CA185926. 13. Zaidi N, Swinnen JV, Smans K. ATP-citrate lyase: a key player in cancer metabolism. Cancer Res. 2012; 72:3709–3714. CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest. 11. Kridel SJ, Axelrod F, Rozenkrantz N, Smith JW. Orlistat is a novel inhibitor of fatty acid synthase with antitumor activity. Cancer Res. 2004; 64:2070–2075. ACKNOWLEDGMENTS We thank members of the Imbalzano lab group for comments. 10. Hatzivassiliou G, Zhao F, Bauer DE, Andreadis C, Shaw AN, Dhanak D, Hingorani SR, Tuveson DA, Thompson CB. ATP citrate lyase inhibition can suppress tumor cell growth. 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https://openalex.org/W2022143936	https://journals.iucr.org/e/issues/2009/02/00/ci2750/ci2750.pdf	English	null	1,2,3,6,7,8-Hexahydrocinnolino[5,4,3-<i>cde</i>]cinnoline	Acta crystallographica. Section E	2,009	cc-by	1,865	organic compounds Monoclinic, P21=c a = 9.698 (5) A˚ b = 5.875 (3) A˚ c = 10.023 (5) A˚ = 117.314 (6) V = 507.4 (4) A˚ 3 Z = 2 Mo K radiation = 0.09 mm1 T = 298 (2) K 0.55 0.41 0.09 mm Data collection Bruker SMART CCD area-detector diffractometer Absorption correction: multi-scan (SADABS; Sheldrick, 1996) Tmin = 0.953, Tmax = 0.992 2508 measured reﬂections 890 independent reﬂections 575 reﬂections with I > 2(I) Rint = 0.028 Reﬁnement R[F 2 > 2(F 2)] = 0.041 wR(F 2) = 0.123 S = 1.01 890 reﬂections 97 parameters All H-atom parameters reﬁned max = 0.16 e A˚ 3 min = 0.15 e A˚ 3 Z = 2 Mo K radiation = 0.09 mm1 T = 298 (2) K 0.55 0.41 0.09 mm Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Zhi-Qiang Gao College of Chemistry and Environmental Engineering, Chongqing University of Arts and Sciences, Chongqing, People’s Republic of China Correspondence e-mail: haowenjuanxz@126.com College of Chemistry and Environmental Engineering, Chongqing University of Arts and Sciences, Chongqing, People’s Republic of China Correspondence e-mail: haowenjuanxz@126.com 97 parameters All H-atom parameters reﬁned max = 0.16 e A˚ 3 min = 0.15 e A˚ 3 Received 23 December 2008; accepted 27 December 2008 Key indicators: single-crystal X-ray study; T = 298 K; mean (C–C) = 0.003 A˚; R factor = 0.041; wR factor = 0.123; data-to-parameter ratio = 9.2. Data collection: SMART (Bruker, 1998); cell reﬁnement: SAINT (Bruker, 1999); data reduction: SAINT; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to reﬁne structure: SHELXL97 (Sheldrick, 2008); molecular graphics: SHELXTL (Sheldrick, 2008); software used to prepare material for publication: SHELXTL. The title compound, C12H12N4, was synthesized by the reaction of hydrazine hydrate and 9-methyl-3,4,6,7-tetra- hydro-2H-xanthene-1,8(5H,9H)-dione in ethanol. In the crystal, the molecule lies across an inversion centre. The pyridazine rings are coplanar and the C6 rings adopt envelope conformations. Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: CI2750). Related literature For the biological properties of cinnoline and its derivatives, see: Abdelrazek et al. (2006); Gomtsyan et al. (2005); Inoue et al. (1993); Lewgowd & Stanczak (2007); Lewgowd et al. (2005); Singh et al. (2003); Stefanska et al. (2003); Tutsumi et al. (1992). 1,2,3,6,7,8-Hexahydrocinnolino[5,4,3- cde]cinnoline Zhi-Qiang Gao S2. Experimental The title compound was prepared by the reaction of 3,4,6,7-tetrahydro -9-methyl-2H-xanthene-1,8(5H,9H)-dione (2 mmol) and hydrazine hydrate 80% (8 mmol) in ethanol (8 ml) stirring at 353 K (yield 84%; m.p. 543–544 K). Single crystals suitable for X-ray diffraction were obtained from an ethanol solution by slow evaporation. S1. Comment It is well known that six-membered nitrogen-containing heterocycles are abundant in numerous natural products that exhibit important biological properties. For example, cinnolines and their derivatives exhibit a diverse range of biological properties (Lewgowd & Stanczak, 2007) such as molluscicidal activity (Abdelrazek et al., 2006), cytotoxic activity (Lewgowd et al., 2005), transient receptor potential vanilloid 1 (TRPV1) receptor antagonists (Gomtsyan et al., 2005), and topoisomerase I (TOP1)-targeting activity and cytotoxicity (Singh et al., 2003). They also acted as anticancer agents active on a multidrug resistant cell line (Stefanska et al., 2003). They can also be used as bactericides in pharmaceutical industry (Inoue et al., 1993; Tutsumi et al.,1992). The chemistry of cinnolines has received much attention based on the above facts. The title molecule lies across an inversion centre (Fig. 1). The two pyridazine rings (C1/C2/C2A/C3A/N2/N1 and C1A/C2A/C2/C3/N2A/N1A) are conjugated and are coplanar. The two cyclohexene rings (C1—C6 and C1A—C6A) adopt envelope conformations, with atoms C5 and C5A deviate from the C1/C2/C3/C4/C6 and C1A/C2A/C3A/C4A/C6A planes by 0.656 (3) and 0.656 (3) Å, respectively. A view of the crystal packing is shown in Fig.2. References Abdelrazek, F. M., Metz, P., Metwally, N. H. & El-Mahrouky, S. F. (2006). Arch. Pharm. 339, 456–460. Bruker (1998). SMART. Bruker AXS Inc., Madison, Wisconsin, USA. Bruker (1999). SAINT. Bruker AXS Inc., Madison, Wisconsin, USA. ( ) , , Gomtsyan, A. et al. (2005). Med. Chem. 48, 744–752. Gomtsyan, A. et al. (2005). Med. Chem. 48, 744–752. Inoue, S., Yasaki, A., Mochizuki, H., Tutsumi, H., Murata, M. & Sakane, K. (1993). Japanese Patent No. 05213951. Experimental Crystal data C12H12N4 Mr = 212.26 Lewgowd, W. & Stanczak, A. (2007). Arch. Pharm. 340, 65–8 Lewgowd, W., Stanczak, A., Ochocki, Z., Krajewska, U. & Rozalski, M. (2005). Acta Pol. Pharm. 62, 105–110. Sheldrick, G. M. (1996). SADABS. University of Go¨ttingen, Germany Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Singh, S. K., Ruchelman, A. L., Zhou, N., Li, T.-K., Liu, An., Liu, L. F. & Lavoie, E. J. (2003). Med. Chem. Res. 12, 1–12. Stefanska, B., Arciemiuk, M., Bontemps-Gracz, M. M., Dzieduszycka, M., Kupiec, A., Martelli, S. & Borowski, E. (2003). Bioorg. Med. Chem. 11, 561– 572. Experimental Tutsumi, H., Terasawa, T., Barret, D., Mutata, M., Sakane, K., Yazaki, A. & Inoue, S. (1992). Japanese Patent No. 9215584; Chem. Abstr. 118, 254944w. Mr = 212.26 Mr = 212.26 Acta Cryst. (2009). E65, o239 Zhi-Qiang Gao o239 doi:10.1107/S1600536808044036 supporting information Acta Cryst. (2009). E65, o239 [doi:10.1107/S1600536808044036] Acta Cryst. (2009). E65, o239 [doi:10.1107/S1600536808044036] Acta Cryst. (2009). E65, o239 [doi:10.1107/S1600536808044036] Acta Cryst. (2009). E65, o239 [doi:10.1107/S1600536808044036] 1,2,3,6,7,8-Hexahydrocinnolino[5,4,3-cde]cinnoline Zhi-Qiang Gao S3. Refinement All H atoms were located in a difference Fourier map and refined freely [C-H = 0.95 (2)-1.04 (2) Å sup-1 Acta Cryst. (2009). E65, o239 supporting information Figure 1 The molecular structure of the title compound, showing 50% probability displacement ellipsoids and the atom-numbering h At l b ll d ith th ffi A t d b th t ti (1 1 ) Figure 2 Figure 2 Molecular packing in the title compound, viewed approximately along the a axis. Figure 2 Molecular packing in the title compound, viewed approximately along the a axis. Molecular packing in the title compound, viewed approximately along the a axis. 1,2,3,6,7,8-Hexahydrocinnolino[5,4,3-cde]cinnoline F(000) = 224 Dx = 1.389 Mg m−3 Melting point = 543–544 K Mo Kα radiation, λ = 0.71073 Å Cell parameters from 734 reflections θ = 2.4–26.3° µ = 0.09 mm−1 T = 298 K Plate, colourless 0.55 × 0.41 × 0.09 mm 2508 measured reflections 890 independent reflections 575 reflections with I > 2σ(I) Rint = 0.028 θmax = 25.0°, θmin = 4.1° h = −11→11 k = −6→6 l = −11→11 Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.041 wR(F2) = 0.123 S = 1.01 890 reflections 97 parameters 0 restraints Primary atom site location: structure-invarian direct methods Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites All H-atom parameters refined w = 1/[σ2(Fo2) + (0.0655P)2 + 0.0552P] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max = 0.001 Δρmax = 0.16 e Å−3 Δρmin = −0.15 e Å−3 Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites All H-atom parameters refined w = 1/[σ2(Fo2) + (0.0655P)2 + 0.0552P] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max = 0.001 Δρmax = 0.16 e Å−3 Δρmin = −0.15 e Å−3 Figure 1 g The molecular structure of the title compound, showing 50% probability displacement ellipsoids and the atom-numbering scheme. Atoms labelled with the suffix A are generated by the symmetry operation (1-x, -y, 1-z). The molecular structure of the title compound, showing 50% probability displacement ellipsoids and the atom-numbering scheme. Atoms labelled with the suffix A are generated by the symmetry operation (1-x, -y, 1-z). sup-2 Acta Cryst. (2009). E65, o239 supporting information Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, conventional R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. sup-3 Acta Cryst. (2009). E65, o239 Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq N1 0.7159 (2) −0.2597 (3) 0.65617 (19) 0.0547 (6) Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) sup-3 Acta Cryst. (2009). E65, o239 supporting information sup-4 Acta Cryst. (2009). Symmetry code: (i) −x+1, −y, −z+1. Special details E65, o239 N2 0.5861 (2) −0.3398 (3) 0.66438 (18) 0.0555 (6) C1 0.7050 (2) −0.0820 (4) 0.5729 (2) 0.0466 (6) C2 0.56377 (19) 0.0384 (3) 0.49395 (18) 0.0390 (5) C3 0.5474 (2) 0.2352 (3) 0.4062 (2) 0.0450 (5) C4 0.6864 (3) 0.3211 (5) 0.3949 (3) 0.0581 (7) C5 0.8354 (3) 0.2567 (4) 0.5326 (3) 0.0647 (7) C6 0.8445 (3) 0.0004 (4) 0.5597 (3) 0.0621 (7) H1 0.682 (2) 0.250 (4) 0.305 (3) 0.065 (6) H2 0.678 (2) 0.487 (4) 0.382 (2) 0.076 (7)* H3 0.930 (3) 0.308 (4) 0.522 (3) 0.086 (8)* H4 0.839 (3) 0.340 (4) 0.623 (3) 0.079 (7)* H5 0.846 (2) −0.086 (4) 0.470 (2) 0.072 (7)* H6 0.936 (3) −0.039 (4) 0.649 (3) 0.081 (7)* Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 N1 0.0567 (12) 0.0508 (12) 0.0500 (10) 0.0164 (9) 0.0187 (9) 0.0061 (8) N2 0.0652 (12) 0.0458 (11) 0.0474 (11) 0.0115 (9) 0.0189 (9) 0.0086 (8) C1 0.0501 (12) 0.0467 (12) 0.0395 (10) 0.0102 (10) 0.0175 (9) −0.0019 (10) C2 0.0445 (11) 0.0386 (11) 0.0306 (9) 0.0072 (8) 0.0146 (8) −0.0029 (8) C3 0.0574 (13) 0.0386 (12) 0.0351 (10) 0.0051 (10) 0.0178 (9) −0.0010 (9) C4 0.0737 (17) 0.0505 (15) 0.0555 (14) −0.0061 (12) 0.0344 (13) −0.0027 (12) C5 0.0595 (15) 0.0680 (17) 0.0681 (16) −0.0087 (13) 0.0306 (13) −0.0101 (13) C6 0.0484 (14) 0.0707 (18) 0.0650 (16) 0.0111 (11) 0.0242 (13) −0.0033 (12) Geometric parameters (Å, º) N1—C1 1.310 (3) C4—C5 1.518 (3) N1—N2 1.381 (2) C4—H1 0.98 (2) N2—C3i 1.309 (3) C4—H2 0.98 (2) C1—C2 1.417 (3) C5—C6 1.526 (3) C1—C6 1.499 (3) C5—H3 1.01 (2) C2—C2i 1.373 (3) C5—H4 1.01 (2) C2—C3 1.417 (3) C6—H5 1.04 (2) C3—N2i 1.309 (3) C6—H6 0.95 (2) C3—C4 1.492 (3) C1—N1—N2 120.01 (17) C5—C4—H2 111.0 (13) C3i—N2—N1 120.67 (18) H1—C4—H2 109.4 (18) N1—C1—C2 121.89 (19) C4—C5—C6 111.2 (2) N1—C1—C6 120.07 (18) C4—C5—H3 111.1 (13) C2—C1—C6 118.0 (2) C6—C5—H3 109.2 (14) C2i—C2—C3 118.3 (2) C4—C5—H4 108.6 (14) C2i—C2—C1 117.8 (2) C6—C5—H4 110.1 (13) C3—C2—C1 123.94 (17) H3—C5—H4 106.5 (19) N2i—C3—C2 121.33 (19) C1—C6—C5 110.70 (19) N2i—C3—C4 120.3 (2) C1—C6—H5 107.1 (12) supporting information Atomic displacement parameters (Å2) sup-4 Acta Cryst. (2009). E65, o239 supporting information C2—C3—C4 118.39 (18) C5—C6—H5 110.5 (12) C3—C4—C5 111.3 (2) C1—C6—H6 109.2 (14) C3—C4—H1 105.8 (12) C5—C6—H6 111.1 (15) C5—C4—H1 110.6 (12) H5—C6—H6 108.2 (19) C3—C4—H2 108.6 (13) supporting information sup-5 Acta Cryst. (2009). E65, o239
https://openalex.org/W2891569200	https://europepmc.org/articles/pmc6156230?pdf=render	English	null	Caspase-1 inhibition alleviates cognitive impairment and neuropathology in an Alzheimer’s disease mouse model	Nature communications	2,018	cc-by	18,938	1 Bloomﬁeld Center for Research in Aging, Lady Davis Institute for Medical Research, Jewish General Hospital, 3755 Ch. Côte Ste-Catherine, Montreal, QC H3T 1E2, Canada. 2 Department of Neurology and Neurosurgery, McGill University, 845 Sherbrooke St West, Montreal, QC H3A 0G4, Canada. 3 Department of Anatomy and Cell Biology, McGill University, 845 Sherbrooke St West, Montreal, QC H3A 0G4, Canada. Correspondence and requests for materials should be addressed to A.C.L. (email: andrea.leblanc@mcgill.ca) ARTICLE Caspase-1 inhibition alleviates cognitive impairment and neuropathology in an Alzheimer’s disease mouse model Joseph Flores 1,2, Anastasia Noël 1,2, Bénédicte Foveau 1, Jeffrey Lynham 1,3, Clotilde Lecrux 2 & Andréa C. LeBlanc 1,2,3 Joseph Flores 1,2, Anastasia Noël 1,2, Bénédicte Foveau 1, Jeffrey Lynham 1,3, Clotilde Lecrux 2 & Andréa C. LeBlanc 1,2,3 Alzheimer's disease (AD) is an intractable progressive neurodegenerative disease char- acterized by cognitive decline and dementia. An inﬂammatory neurodegenerative pathway, involving Caspase-1 activation, is associated with human age-dependent cognitive impair- ment and several classical AD brain pathologies. Here, we show that the nontoxic and blood–brain barrier permeable small molecule Caspase-1 inhibitor VX-765 dose-dependently reverses episodic and spatial memory impairment, and hyperactivity in the J20 mouse model of AD. Cessation of VX-765 results in the reappearance of memory deﬁcits in the mice after 1 month and recommencement of treatment re-establishes normal cognition. VX-765 pre- vents progressive amyloid beta peptide deposition, reverses brain inﬂammation, and nor- malizes synaptophysin protein levels in mouse hippocampus. Consistent with these ﬁndings, Caspase-1 null J20 mice are protected from episodic and spatial memory deﬁcits, neuroin- ﬂammation and Aβ accumulation. These results provide in vivo proof of concept for Caspase- 1 inhibition against AD cognitive deﬁcits and pathologies. 1 Bloomﬁeld Center for Research in Aging, Lady Davis Institute for Medical Research, Jewish General Hospital, 3755 Ch. Côte Ste-Catherine, Montreal, QC H3T 1E2, Canada. 2 Department of Neurology and Neurosurgery, McGill University, 845 Sherbrooke St West, Montreal, QC H3A 0G4, Canada. 3 Department of Anatomy and Cell Biology, McGill University, 845 Sherbrooke St West, Montreal, QC H3A 0G4, Canada. Correspondence and requests for materials should be addressed to A.C.L. (email: andrea.leblanc@mcgill.ca) 1 NATURE COMMUNICATIONS \| (2018) 9:3916 \| DOI: 10.1038/s41467-018-06449-x \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x human Casp1 (IC50 3.68 nM and 9.91 nM) relative to human Casp2−10 (see Supplementary Fig. 1a, b). Similarly, mouse recombinant Casp1 (IC50 52.1 nM and 18 nM) was strongly inhibited compared to inﬂammatory mouse Casp11 (see Sup- plementary Fig. 1c, d). Although variable between individual mice, VX-765 crossed the blood–brain barrier of WT and J20 mice, was metabolized into VRT-043198, and reached physiolo- gically active concentrations in both the hippocampus and cortex (see Supplementary Fig. 1e). C urrently, no treatment signiﬁcantly delays, or reverses AD- associated cognitive decline or pathologies. Targeting earlier degenerative events may be essential to stem the scourge of AD. Caspase-1 inhibition alleviates cognitive impairment and neuropathology in an Alzheimer’s disease mouse model p g g g p g While there are currently no suitable inhibitors for Nlrp1 and Casp6, VX-765, a pro-drug that is rapidly metabolized in vivo to VRT-043198, is a potent bioavailable, blood–brain barrier perme- able, and nontoxic Casp1 small molecule inhibitor25,26. VRT- 043198 is reported to be selective for Casp1 against Casp3−10, Casp14, granzyme B, trypsin and cathepsin B26,27. VX-765 inhibits Il-1β release and Il-18 in activated peripheral blood mononucleated cells and in lipopolysaccharide-induced inﬂammation, oxalozone- induced dermatitis, and rheumatoid arthritis mouse models27. VX- 765 blocks HIV-induced pyroptosis of CD4 T cells28 but lacks signiﬁcant antiapoptotic activity against hypoxia or Fas-induced apoptosis, eliminating the risk of potential inhibition of physiolo- gical apoptotic events in vivo27. VX-765 is safe for humans by oral administration as tested in a phase 2b human clinical trial against epilepsy29. Therefore, VX-765 represents a safe drug that could rapidly be tested against human age-dependent cognitive impair- ment and AD. pp y g g VX-765 was administered at 25 and 10 mg kg−1 to assess the dose-response of J20 to VX-765 (Fig. 2). All groups showed normal motivation (see Supplementary Fig. 3a) and thigmotaxic behaviour (see Supplementary Fig. 3b). NOR discrimination index was normalized with 25 mg kg−1 dose at T1 and T2, deﬁcits reappeared after WO, and returned to normal at T3, similar to the results with the 50 mg kg−1 dose (Fig. 2a and see Supplementary Fig. 3c). Mice treated with 10 mg kg−1 showed normalized NOR behaviour only after T2, the effect was lost with WO, and reappeared after T3. J20 hyperactivity showed a nonsigniﬁcant improvement at T2 with 25, but not 10, mg kg−1 (see Supplementary Fig 3d, e). In the T2 Barnes maze, learning acquisition did not change after VX-765 treatment by the end of the training phase (Fig. 2b and see Supplementary Fig. 3f), and J20 remained impaired in the probe primary latency at both the 25 and 10 mg kg−1 dose (Fig. 2c). However, primary errors were Here, VX-765 was given to the J20 APPSw/Ind transgenic mouse model30. J20 mice show episodic memory deﬁcits by 3−4 months, spatial memory and learning deﬁcits by 6−7 months31, increased total hippocampal Aβ42 levels at 2 months of age, Aβ plaque formation by 7 months30, and synaptophysin loss by 2−6 months30. Microglial inﬂammation and neuronal loss precede plaque deposition32. Caspase-1 inhibition alleviates cognitive impairment and neuropathology in an Alzheimer’s disease mouse model Preclinical involvement of inﬂammation in AD, genetic association of immune-related genes with AD, and increased risk of AD in systemic and CNS conditions that increase inﬂammation support the hypothesis that inﬂammation is an integral part of AD1. In addition, increasing support for the role of inﬂammation in AD is provided from AD mouse models. AD mouse models on complement 32, colony stimulating factor 1 receptor3,4, inﬂammasome Nod-like receptor protein 3 (Nlrp3)5, Caspase-1 (Casp1)5, scavenger receptor class B type I6, poly (ADP-ribose) polymerase 17, or complement 1qa8 null back- ground, show improvements in cognitive behaviour, synaptic pathology, and often amyloid beta peptide (Aβ) pathology. Inhibition of inﬂammasome activator P2X7R decreases Aβ9, and silencing of inﬂammasome Nod-like receptor protein 1 (Nlrp1) improves cognition in AD models10. human Casp1 (IC50 3.68 nM and 9.91 nM) relative to human Casp2−10 (see Supplementary Fig. 1a, b). Similarly, mouse recombinant Casp1 (IC50 52.1 nM and 18 nM) was strongly inhibited compared to inﬂammatory mouse Casp11 (see Sup- plementary Fig. 1c, d). Although variable between individual mice, VX-765 crossed the blood–brain barrier of WT and J20 mice, was metabolized into VRT-043198, and reached physiolo- gically active concentrations in both the hippocampus and cortex (see Supplementary Fig. 1e). Five-month-old mice were behaviourally and longitudinally assessed before treatment (baseline), after 3 injections per week with 50 mg kg−1 VX-765 (Treatment 1; T1), after an additional 2 weeks of injections (Treatment 2; T2), after 4 weeks without treatment (Washout; WO), and after 3 more injections per week (Treatment 3; T3) before sacriﬁcing the mice at 8 months of age (Fig. 1a). At baseline, J20 and littermate WT mice showed normal motivation behaviour determined by % time moving (see Supplementary Fig. 2a) and did not exhibit thigmotaxis indicative of anxiety (see Supplementary Fig. 2b), but J20 mice showed a strong deﬁcit in the novel object recognition (NOR) episodic (retention) memory discrimination index (Fig. 1b). J20 NOR deﬁcits were reversed and reached near-normal levels after VX- 765 T1 and T2, reappeared after WO, and renormalized after T3. Results were consistent across individual mice (see Supplemen- tary Fig. 2c). J20 mice hyperactivity, measured by distance travelled in the open ﬁeld task, was attenuated by VX-765 after T2, reappeared after WO, and again was signiﬁcantly reduced after T3 (Fig. 1c and see Supplementary Fig. 2d). Caspase-1 inhibition alleviates cognitive impairment and neuropathology in an Alzheimer’s disease mouse model At T2, J20 mice showed learning acquisition deﬁcits in the Barnes maze spatial memory training phase evidenced by increased primary errors to identify the target correctly compared to WT mice. This deﬁcit was not signiﬁcantly attenuated by VX-765 (Fig. 1d). Primary latency to ﬁnd the target did not differ between the three groups (see Supplementary Fig. 2e). During the probe test, primary latency, primary errors (Fig. 1e) and the ability to ﬁnd the target (Fig. 1f) were clearly impaired in vehicle-injected J20 compared to WT mice. VX-765 eliminated these spatial memory deﬁcits in J20 mice. After WO, vehicle-injected J20 showed learning deﬁcits early on, but all groups performed equally well by the end of training (Fig. 1g and see Supplementary Fig. 2f). The difference in WO primary latency compared to T2 is likely due to a learning effect by repetition or a reduction of stress to the apparatus. VX- 765-injected, but not vehicle-injected, J20 mice were normal in primary latency and errors during the probe, suggesting spatial memory retention even after 1 month without drug (Fig. 1h). VX- 765-injected mice also performed better than vehicle-injected J20 mice in their ability to ﬁnd the target (Fig. 1i). J20 mice performance in the Y maze working memory task was consistently low but not always statistically different from WT or treated J20 mice (see Supplementary Fig. 2g). 1 g We identiﬁed a human neurodegenerative pathway, mediated by neuronal Nlrp1 inﬂammasome activation of Casp1, which then activates Caspase-6 (Casp6), in stressed CNS human primary neuron cultures and AD brains11. Active Casp6 is abundant in neuritic plaques, neuroﬁbrillary tangles, and neuropil threads of sporadic and familial AD brains12,13, and Nlrp1 immunopositive neurons are increased 20−25-fold in AD11. Entorhinal cortex and hippocampal CA1 Casp6 activation is associated with age- dependent cognitive impairment in humans14,15 and in transgenic mice in the absence of other AD pathologies16. Active Casp6 is associated with axonal degeneration17–19, cleaves several cytoske- leton or cytoskeletal-associated proteins including Tau, αTubulin, Drebrin, Spinophillin, Actinin-1 and -4 synaptic proteins20, increases Aβ production21–23 and impairs valosin-containing pro- tein p97 proteasome-mediated misfolded protein degradation24. Consequently, Nlrp1, Casp1, and Casp6 represent feasible ther- apeutic targets against age-dependent cognitive deﬁcits and AD. Caspase-1 inhibition alleviates cognitive impairment and neuropathology in an Alzheimer’s disease mouse model The early mani- festation of these symptoms and pathologies allows rapid analyses of the VX-765 effects. NATURE COMMUNICATIONS \| (2018) 9:3916 \| DOI: 10.1038/s41467-018-06449-x \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x Target preference: # of pokes of each hole labelled +1 to +9 to the right or −1 to −9 to the left of the target (T a b c e d f ** * * * ** WT+veh WT+veh J20+veh J20+veh J20+VX WT+veh J20+veh J20+VX J20+VX Baseline Distance (m) Washout Treatment Treatment Treatment 2 Treatment 2 WT/J20 1.0 0.8 0.6 0.4 0.2 –0.2 –0.4 15 10 5 0 0 + – 0 0 5 15 J20 J20 WT Veh VX WT Veh VX 10 10 20 20 30 40 Primary errors Primary errors Primary latency (s) NOR discrimination index 0 1 1 1 1 2 2 2 2 2 3 3 3 3 T 4 4 4 5 5 6 6 7 7 8 8 9 9 4 6 8 10 # of pokes 0 2 4 6 8 10 # of pokes 0 2 4 6 8 10 # of pokes Training day b ** WT+veh J20+veh J20+VX Baseline Washout Treatment Treatment WT/J20 1.0 0.8 0.6 0.4 0.2 –0.2 –0.4 NOR discrimination index 0 1 2 3 b c e d ** * * * WT+veh J20+veh J20+VX Baseline Treatment Treatment 2 15 10 5 0 0 0 5 15 J20 J20 WT Veh VX WT Veh VX 10 10 20 20 30 40 Primary errors Primary errors Primary latency (s) 1 2 3 4 Training day i WT+veh J20+veh Washout 12 0 3 6 9 12 # of pokes 0 3 6 9 12 # of pokes d g * * WT+veh J20+veh J20+VX Washout 0 6 12 18 Primary errors 1 2 3 4 Training day f d g g h * * * * 15 0 J20 WT Veh VX J20 WT Veh VX 30 45 60 0 5 15 10 20 Primary errors Primary latency (s) g y h * * * * J20+VX 15 0 J20 WT Veh VX J20 WT Veh VX 30 45 60 0 5 15 10 20 Primary errors Primary latency (s) – 1 2 3 T 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 0 3 6 9 12 0 3 # of # of pokes + J20 J20 Fig. 1 VX-765 treatment restores J20 mice cognitive function. a Experimental treatment paradigm. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x b NOR Discrimination index from vehicle-treated WT (grey squares), vehicle-treated J20 (black circles) and VX-765-treated J20 (blue triangles). Each mouse tested is represented by one symbol. Data represent mean and s.e.m. (Treatment, F(2,20) = 85.8, p < 0.0001; Time, F(4,80) = 4.188, p = 0.0039; Treatment × Time, F(8,80) = 3.599, p = 0013). c Distance travelled during open ﬁeld task (Treatment, F(2,19) = 11.47, p = 0.0005; Treatment × Time, F(8,76) = 5.69, p < 0.0001). b, c Two-way repeated- measures ANOVA and Dunnett’s post-hoc versus J20 + vehicle, p < 0.05, p < 0.01, p < 0.001, *p < 0.0001. d–i Barnes maze: learning acquisition # of primary errors during d T2 (Treatment, F(2,212) = 19.93, p < 0.0001) and g WO (Treatment, F(2,56) = 10.86, p = 0.001; Training day, F(3,56) = 3.392, p < 0.0241). d, g Two-way repeated-measures ANOVA and Dunnett’s post-hoc versus J20 + vehicle p < 0.05, *p < 0.001, *p < 0.0001. Probe primary latency and errors during e T2 and h WO. Target preference: # of pokes of each hole labelled +1 to +9 to the right or −1 to −9 to the left of the target (T) during the probe after f T2 and i WO (T2 primary latency, F(2,52) = 5.879, p = 0.0050; T2 primary errors, F(2,52) = 9.998, p = 0.0002; WO primary latency, F(2,22) = 4.076, p = 0.0312; WO primary errors, F(2,22) = 10.84, p = 0.0005). e, h ANOVA, Tukey’s post-hoc, p < 0.05, p < 0.01, p < 0.001 20 mice cognitive function. a Experimental treatment paradigm. b NOR Discrimination index from vehicle-treated WT (Fig. 2f). However, mice treated with 25 mg kg−1, but not 10 mg kg−1, retained their ability to recognize the target (Fig. 2g). Together, these results demonstrate a VX-765 dose-dependent effect in reversing J20 episodic and spatial memory deﬁcits. decreased with 25 mg kg−1 and both doses showed that the J20 mice had improved ability to recognize the target during the probe (Fig. 2d). After WO, primary latency was shorter initially during training (see Supplementary Fig. 3g), but no differences in learning were observed among the three groups by the end of the training (Fig. 2e). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x Week 1 a b c g i h e d f * * * * * * * * * * * ** * ** ** ** Week 4 Week 6 Week 8 WT+veh WT+veh J20+veh J20+VX WT+veh WT+veh J20+veh J20+veh J20+VX WT+veh J20+veh J20+VX J20+VX WT+veh J20+veh J20+veh J20+VX J20+VX 250 200 150 100 50 25 0 Baseline Baseline Treatment Distance (m) Washout Treatment Treatment Treatment Treatment 2 Treatment 2 Washout Washout Washout WT/J20 WT/J20 1.0 0.8 0.6 0.4 0.2 –0.2 –0.4 15 15 10 5 0 0 0 6 12 18 0 + – 0 0 5 15 J20 J20 WT Veh VX J20 WT Veh VX J20 WT Veh VX WT Veh VX 10 10 20 20 30 30 45 60 40 Primary errors Primary errors Primary errors 0 5 15 10 20 Primary errors Primary latency (s) Primary latency (s) NOR discrimination index 0 1 1 1 1 1 2 2 2 2 2 2 3 3 3 3 3 T 4 4 4 5 5 6 6 7 7 8 8 9 9 – 1 2 3 T 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 4 6 8 10 # of pokes 0 0 3 6 9 12 0 3 6 9 12 2 4 6 8 10 # of pokes # of pokes 0 3 6 9 12 # of pokes # of pokes 0 2 4 6 8 10 # of pokes Training day 1 2 3 4 Training day Week 12 VX-765 (3 injections per week) Age of mice 5 months 6 months Washout 4 weeks 8 months 7 months Histological and biochemical analyses Baseline Treatment 1 Treatment 2 Treatment 3 Washout Behavioural analyses + Fig. 1 VX-765 treatment restores J20 mice cognitive function. a Experimental treatment paradigm. b NOR Discrimination index from vehicle-treated WT (grey squares), vehicle-treated J20 (black circles) and VX-765-treated J20 (blue triangles). Each mouse tested is represented by one symbol. Data represent mean and s.e.m. (Treatment, F(2,20) = 85.8, p < 0.0001; Time, F(4,80) = 4.188, p = 0.0039; Treatment × Time, F(8,80) = 3.599, p = 0013). c Distance travelled during open ﬁeld task (Treatment, F(2,19) = 11.47, p = 0.0005; Treatment × Time, F(8,76) = 5.69, p < 0.0001). Results VX-765 rescues cognitive deﬁcits in symptomatic J20 mice. VX-765 and VRT-043198 potently and speciﬁcally inhibited NATURE COMMUNICATIONS \| (2018) 9:3916 \| DOI: 10.1038/s41467-018-06449-x \| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS \| (2018) 9:3916 \| DOI: 10.1038/s41467-018-06449-x \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x b, c Two-way repeated measures ANOVA and Dunnett’s post-hoc versus J20 + vehicle, p < 0.05, p < 0.01, p < 0.001, *p < 0.0001. d–i Barnes maze: learning acquisition # of primary errors during d T2 (Treatment, F(2,212) = 19.93, p < 0.0001) and g WO (Treatment, F(2,56) = 10.86, p = 0.001; Training day, F(3,56) = 3.392 p < 0.0241). d, g Two-way repeated-measures ANOVA and Dunnett’s post-hoc versus J20 + vehicle p < 0.05, *p < 0.001, p < 0.0001. Probe primary latency and errors during e T2 and h WO. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x 0.8 a b d e g f c Treatment 1 Treatment 2 Primary errors Primary errors Treatment 2 Treatment 3 Washout Washout Washout Treatment 2 0.6 0.4 0.2 0 0 NOR discrimination index NOR discrimination index –0.2 0.8 0.6 0.4 0.2 0 –0.2 NOR discrimination index 0.8 0.6 0.4 0.2 0 –0.2 NOR discrimination index 0.8 0.6 0.4 0.2 0 –0.2 50 25 10 VX-765 (mg kg–1) 0 50 25 10 VX-765 (mg kg–1) 0 50 25 10 0 50 25 10 VX-765 (mg kg–1) VX-765 (mg kg–1) 25 mg kg–1 25 mg kg–1 25 mg kg–1 10 mg kg–1 10 mg kg–1 10 mg kg–1 Veh Veh 25 mg kg–1 10 mg kg–1 Veh 1 10 40 30 20 10 10 10 8 6 4 2 # of pokes Primary latency (s) 40 30 20 Primary latency (s) 0 0 3 6 9 12 Primary errors 0 3 6 9 12 0 25 VX-765 (mg kg–1) VX-765 (mg kg–1) 10 0 25 10 0 25 10 0 25 10 15 5 0 0 0 10 8 6 4 2 # of pokes 0 10 8 6 4 2 # of pokes 0 Primary errors 10 15 5 0 2 3 4 1 1 T 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 Training day 12 9 6 3 0 # of pokes 12 9 6 3 0 # of pokes 12 9 – + 1 1 T 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 – + 6 3 0 # of pokes 1 2 3 4 Training day Veh * * *** * **** * * Fig. 2 VX-765 dose-dependently restores cognitive function in J20 mice. a NOR discrimination index at T1 (F(3,24) = 20.42, p < 0.0001), T2 (F(3,23) = 12.83, p < 0.0001), WO, and T3 (F(3,23) = 8.828, p = 0.0004). Vehicle-treated (black circles), and 50 mg kg−1 (blue triangles), 25 mg kg−1 (pink triangles), or 10 mg kg−1 (purple hexagon) VX-765-treated J20 mice. Each mouse tested is represented by one symbol. Data represent mean and s.e.m. b −g Barnes maze during b−d T2 and e−g WO. Learning acquisition at b T2 and e WO; probe and target preference at c, d T2 and f, g WO. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x a, c, f ANOVA and b, e Two-way repeated-measures ANOVA, Dunnett’s post-hoc versus J20 + vehicle, p < 0.05, p < 0.01, p < 0.001, p < 0.0001 0.8 a Treatment 1 0.6 0.4 0.2 0 0 NOR discrimination index –0.2 50 25 10 VX-765 (mg kg–1) Washout NOR discrimination index 0.8 0.6 0.4 0.2 0 –0.2 0 50 25 10 VX-765 (mg kg–1) Treatment 3 NOR discrimination index 0.8 0.6 0.4 0.2 0 –0.2 0 50 25 10 VX-765 (mg kg–1) * *** * Treatment 2 NOR discrimination index 0.8 0.6 0.4 0.2 0 –0.2 0 50 25 10 VX-765 (mg kg–1) ** * a b d c Treatment 2 Primary errors Primary errors Treatment 2 ( g g ) ( g g ) 25 mg kg–1 10 mg kg–1 25 mg kg–1 10 mg kg–1 Veh 1 10 40 30 20 10 10 Primary latency (s) 0 0 3 6 9 12 0 25 VX-765 (mg kg–1) 10 0 25 15 5 0 2 3 4 1 1 T 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 Training day 12 9 6 3 0 # of pokes 12 9 6 3 0 # of pokes 12 9 – + 6 3 0 # of pokes Veh * * e Washout 25 mg kg–1 10 mg kg–1 Veh Primary errors 10 15 5 0 1 2 3 4 Training day ** b Treatment 2 Primary errors 25 mg kg–1 10 mg kg–1 Veh 1 10 15 5 0 2 3 4 Training day * d Treatment 2 25 mg kg–1 10 mg kg–1 1 1 T 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 12 9 6 3 0 # of pokes 12 9 6 3 0 # of pokes 12 9 – + 6 3 0 # of pokes Veh g Washout 25 mg kg–1 10 mg kg–1 Veh 10 8 6 4 2 # of pokes 0 10 8 6 4 2 # of pokes 0 10 8 6 4 2 # of pokes 0 1 1 T 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 – + e g f Washout Washout 25 mg kg–1 25 mg kg–1 10 mg kg–1 10 mg kg–1 Veh Veh 10 8 6 4 2 # of pokes 40 30 20 Primary latency (s) Primary errors 0 3 6 9 12 VX-765 (mg kg–1) 10 0 25 10 0 25 10 0 0 10 8 6 4 2 # of pokes 0 10 8 6 4 2 # of pokes 0 Primary errors 10 15 5 0 1 1 T 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 – + 1 2 3 4 Training day ** b d g e f 10 mg kg–1 10 8 6 4 2 # of pokes 40 30 20 Primary latency (s) Primary errors 0 3 6 9 12 VX-765 (mg kg–1) 10 0 25 10 0 25 10 0 0 4 2 # of 0 1 1 T 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 – + Training day c Primary errors 40 30 20 10 10 Primary latency (s) 0 0 3 6 9 12 0 25 VX-765 (mg kg–1) 10 0 25 Training day * c Primary errors 10 mg kg 40 30 20 10 10 Primary latency (s) 0 0 3 6 9 12 0 25 VX-765 (mg kg–1) 10 0 25 1 1 T 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 Training day 3 0 # of 12 9 – + 6 3 0 # of pokes * c Fig. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x behaviourally assessed at 8 months of age, corresponding to the age of J20 mice after VX-765 T3 (Fig. 3). Similar to 5-month-old J20 mice, 8-month-old J20/Casp1−/−showed normal locomotor activity and thigmotaxis (see Supplementary Fig. 4a, b) compared to J20/WT, J20/Casp1+/−, WT/WT, WT/Casp1−/−, or WT/Casp1 +/−. NOR discrimination index, which was strongly decreased in J20//WT mice, was normalized in J20/Casp1−/−and J20/Casp1 +/−(Fig. 3a and see Supplementary Fig. 4c). Mice hyperactivity was similar in J20/WT, J20/Casp1−/−, and J20/Casp1+/−mice, in contrast to observations in the VX-765 studies (Fig. 3b). This indicates that the J20 hyperactivity is exacerbated by the extra manipulations or injections in the VX-765 study. Partial or complete Casp1 knockout (KO) ameliorated J20 mice learning deﬁcits at days 3 and 4 of the Barnes maze learning acquisition phase (Fig. 3c), but there were no differences in primary latency to ﬁnd the target between groups (see Supplementary Fig. 4d). During the probe, primary latency did not differ between the different genotypes (Fig. 3d); however, primary errors and the ability to ﬁnd the target was impaired in J20/WT mice and normalized after partial or complete Casp1 KO (Fig. 3d, e). Together, these results suggest that VX-765 reverses episodic and spatial memory deﬁcits by inhibiting Casp1 in J20 mice. Iba1-positive microglia in the hippocampus and cortex of 8- month-old vehicle-treated J20 mice, compared to vehicle- treated WT mice, were normalized in VX-765-treated J20 mice, indicating that VX-765 reversed inﬂammation (Fig. 4a). This was conﬁrmed quantitatively as the number of Iba1- immunopositive microglia measured from the pyramidal cell layer to the stratum lacunosum moleculare (SLM) layer of the hippocampal CA1 region, or from the cortical retrosplenial area to the S1, returned to normal after VX-765 treatment (Fig. 4b). Quantitation of microglial activation levels based on morpho- logical measurements34 (see Supplementary Fig. 5a) showed decreased type I resting microglia and increased type II and III activated microglia in vehicle-treated J20 hippocampus and cortex compared to vehicle-treated WT and VX-765-treated J20 mice (Fig. 4b). Hippocampal Il-1β tended towards an increase in vehicle-treated J20 compared to VX-765-treated J20, vehicle- treated WT, and 5-month-old J20 baseline hippocampus (Fig. 4b). Cortical Il-1β tended towards an increase in vehicle- treated WT and J20 compared to baseline or VX-765-treated J20. No signiﬁcant difference was observed in plasma Il-1β level in VX-765-treated mice (see Supplementary Fig. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x 2 VX-765 dose-dependently restores cognitive function in J20 mice. a NOR discrimination index at T1 (F(3,24) = 20.42, p < 0.0001), T2 (F(3,23) = 12.83, p < 0.0001), WO, and T3 (F(3,23) = 8.828, p = 0.0004). Vehicle-treated (black circles), and 50 mg kg−1 (blue triangles), 25 mg kg−1 (pink triangles), or 10 mg kg−1 (purple hexagon) VX-765-treated J20 mice. Each mouse tested is represented by one symbol. Data represent mean and s.e.m. b −g Barnes maze during b−d T2 and e−g WO. Learning acquisition at b T2 and e WO; probe and target preference at c, d T2 and f, g WO. Learning acquisition # of primary errors during b T2 (Treatment, F(2,56) = 3.377, p = 0.0412; Training day, F(3,56) = 7.102, p = 0.0004), and e WO (Treatment, F (2,56) = 5.434, p = 0.007; Training day, F(3,56) = 11.52, p < 0.0001), probe primary errors at c T2 (F(2,14) = 5.69, p = 0.0155). a, c, f ANOVA and b, e Two-way repeated-measures ANOVA, Dunnett’s post-hoc versus J20 + vehicle, p < 0.05, p < 0.01, p < 0.001, *p < 0.0001 behaviourally assessed at 8 months of age, corresponding to the age of J20 mice after VX-765 T3 (Fig. 3). Similar to 5-month-old J20 mice, 8-month-old J20/Casp1−/−showed normal locomotor activity and thigmotaxis (see Supplementary Fig. 4a, b) compared to J20/WT, J20/Casp1+/−, WT/WT, WT/Casp1−/−, or WT/Casp1 +/−. NOR discrimination index, which was strongly decreased in J20//WT mice, was normalized in J20/Casp1−/−and J20/Casp1 +/−(Fig. 3a and see Supplementary Fig. 4c). Mice hyperactivity was similar in J20/WT, J20/Casp1−/−, and J20/Casp1+/−mice, in contrast to observations in the VX-765 studies (Fig. 3b). This indicates that the J20 hyperactivity is exacerbated by the extra manipulations or injections in the VX-765 study. Partial or complete Casp1 knockout (KO) ameliorated J20 mice learning deﬁcits at days 3 and 4 of the Barnes maze learning acquisition phase (Fig. 3c), but there were no differences in primary latency to ﬁnd the target between groups (see Supplementary Fig. 4d). During the probe, primary latency did not differ between the different genotypes (Fig. 3d); however, primary errors and the ability to ﬁnd the target was impaired in J20/WT mice and normalized after partial or complete Casp1 KO (Fig. 3d, e). Together, these results suggest that VX-765 reverses episodic and spatial memory deﬁcits by inhibiting Casp1 in J20 mice. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x No differences were observed in primary latency and errors, with or without drug during the probe To conﬁrm that VX-765’s effect on re-establishing normal cognition in J20 mice was due to Casp1 inhibition, J20 mice were generated with a Casp1 null background (J20/Casp1−/−) and NATURE COMMUNICATIONS \| (2018) 9:3916 \| DOI: 10.1038/s41467-018-06449-x \| www.nature.com/naturecommunication 3 3 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x Learning acquisition # of primary errors during b T2 (Treatment, F(2,56) = 3.377, p = 0.0412; Training day, F(3,56) = 7.102, p = 0.0004), and e WO (Treatment, F (2,56) = 5.434, p = 0.007; Training day, F(3,56) = 11.52, p < 0.0001), probe primary errors at c T2 (F(2,14) = 5.69, p = 0.0155). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x p < 0.05, p < 10 8 6 4 Primary errors 2 0 1 2 3 4 Training day J20/Casp1 WT/WT WT/Het WT/KO J20/Het J20/KO J20/WT c b a c 90 60 Primary latency (s) Primary errors 30 0 20 15 10 5 0 J20: WT WT WT WT WT KO KO Het Het J20 J20 J20 Casp1: d + – 9 9 8 8 7 7 6 6 5 5 4 4 3 3 2 2 1 1 T 6 4 2 0 # of pokes 6 4 2 0 # of pokes 6 4 2 0 # of pokes J20/WT J20/Het J20/KO WT/WT ( ) J20 / /C 1+/ WT/H t ( i k 6 4 2 0 6 4 2 0 # of pokes # of pokes 6 4 2 0 9 9 8 8 7 7 6 6 5 5 4 4 3 3 2 – + 2 1 1 T # of pokes WT/WT WT/Het WT/KO e 90 60 Primary latency (s) 30 0 20 d d + – 9 9 8 8 7 7 6 6 5 5 4 4 3 3 2 2 1 1 T 6 4 2 0 # of pokes 6 4 2 0 # of pokes 6 4 2 0 # of pokes J20/WT J20/Het J20/KO e Primary errors 20 15 10 5 0 J20: WT WT WT WT WT KO KO Het Het J20 J20 J20 Casp1: Fig. 3 Casp1 KO restores cognitive function in J20 mice. a−e Genotypes: J20−/−/Casp1−/−WT/WT (grey squares), J20−/−/Casp1+/−WT/Het (pink diamonds), J20−/−/Casp1−/−WT/KO (blue symbol), J20−/+/Casp1+/+ J20/WT (black circles), J20−/+/Casp1−/+ J20/Het (purple triangles), J20 −/+/Casp1−/−J20/KO (blue triangles). Each mouse tested is represented by one symbol. Data represent mean and s.e.m. a NOR discrimination index (F (5,67) = 16.22, p < 0.0001) and b distance travelled during open ﬁeld task (F(5,67) = 3.717, p = 0.005). c−e Barnes maze: c # of primary errors during learning acquisition (Genotype, F(5,263) = 6.469, p < 0.0001; Training day, F(3,263) = 29.44, p < 0.0001), d probe primary latency and errors (Primary errors, F(5,66) = 4.8, p = 0008) and e target preference. a, b, d ANOVA and c two-way ANOVA, Dunnett’s post-hoc versus WT/WT. p < 0.05, p < 0.01, **p < 0.0001 Analyses of activated microglia were conducted in the J20/WT, WT/Casp1−/−and J20/Casp1−/−to conﬁrm that VX-765 effects occurred through inhibition of Casp1. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x p < 0.05, p < 0 01 ** 0 0001 80 60 Distance (m) 40 20 0 * * WT WT WT WT WT KO KO Het Het J20 J20 J20 J20: Casp1: b 0.8 0.6 * **** NOR discrimination index 0.4 0.2 –0.2 J20: WT WT WT WT WT KO KO Het Het J20 J20 J20 Casp1: 0 a 10 8 6 4 Primary errors 2 0 1 2 3 4 Training day * 9 8 + – 9 9 8 8 7 7 6 6 5 5 4 4 3 3 2 2 1 1 T 6 4 2 0 # of pokes 6 4 2 0 # of pokes 6 4 2 0 # of pokes J20/WT J20/Het J20/KO KO 20 J20/Casp1 WT/WT WT/Het WT/KO J20/Het J20/KO J20/WT c asp1−/−WT/WT (grey squares), J20−/−/Casp1+/−WT/Het (pink black circles), J20−/+/Casp1−/+ J20/Het (purple triangles), J20 mbol. Data represent mean and s.e.m. a NOR discrimination index (F = 3.717, p = 0.005). c−e Barnes maze: c # of primary errors during ) = 29.44, p < 0.0001), d probe primary latency and errors (Primary -way ANOVA, Dunnett’s post-hoc versus WT/WT. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x 5b) or other inﬂammatory brain protein levels, although TNF-α, KC-GRO, and IFN-γ were slightly elevated in vehicle-treated J20 and normalized after VX-765 treatment (see Supplementary Fig. 5c). VX-765 reverses neuroinﬂammation in J20 mice brains. Iba1 is a marker of activated microglia and inﬂammation33. Increased NATURE COMMUNICATIONS \| (2018) 9:3916 \| DOI: 10.1038/s41467-018-06449-x \| www.nature.com/naturecommunications 4 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x 0.8 80 10 8 6 4 Primary errors 2 0 1 2 3 4 Training day 60 Distance (m) 40 20 0 0.6 * * * ** NOR discrimination index 0.4 0.2 –0.2 90 60 6 4 2 0 6 4 2 0 # of pokes # of pokes 6 4 2 0 9 9 8 8 7 7 6 6 5 5 4 4 3 3 2 – + + – 2 1 1 T 9 9 8 8 7 7 6 6 5 5 4 4 3 3 2 2 1 1 T # of pokes 6 4 2 0 # of pokes 6 4 2 0 # of pokes 6 4 2 0 # of pokes Primary latency (s) Primary errors 30 0 20 15 10 5 0 J20: WT WT WT WT WT WT WT/WT WT/Het J20/WT J20/Het J20/KO WT/KO WT WT WT WT KO KO KO KO Het Het Het Het J20 J20 J20 J20 J20 J20 J20/Casp1 WT/WT WT/Het WT/KO J20/Het J20/KO J20/WT Casp1: J20: WT WT WT WT WT KO KO Het Het J20 J20 J20 Casp1: J20: Casp1: 0 a b c d e Fig. 3 Casp1 KO restores cognitive function in J20 mice. a−e Genotypes: J20−/−/Casp1−/−WT/WT (grey squares), J20−/−/Casp1+/−WT/Het (pink diamonds), J20−/−/Casp1−/−WT/KO (blue symbol), J20−/+/Casp1+/+ J20/WT (black circles), J20−/+/Casp1−/+ J20/Het (purple triangles), J20 −/+/Casp1−/−J20/KO (blue triangles). Each mouse tested is represented by one symbol. Data represent mean and s.e.m. a NOR discrimination index (F (5,67) = 16.22, p < 0.0001) and b distance travelled during open ﬁeld task (F(5,67) = 3.717, p = 0.005). c−e Barnes maze: c # of primary errors during learning acquisition (Genotype, F(5,263) = 6.469, p < 0.0001; Training day, F(3,263) = 29.44, p < 0.0001), d probe primary latency and errors (Primary errors, F(5,66) = 4.8, p = 0008) and e target preference. a, b, d ANOVA and c two-way ANOVA, Dunnett’s post-hoc versus WT/WT. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x J20 baseline Iba1 Hippocampus Cortex 5 months old Hippo Hippo 8 months old Cortex Cortex 18 15 12 9 6 3 0 100 4 3 2 1 0 80 Activation level (% total microglia) Activation level (% total microglia) Activation level (% total microglia) Activation level (% total microglia) 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 0.12 0.08 II-1β (pg mg–1 tissue) II-1β (pg mg–1 tissue) II-1β (pg mg–1 tissue) 0.04 J20 baseline GFAP Cortex Hippocampus J20 + vehicle J20 + VX-765 J20/KO WT + vehicle 0 0.12 0.08 0.04 0.15 0.10 0.05 0 II-1β (pg mg–1 tissue) 0.15 0.10 0.05 0 0 I II III IV I II III IV I II III IV I II III IV 18 15 12 9 6 3 0 18 15 12 9 6 3 0 WT+veh J20+veh J20+VX WT+veh WT+veh WT+veh J20–BL WT/WT WT/WT WT/KO J20/WT J20/KO WT/KO J20/WT J20/KO WT/WT WT/KO J20/WT J20/KO WT/WT WT/KO J20/WT J20/KO WT/WT WT/KO J20/WT J20/KO J20+veh J20+veh J20+VX J20+veh J20+VX J20+VX WT+veh J20–BL J20+veh J20+VX Iba1+ mm–3 (×103) Iba1+ mm–3 (×103) Iba1+ mm–3 (×103) 18 15 12 9 6 3 0 Iba1+ mm–3 (×103) * * * * ** J20 + vehicle J20 + VX-765 J20/KO WT + vehicle a b c d J20 baseline Iba1 Hippocampus Cortex 5 months old 8 months old J20 + vehicle J20 + VX-765 J20/KO WT + vehicle a a Iba1 Hippocampus Cortex 5 months old Hippo Hippo 8 months old Cortex Cortex 18 15 12 9 6 3 0 100 4 3 2 1 0 80 Activation level (% total microglia) Activation level (% total microglia) Activation level (% total microglia) Activation level (% total microglia) 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 0.12 0.08 II-1β (pg mg–1 tissue) II-1β (pg mg–1 tissue) II-1β (pg mg–1 tissue) 0.04 0 0.12 0.08 0.04 0.15 0.10 0.05 0 II-1β (pg mg–1 tissue) 0.15 0.10 0.05 0 0 I II III IV I II III IV I II III IV I II III IV 18 15 12 9 6 3 0 18 15 12 9 6 3 0 WT+veh J20+veh J20+VX WT+veh WT+veh WT+veh J20–BL WT/WT WT/WT WT/KO J20/WT J20/KO WT/KO J20/WT J20/KO WT/WT WT/KO J20/WT J20/KO WT/WT WT/KO J20/WT J20/KO WT/WT WT/KO J20/WT J20/KO J20+veh J20+veh J20+VX J20+veh J20+VX J20+VX WT+veh J20–BL J20+veh J20+VX Iba1+ mm–3 (×103) Iba1+ mm–3 (×103) Iba1+ mm–3 (×103) 18 15 12 9 6 3 0 Iba1+ mm–3 (×103) * * * * ** b c Aβ42/total Aβ38+40+42 ratios were reduced to in the hippocampus and less obviously in the -treated mice (Fig. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x 7a) or protein levels measured with the 9 6 3 0 100 4 3 2 1 0 80 Activation level (% total microglia) Activation level (% total microglia) Activation level (% total microglia) Activation level (% total microglia) 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 0.12 0.08 II-1β (pg mg–1 tissue) II-1β (pg mg–1 tissue) II-1β (pg mg–1 tissue) 0.04 J20 baseline GFAP Cortex 5 months old 120 180 120 60 40 30 20 10 0 0 Hippo Hippo Cortex Cortex * * * * GFAP staining (μm2 mm–2 ×103) GFAP staining (μm2 mm–2 ×103) GFAP staining (μm2 mm–2 ×103) GFAP staining (μm2 mm–2 ×103) 90 8 6 4 2 0 * 60 30 0 WT+veh J20+veh J20+VX WT+veh J20+veh J20+VX 8 months old Hippocampus J20 + vehicle J20 + VX-765 J20/KO WT + vehicle 0 0.12 0.08 0.04 0.15 0.10 0.05 0 II-1β (pg mg–1 tissue) 0.15 0.10 0.05 0 0 I II III IV I II III IV I II III IV I II III IV 9 6 3 0 9 6 3 0 WT+veh J20+veh J20+VX WT+veh WT+veh WT+veh J20–BL WT/WT WT/WT WT/KO J20/WT J20/KO WT/KO J20/WT J20/KO WT/WT WT/KO J20/WT J20/KO WT/WT WT/KO J20/WT J20/KO WT/WT WT/KO J20/WT J20/KO WT/WT WT/KO J20/WT J20/KO WT/WT WT/KO J20/WT J20/KO J20+veh J20+veh J20+VX J20+veh J20+VX J20+VX WT+veh J20–BL J20+veh J20+VX Iba1+ mm– Iba1+ mm– Iba1+ mm– 9 6 3 0 Iba1+ mm– * ** d e f 6 NATURE COMMUNICATIONS \| (2018) 9:3916 \| DOI: 10.1038/s41467-018-06449-x \| www.nature.com/naturecommunications 0.12 0.08 II-1β (pg mg–1 tissue) II-1β (pg mg–1 tissue) II-1β (pg mg–1 tissue) 0.04 0 0.12 0.08 0.04 0.15 0.10 0.05 0 II-1β (pg mg–1 tissue) 0.15 0.10 0.05 0 0 WT+veh J20–BL WT/WT WT/KO J20/WT J20/KO WT/WT WT/KO J20/WT J20/KO J20+veh J20+VX WT+veh J20–BL J20+veh J20+VX J20 baseline GFAP Cortex 5 months old 8 months old Hippocampus J20 + vehicle J20 + VX-765 J20/KO WT + vehicle W W W J J W W J J J J W J J J d d 5 months old 120 180 120 60 40 30 20 10 0 0 Hippo Hippo Cortex Cortex * * * * GFAP staining (μm2 mm–2 ×103) GFAP staining (μm2 mm–2 ×103) GFAP staining (μm2 mm–2 ×103) GFAP staining (μm2 mm–2 ×103) 90 8 6 4 2 0 * 60 30 0 WT+veh J20+veh J20+VX WT+veh J20+veh J20+VX 8 months old WT/WT WT/KO J20/WT J20/KO WT/WT WT/KO J20/WT J20/KO e f e e RIPA-soluble Aβ42/total Aβ38+40+42 ratios were reduced to baseline J20 levels in the hippocampus and less obviously in the cortex of VX-765-treated mice (Fig. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x The microglia in J20/Casp1 −/−were as activated as those of J20/WT (Fig. 4a and see Supplementary Fig. 5d) but as observed in VX-765-treated J20 (Fig. 4b), the number of Iba1-positive microglia increased in J20/ WT compared to WT and were normalized in J20/Casp1−/− (Fig. 4c). However, in contrast to VX-765 treatments, the absence of Casp1 did not modify the distribution of the subtypes of microglia; fewer resting microglia at the expense of increased activated microglia was observed in J20/Casp1−/−mimicking levels of the J20/WT. Il-1β levels were unchanged in J20/Casp1 −/−compared to J20/WT and surprisingly these were not different from WT mice. These results suggest that cremophor vehicle injections in the VX-765 study may have an inﬂammatory effect that exacerbates APP-mediated inﬂammation in J20. VX-765 prevents Aβ accumulation in J20 mice brains. To determine if APP or Aβ levels were altered by VX-765 treatment or the absence of Casp1 in J20 mice, western blots against full length and C-terminal fragments (CTF) of APP, immunohis- tochemistry with an anti-Aβ1-40 antiserum, and ELISA mea- surement of Aβ38, 40, and 42 were conducted. Thioﬂavin S staining of histological brain sections conﬁrmed the amyloid status of Aβ immunopositivity. Thioﬂavin S and immunopositive Aβ levels were substantially reduced in VX-765-treated compared to vehicle-injected J20 hippocampus and cortex (Fig. 5a, b and see Supplementary Fig. 6a). However, Aβ deposits did not completely disappear and were comparable to those observed in baseline J20 brains (Fig. 5a). These remaining Aβ deposits were mainly loca- lized in the SLM and dentate gyrus regions of the hippocampus (see Supplementary Fig. 6a). Levels of immunopositive Aβ pla- ques were similarly reduced in the J20/Casp1−/−hippocampus and cortex (Fig. 5a, c and see Supplementary Fig. 6b). In addition, a much lower level of Aβ was observed in untreated J20 (J20/WT; Fig. 5c) compared to vehicle-injected J20 (Fig. 5b), suggesting an exacerbation of Aβ deposition with the cremophor vehicle injections. Increased GFAP immunopositive astrogliosis in the hippo- campus and cortex of J20 mice was normalized to WT levels with VX-765 treatment and absence of Casp1 (Fig. 4d–f). Overall, these results indicate that VX-765 treatment reversed both microglial and astroglial activation in J20 brains. 5 5 NATURE COMMUNICATIONS \| (2018) 9:3916 \| DOI: 10.1038/s41467-018-06449-x \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x 5d), whereas total Aβ levels did not change (Fig. 5e). Formic acid-soluble Aβ42/total Aβ38 + 40 + 42 ratios were reduced in the hippocampus, but not in the cortex (Fig. 5f), whereas total Aβ levels were more variable than RIPA Aβ levels but tended towards a reduction in both hippocampus and cortex (Fig. 5g). VX-765 treatment had no signiﬁcant effect on Aβ38 or Αβ40 subtypes in hippocampus and cortex (see Supplementary Fig. 6c, d). The reduction in Aβ42 levels with VX- 765 treatment was not due to decreased APP mRNA (see Supplementary Fig. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x 5d), whereas total Aβ levels ig. 5e). Formic acid-soluble Aβ42/total Aβ38 + 40 duced in the hippocampus, but not in the cortex total Aβ levels were more variable than RIPA Aβ levels but tended towards a reduction in bo and cortex (Fig. 5g). VX-765 treatment had no on Aβ38 or Αβ40 subtypes in hippocampus Supplementary Fig. 6c, d). The reduction in Aβ4 765 treatment was not due to decreased A Supplementary Fig. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x 7a) or protein levels me Hippocampu Cortex 5 months old Hippo Hippo 8 months old Cortex Cortex 18 15 12 9 6 3 0 100 4 3 2 1 0 80 Activation level (% total microglia) Activation level (% total microglia) Activation level (% total microglia) Activation level (% total microglia) 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 0.12 0.08 II-1β (pg mg–1 tissue) II-1β (pg mg–1 tissue) II-1β (pg mg–1 tissue) 0.04 J20 baseline GFAP Cortex 5 months old 120 180 120 60 40 30 20 10 0 0 Hippo Hippo Cortex Cortex * * * * GFAP staining (μm2 mm–2 ×103) GFAP staining (μm2 mm–2 ×103) GFAP staining (μm2 mm–2 ×103) GFAP staining (μm2 mm–2 ×103) 90 8 6 4 2 0 * 60 30 0 WT+veh J20+veh J20+VX WT+veh J20+veh J20+VX 8 months old Hippocampus J20 + vehicle J20 + VX-765 J20/KO WT + vehicle 0 0.12 0.08 0.04 0.15 0.10 0.05 0 II-1β (pg mg–1 tissue) 0.15 0.10 0.05 0 0 I II III IV I II III IV I II III IV I II III IV 18 15 12 9 6 3 0 18 15 12 9 6 3 0 WT+veh J20+veh J20+VX WT+veh WT+veh WT+veh J20–BL WT/WT WT/WT WT/KO J20/WT J20/KO WT/KO J20/WT J20/KO WT/WT WT/KO J20/WT J20/KO WT/WT WT/KO J20/WT J20/KO WT/WT WT/KO J20/WT J20/KO WT/WT WT/KO J20/WT J20/KO WT/WT WT/KO J20/WT J20/KO J20+veh J20+veh J20+VX J20+veh J20+VX J20+VX WT+veh J20–BL J20+veh J20+VX Iba1+ mm–3 (×103) Iba1+ mm–3 (×103) Iba1+ mm–3 (×103) 18 15 12 9 6 3 0 Iba1+ mm–3 (×103) * * * * ** b c d e f NATURE COMMUNICATIONS \| (2018) 9:3916 \| DOI: 10.1038/s41467-018-06449-x \| www.nature.com/naturecommunicatio Hippo 8 months old Cortex 18 15 12 9 6 3 0 WT/WT WT/KO J20/WT J20/KO WT/WT WT/KO J20/WT J20/KO Iba1+ mm–3 (×103) 18 15 12 9 6 3 0 Iba1+ mm–3 (×103) c 5 months old Hippo Hippo 8 months old Cortex Cortex 18 15 12 9 6 3 0 100 4 3 2 1 0 80 Activation level (% total microglia) Activation level (% total microglia) Activation level (% total microglia) Activation level (% total microglia) 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 0.12 0.08 II-1β (pg mg–1 tissue) II-1β (pg mg–1 tissue) II-1β (pg mg–1 tissue) 0.04 0 0.12 0.08 0.04 0.15 0.10 0.05 0 II-1β (pg mg–1 tissue) 0.15 0.10 0.05 0 0 I II III IV I II III IV I II III IV I II III IV 18 15 12 9 6 3 0 18 15 12 9 6 3 0 WT+veh J20+veh J20+VX WT+veh WT+veh WT+veh J20–BL WT/WT WT/WT WT/KO J20/WT J20/KO WT/KO J20/WT J20/KO WT/WT WT/KO J20/WT J20/KO WT/WT WT/KO J20/WT J20/KO WT/WT WT/KO J20/WT J20/KO J20+veh J20+veh J20+VX J20+veh J20+VX J20+VX WT+veh J20–BL J20+veh J20+VX Iba1+ mm–3 (×103) Iba1+ mm–3 (×103) Iba1+ mm–3 (×103) 18 15 12 9 6 3 0 Iba1+ mm–3 (×103) * * * * b c 5 months old Hippo Cortex 18 15 12 9 6 3 0 18 15 12 9 6 3 0 WT+veh J20+veh J20+VX WT+veh J20+veh J20+VX Iba1+ mm–3 (×103) Iba1+ mm–3 (×103) * * * * b b RIPA-soluble Aβ42/total Aβ38+40+42 ratios were reduced to baseline J20 levels in the hippocampus and less obviously in the cortex of VX-765-treated mice (Fig. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x These reduced ratios were due to increased Aβ38 and Aβ40 rather than decreased Aβ42 levels in J20/Casp1−/−(see Supplementary Fig. 6e). J20/Casp1+/−and J20/Casp1−/−mice had similar formic acid-soluble Aβ42/total Aβ38 + 40 + 42 ratios, total Aβ levels (Fig. 5j, k), and Aβ subtypes in the hippocampus or cortex (see Supplementary Fig. 6f). The absence of Casp1 in J20 mice had no effect on APP protein levels (Fig. 5n, o and see Supplementary Fig. 7e, f). VX-765 normalizes synaptophysin in J20 mice brains. At 5 months of age, J20 hippocampi showed decreased immuno- positive synaptophysin levels (Fig. 6a). Synaptophysin levels remained low in 8-month-old vehicle-treated J20 hippocampus but were signiﬁcantly increased and returned to normal levels in VX-765-treated mice hippocampi (Fig. 6a, b). There was no signiﬁcant difference in the cortex. Measures of 84 different synaptic gene mRNA levels in vehicle-treated J20 hippocampi revealed signiﬁcantly increased mRNA levels in three genes involved in synaptic function: Camk2a, Grin2b, Kif17 (Fig. 6c). Levels were decreased with VX-765 treatment. Additionally, TNF-α mRNA was increased in J20 and VX-765-treated J20 hippocampi (Fig. 6c), whereas TNFα protein levels increased slightly in J20 mice hippocampi and returned to normal with the VX-765 treatment (see Supplementary Fig. 5c). These results indicate a normalizing effect of VX-765 on several synaptic components that may account for the reversal to normal cognition. To determine if Aβ clearance might have increased with VX- 765 treatment or the absence of Casp1, we measured the levels of Aβ-degrading neprilysin (Nep) and insulin degrading (IDE) enzymes. Increased degradation of Aβ was unlikely since IDE or Nep protein (see Supplementary Fig. 8a, b) and mRNA levels (see Supplementary Fig. 8c, d) were unchanged with VX-765 treatment or the absence of Casp1 (see Supplementary Fig. 8e, f). Together, these results indicate that VX-765 treatment and the absence of Casp1 stopped the progressive deposition of Aβ in J20 brains. VX-765 does not alter Casp8 or Casp9 processing in J20 mice. Casp5, Casp8, Casp9 and Casp10, which were inhibited in the nanomolar range in vitro by VX-765 and VRT-043198, were considered to determine whether VX-765 could affect other caspases in J20 mice (see Supplementary Fig. 9). Since Casp5 and Casp10 are not expressed in mouse35, they were not examined. Pro-Casp9 levels were signiﬁcantly reduced in the hippocampus, but not in cortex, of vehicle-treated J20 com- pared to WT mice, but normalized after VX-765 treatment (see Supplementary Fig. 9a). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x Fig. 4 VX-765 reverses neuroinﬂammation in J20 mice. a Iba1-immunopositive microglia in hippocampal SLM and cortical S1 regions. b, c, e Vehicle- treated J20−/−/Casp1−/−WT (grey squares), vehicle-treated J20−/−/Casp1−/−WT/KO (blue hexagons), J20−/+ at 5 months baseline (J20-BL; dark grey hexagons), vehicle-treated J20−/+ (black circles), and VX-765-treated J20−/+ (purple triangles). b Stereological quantiﬁcation of Iba1-positive microglia from hippocampal pyramidal cell layer to the SLM (F(2,11) = 58, p < 0.0001) and cortex (F(2,11) = 39.95, p < 0.0001) (top panels), average % distribution of morphological microglial subtypes I, II, III and IV (middle panels), and Il-1-β levels (bottom panels) in 5-month-old baseline J20 and in 8- month-old WT + vehicle, J20 + vehicle, and J20 + VX-765 WT mice. ANOVA, Dunnett’s post-hoc versus J20 + vehicle, p < 0.001. c Stereological quantiﬁcation of Iba1-positive microglia in the hippocampal CA1 (F(3,21) = 50.53, p < 0.0001) and cortex (F(3,21) = 96.21, p < 0.0001) (top panels), average % distribution of morphological microglial subtypes I, II, III and IV (middle panels), and Il-1-β levels (bottom panels) in WT/WT, WT/Casp1−/− (WT/KO), J20/WT, and J20/Casp1−/−(J20/KO) mice. ANOVA, Dunnett’s post-hoc versus J20/WT, **p < 0.0001. d Micrographs of GFAP immunopositive astrocytes. e GFAP immunostaining density in vehicle-treated WT and J20 and in VX-765-treated J20 brain hippocampi (F(2,12) = 5.234, p = 0.0232) and cortex (F(2,12) = 8.582, p = 0.0049). f GFAP immunostaining density in J20/KO and littermate control brain hippocampi (F(3,21) = 41.15, p < 0.0001) and cortex (F(3,21) = 4.746, p = 0.0111). ANOVA, Dunnett’s post-hoc versus J20 + vehicle (e) or J20/WT (f), p < 0.05, *p < 0.001, *p < 0.0001. Scale bar in a, d = 50 µm human-speciﬁc 6E10 antibody (Fig. 5l, m) or the mouse and human anti-CTF antibody (see Supplementary Fig. 7c, d). of these enzymes and is known to naturally contain abundant active Casp8 (see Supplementary Fig. 9b, c). There were no differences in active Casp8 subunits, pro-Casp9 levels, or Casp8 or Casp9 substrate pro-Casp3 and active Casp3 20 kDa subunit, among the three groups (see Supplementary Fig. 9c). Together, these results suggest that VX-765 did not inhibit Casp8 and Casp9 in J20 mice. RIPA-soluble Aβ42/total Aβ38+40+42 ratios were reduced in the hippocampus and cortex in J20/Casp1+/−and J20/Casp1−/− mice, with no changes in total Aβ levels although there was a trend towards an increase in the hippocampus (Fig. 5h, i). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x 5d), whereas total Aβ levels did not change (Fig. 5e). Formic acid-soluble Aβ42/total Aβ38 + 40 + 42 ratios were reduced in the hippocampus, but not in the cortex (Fig. 5f), whereas total Aβ levels were more variable than RIPA Aβ levels but tended towards a reduction in both hippocampus and cortex (Fig. 5g). VX-765 treatment had no signiﬁcant effect on Aβ38 or Αβ40 subtypes in hippocampus and cortex (see Supplementary Fig. 6c, d). The reduction in Aβ42 levels with VX- 765 treatment was not due to decreased APP mRNA (see Supplementary Fig. 7a) or protein levels measured with the NATURE COMMUNICATIONS \| (2018) 9:3916 \| DOI: 10.1038/s41467-018-06449-x \| www.nature.com/naturecommunications 6 6 ARTICLE NATURE COMMUNICATIONS \| (2018) 9:3916 \| DOI: 10.1038/s41467-018-06449-x \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x 8 months old 5 months old Cortex Hippocampus J20 baseline J20 + vehicle J20 + VX-765 WT + vehicle J20/KO a 150 120 90 60 30 0 Aβ staining (μm2 mm–2) 150 120 90 60 30 0 J20+veh J20+veh J20+VX J20+VX * Hippo Cortex b b b a * Hippo Hippo Cortex Cortex Hippo Hippo Cortex Cortex Aβ42/Aβ38,40,42 Aβ42Aβ38,40,42 –1 Total Aβ (pg mg–1) Total Aβ (pg mg–1 ×103) 0.6 0.4 0.2 0 J20-BL J20+veh J20+VX J20-BL J20+veh J20+VX J20-BL J20+veh J20+VX J20-BL J20+veh J20+VX J20-BL J20+veh J20+VX J20-BL J20+veh J20+VX J20-BL J20+veh J20+VX J20-BL J20+veh J20+VX 500 400 300 200 100 0 1.0 0.8 0.6 0.4 0.2 0 12 8 4 0 12 8 4 0 1.0 0.8 0.6 0.4 0.2 0 500 400 300 200 100 0 0.6 0.4 0.2 0 150 120 90 60 30 0 30 20 10 0 30 20 10 0 Aβ staining (μm2 mm–2) Aβ staining (μm2 mm–2) 150 120 90 60 30 0 J20/WT J20/KO J20/WT J20+veh J20+veh J20+VX J20+VX J20/KO ** * * * 8 months old 5 months old Cortex Hippocampus J20 baseline J20 + vehicle J20 + VX-765 WT + vehicle J20/KO c d e f g (Fig. 7d). Il-1β and TNF-α levels were signiﬁcantly increased after serum deprivation but reduced after VX-765 (Fig. 7e). These results indicate that VX-765 can prevent human neuronal degeneration. Discussion These results strongly indicate that VX-765 is a promising drug against AD. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x This could indicate that pro-Casp9 has been converted to active subunits, and that activation was inhibited by VX-765. Yet, activated Casp9 subunits could not be detected in the mouse brain. Since Casp3 is an excellent sub- strate of Casp9, we veriﬁed if Casp3 was processed into its active subunits in the hippocampal tissues. No active Casp3 subunit was observed in vehicle- or VX-765-treated J20 hippocampus, and there was also no effect of VX-765 on pro- Casp3 levels. Cleaved Casp8 20 kDa active subunit could not be detected in the hippocampus in either WT or J20 mice. A faint band corresponding to 43 kDa cleaved Casp8 active subunit (p43) was detected in the cortex, but its levels remained unaf- fected after VX-765 treatment (see Supplementary Fig. 9a). d d l l h d l VX-765 prevents axonal degeneration in human neurons. To determine if VX-765 can protect neurons against neurodegen- eration, VX-765 was assessed in human CNS human primary neuron cultures (HPN). Treatment of HPN with 25, 50, 100, or 200 μM VX-765 for 72 h was not toxic (Fig. 7a). As pre- viously described19, EGFP was homogeneously distributed within the cell body and neurites of HPN, whereas coexpression with APPWT resulted in EGFP-positive beading indicative of neuro- degeneration (Fig. 7b). HPN were pretreated with VX-765 for 1 h before APP transfection and treatment continued thereafter. VX- 765 treatment at 25 and 50 µM prevented neuritic beading in APPWT-transfected neurons at 48 and 72 h post transfection, similar to the Casp1 Z-YVAD-fmk peptide inhibitor (Fig. 7c). Similarly, VX-765 protected against, albeit less strongly, serum- deprivation-induced neuritic beading. To assess if neuritic bead- ing was reversible, VX-765 was administered 48 h after APPWT transfection or serum deprivation. VX-765 treatment did not reverse but prevented further neuritic beading in APP-transfected neurons. In contrast, Z-YVAD-fmk was unable to reverse or prevent neuritic beading at any time point. The 50 µM VX-765 reduced secreted and cellular Aβ42/Aβ38 + 40 + 42 levels, whereas 25 µM reduced secreted, but not cellular, Aβ42/total Aβ levels y g Because processed Casp8 and Casp9 levels in the adult mouse brain were low, VX-765’s potential effects on Casp8 and Casp9 were further analysed in the spleen, which expresses higher levels NATURE COMMUNICATIONS \| (2018) 9:3916 \| DOI: 10.1038/s41467-018-06449-x \| www.nature.com/naturecommunication 7 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x Our idea of using Casp1 as a therapeutic target is based on the identiﬁcation of the Nlrp1-Casp1-Casp6 neuronal * * Hippo Hippo Cortex Cortex Hippo Hippo Cortex Cortex 0.4 0.3 0.2 0.1 0 800 600 400 200 0 800 600 400 200 0 0.4 0.3 0.2 0.1 0 Aβ42/Aβ38,40,42 Aβ42/Aβ38,40,42 Aβ42Aβ38,40,42 –1 Aβ42/Aβ38,40,42 –1 Total Aβ (pg mg–1) Total Aβ (pg mg–1) Total Aβ (pg mg–1 ×103) Total Aβ (pg mg–1 ×103) 0.6 0.4 0.2 0 J20-BL J20+veh J20+VX J20-BL J20+veh J20+VX J20-BL J20+veh J20+VX J20-BL J20+veh J20+VX J20-BL J20+veh J20+VX J20-BL J20+veh J20+VX J20-BL J20+veh J20+VX J20-BL J20+veh J20+VX J20/WT J20/het J20/KO J20/WT J20/het J20/KO J20/WT J20/het J20/KO J20/WT J20/het J20/KO J20/WT J20/het J20/KO J20/WT J20/het J20/KO J20/WT J20/het J20/KO J20/WT J20/het J20/KO 500 400 300 200 100 0 1.0 0.8 0.6 0.4 0.2 0 1.0 0.8 0.6 0.4 0.2 0 250 200 150 100 50 0 250 200 150 100 50 0 400 300 200 100 0 150 kDa 100 kDa 50 kDa 150 kDa 50 kDa 37 kDa 50 kDa 50 kDa 100 kDa 100 kDa 100 kDa 37 kDa hAPP levels (% J20+veh) hAPP levels (% J20/WT) 400 300 200 100 0 12 8 4 0 6 4 2 0 4 3 2 1 0 12 8 4 0 1.0 0.8 0.6 0.4 0.2 0 1.0 0.8 0.6 0.4 0.2 0 500 400 300 200 100 0 0.6 0.4 0.2 0 Formic acid-soluble RIPA-soluble 120 90 60 30 0 30 20 10 0 30 20 10 0 Aβ staining (μm2 mm–2 Aβ staining (μm2 mm–2) 120 90 60 30 0 J20/WT J20/KO J20/WT J20+veh J20+veh J20+VX J20+VX J20/KO * 8 months old 5 months old Cortex Hippocampus J20/WT J20/Het J20/KO J20/WT J20/Het J20/KO J20+VX J20+VX J20+veh J20+veh Vehicle J20+VX J20 WT J20 WT WT/WT J20/WT J20/Het J20/KO hAPP hAPP β-actin β-actin hAPP Hippo Cortex Hippo Cortex hAPP β-actin β-actin c d e f g h i j k l m n o 30 20 10 0 30 20 10 0 Aβ staining (μm2 mm–2) J20/WT J20/KO J20/WT J20/KO ** * c c ** * Hippo Hippo Cortex Cortex Hippo Hippo Cortex Cortex Aβ42/Aβ38,40,42 Aβ42Aβ38,40,42 –1 Total Aβ (pg mg–1) Total Aβ (pg mg–1 ×103) 0.6 0.4 0.2 0 J20-BL J20+veh J20+VX J20-BL J20+veh J20+VX J20-BL J20+veh J20+VX J20-BL J20+veh J20+VX J20-BL J20+veh J20+VX J20-BL J20+veh J20+VX J20-BL J20+veh J20+VX J20-BL J20+veh J20+VX 500 400 300 200 100 0 1.0 0.8 0.6 0.4 0.2 0 12 8 4 0 12 8 4 0 1.0 0.8 0.6 0.4 0.2 0 500 400 300 200 100 0 0.6 0.4 0.2 0 J20/WT J20/KO J20/WT J20/KO 8 months old 5 months old d e f g g d Aβ42/Aβ38,40,42 –1 Total Aβ (pg mg–1 ×103) J J J J J20/WT J20/het J20/KO J20/WT J20/het J20/KO J20/WT J20/het J20/KO J20/WT J20/het J20/KO 1.0 0.8 0.6 0.4 0.2 0 6 4 2 0 4 3 2 1 0 1.0 0.8 0.6 0.4 0.2 0 j k ** * 0.4 0.3 0.2 0.1 0 800 600 400 200 0 800 600 400 200 0 0.4 0.3 0.2 0.1 0 Aβ42/Aβ38,40,42 Aβ42/Aβ38,40,42 –1 Total Aβ (pg mg–1) Total Aβ (pg mg–1 ×103) J20/WT J20/het J20/KO J20/WT J20/het J20/KO J20/WT J20/het J20/KO J20/WT J20/het J20/KO J20/WT J20/het J20/KO J20/WT J20/het J20/KO J20/WT J20/het J20/KO J20/WT J20/het J20/KO 1.0 0.8 0.6 0.4 0.2 0 6 4 2 0 4 3 2 1 0 1.0 0.8 0.6 0.4 0.2 0 h i j k j j i h k 150 kDa 100 kDa 50 kDa 150 kDa 50 kDa 37 kDa 100 kDa 37 kDa RIPA-soluble Vehicle J20+VX J20 WT J20 WT hAPP hAPP β-actin β-actin Hippo Cortex l l l 400 300 200 100 0 hAPP levels (% J20+veh) 400 300 200 100 0 Formic acid soluble J20+VX J20+VX J20+veh J20+veh m m m 250 200 150 100 50 0 250 200 150 100 50 0 hAPP levels (% J20/WT) J20/WT J20/Het J20/KO J20/WT J20/Het J20/KO o 50 kDa 50 kDa 100 kDa 100 kDa WT/WT J20/WT J20/Het J20/KO hAPP Hippo Cortex hAPP β-actin β-actin n o n (Fig. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x c Signiﬁcantly altered (Kruskall−Wallis) ynaptic protein mRNA levels in vehicle or VX-765-treated J20 mice hippocampi (n = 3 per group) Hippocampus Cortex J20 baseline J20 + vehicle J20 + VX-765 WT + vehicle 5 months old a a 8 months old Fold change (relative to WT) 1.5 1.0 0.5 0 1.5 1.0 0.5 0 1.5 1.0 2.5 7 6 5 4 3 2 1 0 2.0 0.5 0 J20+veh J20+VX J20+veh J20+VX J20+veh J20+VX J20+veh J20+VX Camk2a 0.044 p Grin2b 0.006 Kif17 0.044 TNF-α 0.045 c 8 months old 5 months old Synaptophysin staining (μm2 mm–2 ×103) 250 200 150 100 50 0 WT+veh J20+veh J20+VX WT+veh J20+veh J20+VX 250 200 150 100 50 0 Hippo Cortex b Fold change (relative to WT) 1.5 1.0 0.5 0 1.5 1.0 0.5 0 1.5 1.0 2.5 7 6 5 4 3 2 1 0 2.0 0.5 0 J20+veh J20+VX J20+veh J20+VX J20+veh J20+VX J20+veh J20+VX Camk2a 0.044 p Grin2b 0.006 Kif17 0.044 TNF-α 0.045 c Fig. 6 VX-765 reverses loss of synaptophysin in J20 mice. a Synaptophysin immunopositive micrographs. Scale bar = 200 µm for hippocampus and 50 µm for cortex. b Synaptophysin immunopositive density in WT + vehicle (grey squares, n = 4), J20 + vehicle (black circles, n = 4), and J20 + VX-765 (blue triangles, n = 4) hippocampus (F(2,9) = 7.974, p = 0.0102, ANOVA, Tukey’s post-hoc, p < 0.01) and cortex. c Signiﬁcantly altered (Kruskall−Wallis) synaptic protein mRNA levels in vehicle or VX-765-treated J20 mice hippocampi (n = 3 per group) Synaptophysin staining (μm2 mm–2 ×103) 250 200 150 100 50 0 WT+veh J20+veh J20+VX WT+veh J20+veh J20+VX 250 200 150 100 50 0 Hippo Cortex b Synaptophysin staining (μm2 mm–2 ×103) WT+veh J20+veh J20+VX 250 200 150 100 50 0 Hippo b b p Fig. 6 VX-765 reverses loss of synaptophysin in J20 mice. a Synaptophysin immunopositive micrographs. Scale bar = 200 µm for hippocampus and 50 µm for cortex. b Synaptophysin immunopositive density in WT + vehicle (grey squares, n = 4), J20 + vehicle (black circles, n = 4), and J20 + VX-765 (blue triangles, n = 4) hippocampus (F(2,9) = 7.974, p = 0.0102, ANOVA, Tukey’s post-hoc, p < 0.01) and cortex. c Signiﬁcantly altered (Kruskall−Wallis) synaptic protein mRNA levels in vehicle or VX-765-treated J20 mice hippocampi (n = 3 per group) inﬂammation and Aβ accumulation. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x ARTICLE Fig. 5 VX-765 prevents progressive Aβ accumulation in J20 mice. a Aβ micrographs of hippocampal SLM and S1 cortex (scale bar = 50 µm). b Quantitative analysis comparing Aβ immunostaining density between J20 + vehicle (black circles) and J20 + VX-765 (blue triangles) mice in the CA1 hippocampal pyramidal cell layer to the SLM (p = 0.0029) and cortex (p = 0.0314, unpaired t test). c Quantitative analysis comparing Aβ immunostaining density between J20−/+/Casp1+/+ J20/WT (black circles) and J20−/+/Casp1−/−J20/KO (blue triangles) mice in the CA1 hippocampal cell layer (p = 0.0040) and cortex (0.0276, unpaired t test). d−g RIPA- (d, e) and formic acid- (f, g) soluble Aβ levels in the hippocampus and cortex of 5-month baseline J20−/+ (black diamonds), J20 + vehicle (black circles), and J20 + VX-765 (blue triangles) mice. d, f Aβ42/Aβ38 + Aβ40 + Aβ42 levels (RIPA soluble, hippo: F(2,17) = 27.85, p < 0.0001) and e, g total Aβ ANOVA, Dunnett’s post-hoc versus J20 + vehicle, p < 0.001, *p < 0.0001. h−k J20−/+/Casp1+/+ J20/WT (black circles), J20−/+/Casp1−/+ J20/Het (purple triangles), and J20−/+/Casp1−/−J20/KO (blue triangles) RIPA- (h, i) and formic acid- (j, k) soluble Aβ levels in the hippocampus and cortex of J20/WT, J20/Casp1+/−(J20/Het), and J20/Casp1−/−J20/KO mice. h, j Aβ42/Aβ38 + Aβ40 + Aβ42 levels (RIPA- soluble, hippo: F(2,26) = 16.03, p < 0.0001; RIPA-soluble, cortex: F(2,26) = 6.602, p < 0.0048) and i, k total Aβ ANOVA, Dunnett’s post-hoc versus J20/ WT, p < 0.01, *p < 0.001, p < 0.0001. l−o Human APP protein levels (6E10 immunostaining) and quantiﬁcation in the hippocampus and cortex of l, m J20 + vehicle (black circles) and J20 + VX-765 mice (blue triangles), and n−o J20/WT (black circles), J20/Het (purple triangles), and J20/KO (blue triangles) mice Fig. 5 VX-765 prevents progressive Aβ accumulation in J20 mice. a Aβ micrographs of hippocampal SLM and S1 cortex (scale bar = 50 µm). b Quantitative analysis comparing Aβ immunostaining density between J20 + vehicle (black circles) and J20 + VX-765 (blue triangles) mice in the CA1 hippocampal pyramidal cell layer to the SLM (p = 0.0029) and cortex (p = 0.0314, unpaired t test). c Quantitative analysis comparing Aβ immunostaining density between J20−/+/Casp1+/+ J20/WT (black circles) and J20−/+/Casp1−/−J20/KO (blue triangles) mice in the CA1 hippocampal cell layer (p = 0.0040) and cortex (0.0276, unpaired t test). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x d−g RIPA- (d, e) and formic acid- (f, g) soluble Aβ levels in the hippocampus and cortex of 5-month baseline J20−/+ (black diamonds), J20 + vehicle (black circles), and J20 + VX-765 (blue triangles) mice. d, f Aβ42/Aβ38 + Aβ40 + Aβ42 levels (RIPA soluble, hippo: F(2,17) = 27.85, p < 0.0001) and e, g total Aβ ANOVA, Dunnett’s post-hoc versus J20 + vehicle, p < 0.001, *p < 0.0001. h−k J20−/+/Casp1+/+ J20/WT (black circles), J20−/+/Casp1−/+ J20/Het (purple triangles), and J20−/+/Casp1−/−J20/KO (blue triangles) RIPA- (h, i) and formic acid- (j, k) soluble Aβ levels in the hippocampus and cortex of J20/WT, J20/Casp1+/−(J20/Het), and J20/Casp1−/−J20/KO mice. h, j Aβ42/Aβ38 + Aβ40 + Aβ42 levels (RIPA- soluble, hippo: F(2,26) = 16.03, p < 0.0001; RIPA-soluble, cortex: F(2,26) = 6.602, p < 0.0048) and i, k total Aβ ANOVA, Dunnett’s post-hoc versus J20/ WT, p < 0.01, *p < 0.001, p < 0.0001. l−o Human APP protein levels (6E10 immunostaining) and quantiﬁcation in the hippocampus and cortex of l, m J20 + vehicle (black circles) and J20 + VX-765 mice (blue triangles), and n−o J20/WT (black circles), J20/Het (purple triangles), and J20/KO (blue triangles) mice Hippocampus Cortex J20 baseline J20 + vehicle J20 + VX-765 WT + vehicle 8 months old 5 months old Synaptophysin staining (μm2 mm–2 ×103) 250 200 150 100 50 0 WT+veh J20+veh J20+VX WT+veh J20+veh J20+VX 250 200 150 100 50 0 Hippo Cortex a b Fold change (relative to WT) 1.5 1.0 0.5 0 1.5 1.0 0.5 0 1.5 1.0 2.5 7 6 5 4 3 2 1 0 2.0 0.5 0 J20+veh J20+VX J20+veh J20+VX J20+veh J20+VX J20+veh J20+VX Camk2a 0.044 p Grin2b 0.006 Kif17 0.044 TNF-α 0.045 c g. 6 VX-765 reverses loss of synaptophysin in J20 mice. a Synaptophysin immunopositive micrographs. Scale bar = 200 µm for hippocampus and 50 µm or cortex. b Synaptophysin immunopositive density in WT + vehicle (grey squares, n = 4), J20 + vehicle (black circles, n = 4), and J20 + VX-765 (blue iangles, n = 4) hippocampus (F(2,9) = 7.974, p = 0.0102, ANOVA, Tukey’s post-hoc, *p < 0.01) and cortex. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x 7d). Il-1β and TNF-α levels were signiﬁcantly increased after serum deprivation but reduced after VX-765 (Fig. 7e). These results indicate that VX-765 can prevent human neuronal degeneration. Discussion These results strongly indicate that VX-765 is a promising drug against AD. Our idea of using Casp1 as a therapeutic target is based on the identiﬁcation of the Nlrp1-Casp1-Casp6 neuronal 8 NATURE COMMUNICATIONS \| (2018) 9:3916 \| DOI: 10.1038/s41467-018-06449-x \| www.nature.com/naturecommunications (Fig. 7d). Il-1β and TNF-α levels were signiﬁcantly increased after serum deprivation but reduced after VX-765 (Fig. 7e). These results indicate that VX-765 can prevent human neuronal degeneration. Discussion These results strongly indicate that VX-765 is a promising drug against AD. Our idea of using Casp1 as a therapeutic target is based on the identiﬁcation of the Nlrp1-Casp1-Casp6 neuronal NATURE COMMUNICATIONS \| (2018) 9:3916 \| DOI: 10.1038/s41467-018-06449-x \| www.nature.com/naturecommunications 8 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x 0.10 0.08 0.06 0.04 0.02 0 Secreted Cellular EGFP % beaded neurons % beaded neurons MTT abs (560 – 670 nm) EGFP + APP Aβ42/total Aβ 0.10 0.08 0.06 0.04 0.02 0 + Serum 2.5 2.0 1.5 1.0 0.5 0 10 8 6 4 2 0 Serum deprived APP transfected Stress + DMSO Stress + 5 μM YVAD Stress + 25 μM VX-765 Stress + 50 μm VX-765 GFP + DMSO Pretreatment Postbeading treatment + treatment * * 1.0 0.9 0.8 0.7 0.6 0.5 DMSO 25 50 100 200 100 80 60 40 20 0 % beaded neurons 100 80 60 40 20 0 100 80 60 40 20 0 IFN-γ (rel to + serum) % beaded neurons TNF-α (rel to + serum) IL-6 (rel to + serum) IL-1β (rel to + serum) 2.5 2.0 1.5 1.0 0.5 0 * * ** * 5 4 3 2 1 0 – Serum – Serum + 25 μM VX-765 – Serum + 50 μM VX-765 Secreted Secreted Secreted Secreted + treatment * * 24 h 48 h 72 h 96 h 120 h * * * * 100 80 60 40 20 0 24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h 96 h 120 h ** * * * ** VX-765 (μM) a c b d e Fig. 7 VX-765 protects human neurons against stress-mediated neuritic beading. a MTT assay. b EGFP ﬂuorescent micrographs of EGFP- or APP+ EGFP- transfected neurons. c % beading in 25 or 50 µM VX-765 or 5 µM Z-YVAD-fmk Casp1 peptide inhibitor pretreated (1 h) or post-treated (48 h) APP- transfected or serum-deprived neurons. DMSO vehicle treatment of EGFP-transfected neurons (grey squares), DMSO- (black circles), 5 µM YVAD- (purple hexagons), 25 µM VX-765- (blue triangles) or 50 µM VX-765- (purple triangles)-treated stressed (APP transfection in upper panels or serum deprived in bottom panel) neurons. Two-way repeated-measures ANOVA, Dunnett’s post hoc versus APPWT or serum-deprived, p < 0.05, p < 0.01, p < 0.001, *p < 0.0001. Pretreatment: APPWT (Treatment F(4,10) = 10.17, p = 0.0015; Time F(2,20) = 93.32, p < 0.0001; Treatment × Time F(8,20) = 6.736, p = 0.0003), serum-deprived (Treatment F(4,10) = 10.2, p = 0.0015; Time F(2,20) = 37.65, p < 0.0001). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x Pretreatment: APPWT (Treatment F(4,10) = 10.17, p = 0.0015; Time F(2,20) = 93.32, p < 0.0001; Treatment × Time F(8,20) = 6.736, p = 0.0003), serum-deprived (Treatment F(4,10) = 10.2, p = 0.0015; Time F(2,20) = 37.65, p < 0.0001). Postbeading treatment: APPWT (Time F (4,40) = 102.7, p < 0.0001), serum-deprived (Treatment F(4,23) = 14.12, p < 0.0001; Time F(4,23) = 24.35, p < 0.0001). d, e Serum-treated normal (grey square), serum-deprived (black circles), serum-deprived and treated with 25 µM VX-765 (blue triangles), and serum-deprived and treated with 50 µM VX- 765 (pink triangles) neurons. d Secreted or cellular Aβ42/ Aβ38 + Aβ40 + Aβ42. e Secreted Il-1β (F(3,8) = 6.636, p = 0.0146), IFN-γ, TNF-α (F(3,8) = 3.931, p = 0.054), and IL-6 (F(3,8) = 10.24, p = 0.0041) relative to untreated (+Serum) HPN. ANOVA, Dunnett’s post-hoc versus +Serum, p<0.05. a, c, d, e. Each individual neuron preparation tested is represented by one symbol. Data represent mean and s.e.m. AD-like pathologies5. LTP and dendritic spine density are restored, and increased Aβ levels are reduced, in APP/PS1/Casp1 −/−mice5. While the defect in AD immunity is largely attributed to CNS resident microglia, astrocytes and perivascular myeloid immune cells, we have identiﬁed abnormal activation of the Nlrp1 inﬂammasome in AD neurons, resulting in Casp1 activa- tion, which then can activate Casp6-mediated neurodegeneration and Il-1β-mediated glial inﬂammation11. By targeting Casp1 in both neurons and glia, VX-765 provides a double protection against these coexisting degenerative AD features. AD-like pathologies5. LTP and dendritic spine density are restored, and increased Aβ levels are reduced, in APP/PS1/Casp1 −/−mice5. While the defect in AD immunity is largely attributed to CNS resident microglia, astrocytes and perivascular myeloid immune cells, we have identiﬁed abnormal activation of the Nlrp1 inﬂammasome in AD neurons, resulting in Casp1 activa- tion, which then can activate Casp6-mediated neurodegeneration and Il-1β-mediated glial inﬂammation11. By targeting Casp1 in both neurons and glia, VX-765 provides a double protection against these coexisting degenerative AD features. accumulation and deposition of Aβ. Furthermore, it became apparent that cremophor vehicle injections increased Aβ accu- mulation in the J20 mouse of the VX-765 study compared to the Casp1 null study. It is tempting to speculate that either the cre- mophor or the injections mimic increased inﬂammation in the aged brain. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x To our knowledge, this drug provides the most rapid response observed with a preclinical treatment in the J20 AD mouse model40–42. degeneration pathway in CNS primary human neurons, the association of this pathway with memory and cognitive impair- ment in aged humans and mice11–16,36,37, and the genetic asso- ciation between NLRP1 and CASP1 with AD progression or AD38,39. VX-765 effect is exquisitely rapid against episodic and spatial memory impairment, which are normalized after only 1 (3 injections) or 3 (9 injections) weeks of treatment, respectively. Reversal of cognitive impairment is accompanied by normalized microglial and astroglial reactivity and synaptophysin immuno- histochemical staining, a normalization of gene expression of four different synaptic components, and the prevention of progressive amyloid pathology in the brain. Target engagement for VX-765 against Casp1 was conﬁrmed in the J20 Casp1 null mice, which also exhibited normal episodic and spatial memory, and reduced degeneration pathway in CNS primary human neurons, the association of this pathway with memory and cognitive impair- ment in aged humans and mice11–16,36,37, and the genetic asso- ciation between NLRP1 and CASP1 with AD progression or AD38,39. VX-765 effect is exquisitely rapid against episodic and spatial memory impairment, which are normalized after only 1 (3 injections) or 3 (9 injections) weeks of treatment, respectively. Reversal of cognitive impairment is accompanied by normalized microglial and astroglial reactivity and synaptophysin immuno- histochemical staining, a normalization of gene expression of four different synaptic components, and the prevention of progressive amyloid pathology in the brain. Target engagement for VX-765 against Casp1 was conﬁrmed in the J20 Casp1 null mice, which also exhibited normal episodic and spatial memory, and reduced Casp1 represents a feasible therapeutic target against age- dependent cognitive impairment and AD. Rare naturally occur- ring human CASP1 variants associated with very low activity, in vitro and in vivo, suggest that inhibition of Casp1 activity is compatible with normal human life44,45. In these CASP1 variants, proinﬂammatory signalling still occurs through NFKβ46. Casp1 null mice are fertile and healthy indicating that Casp1 is not essential for proper development47. The implication of microglia in AD progression has been nicely demonstrated in the APP/PS1 AD transgenic mouse model where Aβ-mediated activation of microglial Nlrp3 inﬂammasome results in cognitive deﬁcits and NATURE COMMUNICATIONS \| (2018) 9:3916 \| DOI: 10.1038/s41467-018-06449-x \| www.nature.com/naturecommunications 9 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x Postbeading treatment: APPWT (Time F (4,40) = 102.7, p < 0.0001), serum-deprived (Treatment F(4,23) = 14.12, p < 0.0001; Time F(4,23) = 24.35, p < 0.0001). d, e Serum-treated normal (grey square), serum-deprived (black circles), serum-deprived and treated with 25 µM VX-765 (blue triangles), and serum-deprived and treated with 50 µM VX- 765 (pink triangles) neurons. d Secreted or cellular Aβ42/ Aβ38 + Aβ40 + Aβ42. e Secreted Il-1β (F(3,8) = 6.636, p = 0.0146), IFN-γ, TNF-α (F(3,8) = 3.931, p = 0.054), and IL-6 (F(3,8) = 10.24, p = 0.0041) relative to untreated (+Serum) HPN. ANOVA, Dunnett’s post-hoc versus +Serum, p<0.05. a, c, d, e. Each individual neuron preparation tested is represented by one symbol. Data represent mean and s.e.m. MTT abs (560 – 670 nm) 1.0 0.9 0.8 0.7 0.6 0.5 DMSO 25 50 100 200 VX-765 (μM) a MSO M YVAD Stress + 25 μM VX-765 Stress + 50 μm VX 765 Postbeading treatment + treatment * * % beaded neurons 100 80 60 40 20 0 24 h 48 h 72 h 96 h 120 h a c EGFP % beaded neurons EGFP + APP S de Str *** * * * 100 80 60 40 20 0 24 h 48 h 72 h (μ ) b b rum rived 100 80 60 40 20 0 % beaded neurons + treatment * * ** 24 h 48 h 72 h 96 h 120 h 0.10 0.08 0.06 0.04 0.02 0 Secreted Cellular Aβ42/total Aβ 0.10 0.08 0.06 0.04 0.02 0 + Serum – Serum – Serum + 25 μM VX-765 – Serum + 50 μM VX-765 d d d 2.5 2.0 1.5 1.0 0.5 0 10 8 6 4 2 0 IFN-γ (rel to + serum) TNF-α (rel to + serum) IL-6 (rel to + serum) IL-1β (rel to + serum) 2.5 2.0 1.5 1.0 0.5 0 * * ** * 5 4 3 2 1 0 Secreted Secreted Secreted Secreted e e e rons against stress-mediated neuritic beading. a MTT assay. b EGFP ﬂuorescent micrographs of EGFP- or APP+ EGFP Fig. 7 VX-765 protects human neurons against stress-mediated neuritic beading. a MTT assay. b EGFP ﬂuorescent micrographs of EGFP- or APP+ EGFP- transfected neurons. NATURE COMMUNICATIONS \| (2018) 9:3916 \| DOI: 10.1038/s41467-018-06449-x \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x c % beading in 25 or 50 µM VX-765 or 5 µM Z-YVAD-fmk Casp1 peptide inhibitor pretreated (1 h) or post-treated (48 h) APP- transfected or serum-deprived neurons. DMSO vehicle treatment of EGFP-transfected neurons (grey squares), DMSO- (black circles), 5 µM YVAD- (purple hexagons), 25 µM VX-765- (blue triangles) or 50 µM VX-765- (purple triangles)-treated stressed (APP transfection in upper panels or serum deprived in bottom panel) neurons. Two-way repeated-measures ANOVA, Dunnett’s post hoc versus APPWT or serum-deprived, p < 0.05, p < 0.01, p < 0.001, *p < 0.0001. Pretreatment: APPWT (Treatment F(4,10) = 10.17, p = 0.0015; Time F(2,20) = 93.32, p < 0.0001; Treatment × Time F(8,20) = 6.736, p = 0.0003), serum-deprived (Treatment F(4,10) = 10.2, p = 0.0015; Time F(2,20) = 37.65, p < 0.0001). Postbeading treatment: APPWT (Time F (4,40) = 102.7, p < 0.0001), serum-deprived (Treatment F(4,23) = 14.12, p < 0.0001; Time F(4,23) = 24.35, p < 0.0001). d, e Serum-treated normal (grey square), serum-deprived (black circles), serum-deprived and treated with 25 µM VX-765 (blue triangles), and serum-deprived and treated with 50 µM VX- 765 (pink triangles) neurons. d Secreted or cellular Aβ42/ Aβ38 + Aβ40 + Aβ42. e Secreted Il-1β (F(3,8) = 6.636, p = 0.0146), IFN-γ, TNF-α (F(3,8) = 3.931, p = 0.054), and IL-6 (F(3,8) = 10.24, p = 0.0041) relative to untreated (+Serum) HPN. ANOVA, Dunnett’s post-hoc versus +Serum, p<0.05. a, c, d, e. Each individual neuron preparation tested is represented by one symbol. Data represent mean and s.e.m. Fig. 7 VX-765 protects human neurons against stress-mediated neuritic beading. a MTT assay. b EGFP ﬂuorescent micrographs of EGFP- or APP+ EGFP- transfected neurons. c % beading in 25 or 50 µM VX-765 or 5 µM Z-YVAD-fmk Casp1 peptide inhibitor pretreated (1 h) or post-treated (48 h) APP- transfected or serum-deprived neurons. DMSO vehicle treatment of EGFP-transfected neurons (grey squares), DMSO- (black circles), 5 µM YVAD- (purple hexagons), 25 µM VX-765- (blue triangles) or 50 µM VX-765- (purple triangles)-treated stressed (APP transfection in upper panels or serum deprived in bottom panel) neurons. Two-way repeated-measures ANOVA, Dunnett’s post hoc versus APPWT or serum-deprived, p < 0.05, p < 0.01, p < 0.001, **p < 0.0001. Methods S d d i Novel object recognition (NOR): mice were pre-exposed to two identical objects in the open ﬁeld chamber for 5 min. Two hours after pre-exposure, mice were placed back into the chamber and exposed to one familiar object and one novel object for 5 min. Mice were exposed to a speciﬁc object only once across different test sessions, and novel object placement was counterbalanced within each treatment group. Percentage touches of the novel object during the test phase and discrimination index ((# touches novel −# touches familiar)/total touches) were assessed. Casp1 validation study: To validate Casp1-speciﬁc effects of VX-765, J20 mice were generated on a Casp1−/−background. Seventy-three mice were used (n = 12 −13 per genotype, six different genotypes), all of which were bred through in vitro fertilization (IVF) from 35 donor females. Breeding details are described in the Animal Studies section below. All mice were behaviourally assessed between 7 and 8 months of age, and no animals were excluded from this study. All mice were sacriﬁced and processed at 8 months of age. Behavioural scoring was blinded to mouse genotype and treatment, and all animals were randomly assigned for either biochemical or histological analysis and blindly analysed. Barnes maze: the Barnes maze platform (91 cm diameter, elevated 90 cm from the ﬂoor) consisted of 20 holes (each 5 cm in diameter). All holes were blocked except for one target hole that led to a recessed escape box. Spatial cues, bright light, and white noise were used to motivate the mice to ﬁnd the escape during each session. For the adaptation phase, each mouse explored the platform for 60 s. Any mouse that did not ﬁnd the escape box was guided to it and remained there for 90 s. For the acquisition phase, each trial followed the same protocol, with the goal to train each mouse to ﬁnd the target and enter the escape box within 180 s. Mice remained in the box for an additional 60 s. Four trials per day, approximately 15 min apart, were performed for 4 consecutive days. In the probe test (day 5), each mouse performed one 90 s trial. The target was still located in the same position, but the escape box was blocked. The latency and errors to reach the target, plus the number of mouse pokes to each of the holes (target preference) were measured. Methods S d d i The Y-Maze was composed of a symmetrical Y-shaped maze with three arms 120° from each other, designated A, B, and C. Animals were placed at the distal end of arm A and allowed to explore the maze for 5 min. Total arm entries and spontaneous alternation, deﬁned as consecutive entries in three different arms, were recorded. Percentage alternation [(# alternations/total number arm entries – 2) × 100] was determined. VX-765, VRT-043198 IC50 assays on recombinant caspases. Recombinant caspases were commercially obtained (Biovision, Milpitas, CA, USA; K233-10-25) and used according to the manufacturer’s protocol. Caspase activity was measured using a ﬂuorescence-based assay using the following substrates (Enzo Life Sciences, NY, USA): N-Ac-Tyr-Val-Ala-Asp-7-amino-4-triﬂuoromethylcoumarin (Ac- YVAD-AFC) for human Casp1 and mouse Casp1, N-Ac-Val-Asp-Val-Ala-Asp-7- amino-4-triﬂuoromethylcoumarin (Ac-VDVAD-AFC) for human Casp2, N-Ac- AspGlu-Val-Asp-7-amino-4-triﬂuoromethylcoumarin (Ac-DEVD-AFC) for human Casp3 and Casp7, Ac-Trp-Glu-His-Asp-7-amino-4-tri- ﬂuoromethylcoumarin (Ac-WEHD-AFC) for human Casp4 and Casp5, and mouse Casp11, N-Ac-Val-Glu-Ile-Asp-7-amino-4-triﬂuoromethylcoumarin (Ac-VEID- AFC) for human Casp6, Ac-Ile-Glu-Thr-Asp-7-amino-4-triﬂuoromethylcoumarin (Ac-IETD-AFC) for human Casp8 and Casp10, and N-Ac-Leu-Glu-His-Asp-7- amino-4-triﬂuoromethylcoumarin (Ac-LEHD-AFC) for human Casp9. VX-765 (100 µM–100 pM) and VRT-043198 (100 µM–1 pM) (DSK Biopharma, Morrisville, NC) were mixed with 0.25 units of caspase and 10 µM substrate in a buffer con- taining 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; pH 7.2), 50 mM NaCl, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propane- sulfonate (CHAPS), 10 mM ethylenediaminetetraacetic acid (EDTA), 5% glycerol, and 10 mM dithiothreitol. Enzyme activity was measured in a Synergy H4 plate reader (BioTek, Winooski, VT, USA) at 380 nm excitation, 505 nm emission wavelengths every 30 s for 30 min at 37 °C. Cleavage rates were calculated from the linear phase of the assay. Fluorescence units were converted to the molar con- centration of released 7-amino-4-triﬂuoromethylcoumarin (AFC) based on a 0–25.0 µM free AFC standard curve. VX-765 IC50 values were determined by plotting relative activity (% DMSO control) to inhibitor concentrations. Data were ﬁtted to a dose−response curve equation containing a −1 slope with top and bottom constraints of 1 and 0, respectively. Tissue processing. For immunohistochemistry, mice (n = 4−6 per group) were anesthetised with isoﬂuorane and transcardially perfused with ice-cold saline fol- lowed by ice-cold 4% paraformaldehyde (Sigma-Aldrich, St Louis, MO, USA) in 0.1 M phosphate-buffered saline. Brains were post-ﬁxed in 10% neutral-buffered formalin (Thermo Fisher Scientiﬁc, Mississauga, ON, Canada) for 16 h and transferred to 70% ethanol. Brains were parafﬁn embedded and sectioned at 4 μm. Methods S d d i Study design. VX-765 study: Five-month old vehicle-injected WT (WT + vehicle; n = 18) and J20 mice (J20 + vehicle: n = 14), and VX-765-injected J20 mice (J20 + VX-765: n = 13) mice were longitudinally assessed at ﬁve different time points: baseline before treatment (baseline untreated J20 were grouped together; n = 19), after three IP injections per week of VX-765 or vehicle (Treatment 1), after six additional injections over 2 weeks (Treatment 2), after 4-week washout period (WO), and after an additional three injections in 1 week (Treatment 3) (Fig. 1a). All animals tested were included in the analyses unless (a) the animal died during the experiment, or (b) the animal did not behaviourally respond. A total of 25 litters, spread across four different cohorts and tested at different times, were used. Three of these cohorts underwent NOR and open ﬁeld testing, while cohort 4 was used for Barnes maze. After animals were baseline tested to conﬁrm that J20 animals were behaviourally deﬁcit, J20 animals were randomly assigned to either vehicle or VX-765 treatment independent of behavioural performance. Of all animals used, four J20 mice showed no behavioural deﬁcits at the beginning of the experiment and were not used. Two J20 + vehicle mice did not move at all during the experiment and their behavioural data excluded; however, these animals were kept and were still used for post-mortem analysis. Blood–brain barrier permeability analysis. Five-month-old J20 and WT mice were anesthetised with isoﬂurane and the right carotid artery was separated. A small incision was made along the carotid wall and a micro-catheter was inserted into the entrance of the internal carotid artery. The catheter was secured in place using suture thread. VX-765 (50 mg kg−1) was infused at 50 μl min−1 (90−120 μl total volume). The micro-catheter was left an additional 5 min to allow diffusion, after which blood was collected by intra-cardiac puncture. The mouse was trans- cardially perfused with ice-cold saline and the hippocampus and cortex were dis- sected out. Samples were sent to the Biopharmacy platform (Université de Montreal) for liquid chromatography and tandem mass spectrometry analysis. Behavioural analysis. Open ﬁeld: locomotor activity was measured using an HVS2100 automated tracking system (HVS Image, Hamptom, UK). Mice were placed in the open ﬁeld chamber (40 × 40 cm Plexiglas box, no ceiling, and white ﬂoor) and allowed to explore for 5 min. ARTICLE ARTICLE selectivity for Casp125,26, nontoxicity to human neurons and tolerability in phase 2b clinical trials against epilepsy29, blood–brain barrier permeability, and prevention of neurode- generation in cultured human CNS neurons suggest that it is a most promising novel therapy against both sporadic and familial forms of AD. Animals studies. All animal procedures followed the Canadian Council on Animal Care guidelines and were approved by the McGill University Animal Care com- mittee. The J20 transgenic mouse line (JAX Stock No. 006293, Jackson Labora- tories, Bar Harbor, ME, USA) expresses the Swedish (670/671KM→NL) and Indiana (717V→F) human APP mutations under the PDGF-βpromoter. Male J20 mice were used as breeders and their sperm was used for IVF colony expansion. The J20/Casp1 cohort was generated following the animal breeding standard protocols of the Institut de recherche en immunologie et en cancérologie (IRIC) at the Université de Montréal (Montreal, QC, Canada). Casp1 null mice were obtained from Jackson Laboratories (B6N.129S2-Casp1tm1Flv/J). IVF was carried out using fresh sperm from J20/Casp1+/−males and the eggs of WT/Casp1+/− donor females. Fertilized eggs were reimplanted in 20 C57Bl/6 females, generating pups with six possible genotypes: WT/Casp1+/+ (WT/WT), WT/Casp1+/−(WT/ Het), WT/Casp1−/−(WT/KO), J20/Casp1+/+ (J20/WT), J20/Casp1+/−(J20/Het), J20/Casp1−/−(J20/KO). Both males and females were used, aged at the IRIC, and then transferred to our laboratories prior to the start of experiments. It is likely that an efﬁcient treatment against AD will require very early treatment to prevent neuronal degeneration. Similar to antihypertensive or anticholesterol medications that protect against stroke or heart infarcts, we believe that the brain requires preventative early treatments before serious damage occurs. With this in mind, we chose to treat J20 mice only 1 month after the onset of cognitive decline. The outcome was unexpectedly posi- tive. These results suggest that an efﬁcient therapeutic interven- tion with VX-765 is likely to be more successful in mild cognitively impaired individuals or at the very early onset of clinically diagnosed AD. Genotypes were determined by tail biopsy and RT-PCR. Mice were group- housed (2−4 animals per cage) in standard macrolon cages under a 12-h reverse light/dark cycle and controlled environmental conditions. Food and water were available ad libitum. VX-765 (Adooq Bioscience, Irvine, CA) was dissolved in 20% cremophor in dH2O (Sigma-Aldrich, Oakville, ON, Canada) and administered intraperitoneally. Vehicle-treated J20 and WT mice received cremophor only. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x g Since VX-765 inhibits inﬂammatory Casp1, the results indicate that in the J20 mouse model, inﬂammation may be driving Aβ accumulation and contrast with the more popular view that Aβ drives inﬂammation (see Supplementary Fig. 10). These results are consistent with recent evidence indicating that microglia elimination prevents amyloid deposition3,4, and increased per- ipheral inﬂammatory markers in midlife humans are associated with brain atrophy and decreased cognition in ageing43. g g g The reduction of Aβ levels in VX-765-treated mice was unexpected. After only 12 treatments over a 3-month span, RIPA- and formic acid-soluble, immunostained, and thioﬂavin S- positive Aβ levels were considerably lower than in vehicle-treated J20 mice and remained similar to levels in pretreated 5-month- old J20 brains. Similarly, the absence of Casp1 in the J20/Casp1 −/−decreased Aβ deposits in the brain. These results indicate that VX-765 and the absence of Casp1 stop the progressive y g g g Whereas the J20 is a familial AD mouse model, the presence of the Nlrp1-Casp1-Casp6 pathway in mild cognitively impaired and in the early and late stages of sporadic human AD suggests that the treatment will also work in sporadic AD. VX-765’s 10 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x ARTICLE Representative images have only been cropped to conform to size constraints and have not undergone any post-processing. Neuronal cell culture and axonal degeneration assay. Twelve- to sixteen-week- old fetal cortical tissue were obtained from the Birth Defects Research Laboratory (University of Washington, Seattle, USA) in accordance with an approved protocol by the McGill institutional review board. HPN were cultured from brains that were dissociated with trypsin, treated with deoxyribonuclease I, and ﬁltered through 130, 70, and 30 μm nylon mesh21. Dissociated cells were centrifuged for 10 min at 300 × g and resuspended in minimal essential medium (MEM) with Earle’s salts, and supplemented with 5% decomplemented BCS, 1× Penicillin-Streptomycin, 1 mM sodium pyruvate, and 2 mM L-glutamine (Invitrogen, Carlsbad, CA, USA). Cells were seeded at a density of 3×106 cells ml−1 on 5 μg mL−1 poly-L-lysine-coated six- well tissue culture plates (Sigma-Aldrich). MEM medium was changed every 2 days and astrocyte proliferation was limited with ﬂuorodeoxyuridince (FdU) antimitotic treatment (1 mM, Sigma) for the ﬁrst 4 days. Neuronal cultures were grown 7 −10 days before conducting experiments. Four different neuron preparations at different times were tested. One of the neuron preparations was not stable and the culture, including the Serum + control that did not receive any drug, died in the middle of the experiment. Therefore, three different neuron preparations from three genetically different donors were used. Western blotting. Mouse protein samples (25–100 μg) were prepared in Laemmli (5% 2-β-mercaptoethanol, 1 mM Na3VO4, 1 mM NaF, 1 mM PMSF, 10 µl ml−1 of Proteases Inhibitors Cocktail (Sigma-Aldrich)), boiled for 5 min, and separated by polyacrylamide gel electrophoresis (PAGE). Samples were probed with the fol- lowing primary antibodies, diluted in either 5% nonfat dry milk in Tris-buffered saline and 0.1% Tween-20 or PBS blocking buffer (Thermoscientiﬁc): 1:1000 mouse anti-human Aβ1-16 (6E10) (BioLegend 803001, San Diego, CA, USA), 1:2000 rabbit anti-APP C-terminus (Sigma-Aldrich A8717), 1:1000 rabbit anti- neprilysin (Abcam ab79423), 1:500 rabbit anti-IDE (Abcam ab32216), 1:1000 rabbit anti-Caspase-3 (Cell Signaling 9665), 1:1000 mouse antiactive Caspase-8 (Cell Signaling 8592), 1:1000 rabbit anti-Caspase-9 (Cell Signaling 9504), and 1:5000 mouse anti β-actin (Sigma-Aldrich A5441). Blots were detected with 1:5000 HRP-linked anti-mouse (GE Amersham NA9310, Montreal, QC, CA) or 1:5000 anti-rabbit (Dako P0217) secondary antibody followed by ECL (GE Amersham). ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x ice with a tissue homogenizer (OMNI International, Kennesaw, GA, USA). Samples were centrifuged (20,000 × g, 4 °C) for 20 min, the supernatant recovered, and protein quantiﬁed using the BCA method. The RIPA-insoluble pellet was further extracted in 70% formic acid in dH2O, evaporated, and resolubilized in 200 mM Tris-HCl, pH 7.5. ice with a tissue homogenizer (OMNI International, Kennesaw, GA, USA). Samples were centrifuged (20,000 × g, 4 °C) for 20 min, the supernatant recovered, and protein quantiﬁed using the BCA method. The RIPA-insoluble pellet was further extracted in 70% formic acid in dH2O, evaporated, and resolubilized in 200 mM Tris-HCl, pH 7.5. 11 FX+, DeNovix, Wilmington, DE, USA). Five hundred nanograms of total RNA was reverse transcribed for each sample (with avian myeloblastosis reverse tran- scriptase (AMV-RT)) (Roche, Mannheim, Germany). PCR and real-time PCR. HAPP transgene PCR ampliﬁcation was performed using Taq DNA polymerase (New England Biolabs, Whitby, ON, Canada) using the following primers: (hAPP) 5′-AACACAGAAAACGAAGTT-3′ and 5′-CCGATG GGTAGTGAAGCA-3′ (480-bp amplicon)31, (Hypoxanthine-guanine phosphor- ibosyltransferase, HPRT) 5′-GTAATGATCAGTCAACGGGGGAC-3′ and 5′-CCA GCAAGCTTGCAACCTTAACCA-3′ (177-pb amplicon) (Carcinogenesis). Pro- ducts were visualized on RedSafe (FroggaBio, North York, ON, Canada) stained 2% agarose gels. Real-time PCR experiments were performed with SYBR Green Taq Mastermix (Quanta BioSciences, Gaithersburg, MD, USA) on an Applied Biosys- tems 7500 Fast Real-Time PCR system machine (Applied Biosystems, Foster City, CA, USA). Gene ampliﬁcation was performed with the following primers: (18s) 5′- GTAACCCGTTGAACCCCAT-3′ and 5′-CCATCCAATCGGTAGTAGCG-3′; (IDE) 5′- ACTAACCTGGTGGTGAAG-3′ and 5′- GGTCTGGTATGGGAAATG -3′; (Neprilysin) 5′- TCTTGTAAGCAGCCTCAGCC-3′ and 5′- CTCCCCACAG CATTCTCCAT-3′. Results are expressed as fold-induction values normalized to mean HPRT and 18S reference genes using the Pfafﬂ’s method. Immunohistological staining. Epitopes were demasked in either citrate (10 mM Tris-Na Citrate, pH 6.0) or EDTA (10 mM Tris Base, 1 mM EDTA, 0.05% Tween- 20, pH 9.0) antigen retrieval buffer, and immunostained using the Dako Auto- stainer Plus automated slide processor and EnVision Flex system (Dako, Bur- lington, ON, Canada). Following deparafﬁnization and rehydration, sections were peroxidase treated, blocked in serum-free protein block, and immunostained with the following antibodies diluted in EnVision Flex Antibody Diluent: 1:2000 rabbit anti-Iba1 (Wako 019-19741, Richmond, VA, USA), 1:8000 rabbit anti-GFAP (Dako Z-0334), 1:2000 rabbit anti-Aβ1-40 (F25276, laboratory developed), and 1:1000 mouse antisynaptophysin (Sigma-Aldrich S5768). Sections were treated with the kit-supplied appropriate mouse or rabbit HRP-conjugated secondary antibody and visualized with DAB. Sections were hematoxylin counterstained, dehydrated, and coverslipped with Permount (Fisher Scientiﬁc). ARTICLE Immunoreactive bands were visualized using ImageQuant LAS 4000 imaging system (Fujiﬁlm USA, Valhalla, NY, USA), and densitometric analyses were per- formed with Image Gauge analysis software (Fujiﬁlm USA). Brightness/contrast of raw blots were equally adjusted across the entire image with Adobe Photoshop CS5 software (Adobe Systems, Ottawa, ON, CA) to generate representative images. No additional modiﬁcation was made. CanvasTM X software (Acd system, Plan- tation, FL, USA) was used to create ﬁnal ﬁgures. Raw blots for western blots are available in Supplementary Figure 11. VX-765 toxicity was assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT). Neurons were treated with 25−200 μM VX- 765 or 0.1% DMSO (highest DMSO concentration used) for 72 h. Neurons were then incubated with 0.5 μg ml−1 MTT (Sigma-Aldrich) for 4 h at 37 °C (5% CO2). The formazan crystals were dissolved in 100 μl DMSO for 30 min. Absorbance was measured at 560 and 670 nm using the Synergy H4 plate reader. Axonal degeneration was induced either by APPWT transfection or serum deprivation stress. VX-765 was administered either as a pretreatment strategy (1 h prior to stressor) or as a postbeading treatment strategy (48 h after stressor). Neurons were transfected by Gene gun (BioRad, Mississauga, ON, Canada) with gold beads coated with pBudCE41-eGFP for ﬂuorescence visualization. Neurons that were APPWT stressed were transfected with pBudCE41-eGFP/APPWT. Brieﬂy, neurons were treated with medium supplemented with 25 or 50 μM VX-765, 5 μM Casp1 inhibitor Z-YVAD-fmk, or DMSO. Fluorescence images were taken every 24 h (up to 72 h post-treatment) with a Nikon Eclipse Ti microscope (Nikon, Mississauga, ON, CA) and Photometrics coolSNAP HQ2 CCD camera (Photometrics, Tucson, AZ, USA) using NIS Elements AR 3.10 software (Nikon). Percentage neuronal beading was measured by counting the number of beaded eGFP-positive neurons over total number of eGFP-positive neurons at each time point. A minimum of 150 eGFP-positive neurons was counted for each condition. Conditioned medium was sampled (supplemented with 1 mM Na3VO4, 1 mM NaF, 1 mM PMSF, 10 µl ml−1 of Proteases Inhibitors Cocktail (Sigma-Aldrich)), and neurons were harvested in cell lysis buffer (50 mM HEPES, 0.1% CHAPS, 0.1 mM EDTA) for analysis. ELISA. Pro- and anti-inﬂammatory cytokines, and Aβ levels in mouse brain were determined using electrochemiluminescence immune assay kits from Meso Scale Discovery (Rockville, MD, USA). Standards and samples were prepared according to the manufacturer’s protocols and run in duplicate. ARTICLE Sections were digitally scanned with MIRAX SCAN for analysis (Zeiss, Don Mills, ON, Canada). After deparafﬁnization and rehydration, slides were stained with ﬁltered 1% aqu- eous Thioﬂavin-S (Sigma-Aldrich). Mouse synaptic plasticity RT2-Proﬁler PCR array. The mouse synaptic plasticity RT2 Proﬁler PCR Array (PAMM-126Z, Qiagen) proﬁles the expression of 84 genes involved in synaptic alterations during learning and memory and ﬁve housekeeping genes (β-actin; β-2-microglobulin; glyceraldehyde 3-phosphate dehydrogenase, GAPDH; β-glucuronidase, Gusb; Heat shock protein 90 alpha (cytosolic), Hsp90ab1) with real-time PCR. Total RNA (465 ng for each sample) was reverse transcribed (which included a genomic DNA elimination step) following the manufacturer’s protocol (First Strand cDNA Synthesis Kit, Qiagen). The real-time PCR reaction was performed in a real-time cycler ABI 7500 (Fast Block) (Applied Biosystems) using the RT2 SYBR Green/ROX PCR master mix (Qiagen). Cycling conditions were as follows: 2 min at 50 °C, 10 min at 95 °C, 40 cycles, 15 s each at 95 °C, and 1 min at 60 °C. Threshold and baseline were set manually according to the manufacturer’s instructions. Data were normalized to the geometric mean of the ﬁve housekeeping genes and analysed by the comparative Ct-method (2−ΔCT). Quantitative immunohistological analysis. Iba1-positive cells in the cortex and anterior portion of the CA1 region of the hippocampus were quantiﬁed using a modiﬁed version of the areal counting frame48. Brieﬂy, every ﬁfth slide was quantiﬁed (resulting in four sections per brain). Multiple ×80 images were taken with the MIRAX SCAN within the area of interest and the number of Iba1-positive cells were manually counted. The numerical density (number of cells mm−3) in the anterior CA1 region and cortex was estimated. A minimum number of 150 hits per area were needed to make a reliable estimate. Additional sections were counted if we were unable to reach our minimum number. Quantiﬁcation was performed blinded for treatment and genotype. ImageJ software (NIH, Bethesda, MD, USA) was used to measure Aβ accumulation, GFAP and synaptophysin immunopositive staining in the CA1 region and cortex. Four sections per brain were analysed, and multiple images were taken within each section to cover the CA1 region and cortex. Threshold was equally adjusted across all brains to highlight the stained area, and particles were analysed to determine total area of staining. Results are expressed as total area stained per total area analysed (μm2 mm−2). 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(S)-1-((S)-2-{[1-(4-amino-3-chloro-phenyl)- methanoyl]-amino}-3,3-dimethyl-butanoy l)-pyrrolidine-2-carboxylic acid ((2R,3S)-2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide (VX-765), an orally available selective interleukin (IL)-converting enzyme/caspase-1 inhibitor, exhibits potent anti-inﬂammatory activities by inhibiting the release of IL- 1beta and IL-18. J. Pharmacol. Exp. Ther. 321, 509–516 (2007). ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x ARTICLE The ten-plex mouse proin- ﬂammatory panel was used for mouse hippocampal and cortical and samples. A single Il-1β kit was used to measure mouse plasma. The four-plex human proin- ﬂammatory panel was used for HPN samples (see below). The four-panel human Aβ 6E10 kit was used for mouse hippocampus and cortex, and HPN samples. RNA extraction and cDNA synthesis. Total RNA was extracted from mouse hippocampus and cortex (n = 3 per group) with Qiazol (Qiagen, Valencia, CA, USA) followed by homogenization with a 20-gauge needle. The miRNeasy mini kit, including on-column DNase treatment step, was used to purify total RNA (Qia- gen). RNA quality and quantity were determined using a spectrophotometer (DS- NATURE COMMUNICATIONS \| (2018) 9:3916 \| DOI: 10.1038/s41467-018-06449-x \| www.nature.com/naturecommunications 12 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06449-x ARTICLE Competing interests: The authors declare no competing interests. Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ Additional information 47. Kuida, K. et al. Altered cytokine export and apoptosis in mice deﬁcient in interleukin-1 beta converting enzyme. Science 267, 2000–2003 (1995). 47. Kuida, K. et al. Altered cytokine export and apoptosis in mice deﬁcient in interleukin-1 beta converting enzyme. Science 267, 2000–2003 (1995). Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- 018-06449-x. 48. Gundersen, H. 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Proteom. 7, 1541–1555 (2008). 13 NATURE COMMUNICATIONS \| (2018) 9:3916 \| DOI: 10.1038/s41467-018-06449-x \| www.nature.com/naturecommunications Acknowledgements We are indebted to Edith Hamel for sharing her laboratory for the blood–brain barrier experiments. We would like to thank the animal quarters staff, especially Véronique Michaud, for maintaining the mice, the IRIC Université de Montréal pathology core for embedding and cutting brain tissue sections. A.N. received a postdoctoral scholarship from the Alzheimer Society of Canada. This work was supported by funds from the Canadian Institutes of Health Research (CIHR) MOP-243413-BCA-CGAG-45097, CIHR project grant 153097, and the JGH Foundation to A.C.L. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Author contributions J.F.: animal behaviour and analyses, immunohistochemistry analyses, ELISA, cell culture experiments, blood–brain barrier permeability assays, designed experimental paradigm, made ﬁgures, wrote methods, results and revised manuscript. B.F.: molecular biology, immunostaining, prepared ﬁgures, wrote methods and results, revised the manuscript. A. N.: western blot analyses, prepared ﬁgures, wrote methods and results, revised the manuscript. J.L.: performed experiments for IC50 on recombinant enzymes, wrote methods and results. Revised the manuscript. C.L.: performed blood–brain barrier per- meability experiments, revised the manuscript. A.C.L.: Conception of the experimental idea and design, supervision of data collection and analysis, wrote manuscript. © The Author(s) 2018 14 NATURE COMMUNICATIONS \| (2018) 9:3916 \| DOI: 10.1038/s41467-018-06449-x \| www.nature.com/naturecommunications
https://openalex.org/W4361187470	https://aacr.figshare.com/articles/journal_contribution/Supplemental_Table_ST1_Detection_rates_of_somatic_exomic_TMB_10_t_based_on_stratification_by_panel-derived_TMB_measures_from_Probabilistic_Mixture_Models_Improve_Calibration_of_Panel-derived_Tumor_Mutational_Burden_in_the_Context_of_both_Tu/22348727/1/files/39763571.pdf	Armenian	null	Supplemental Table ST1. Detection rates of somatic, exomic TMB > 10 (t) based on stratification by panel-derived TMB measures. from Probabilistic Mixture Models Improve Calibration of Panel-derived Tumor Mutational Burden in the Context of both Tumor-normal and Tumor-only Sequencing	null	2,023	cc-by	239	Tumor Normal Tumor Only Exomic TMB > t n Exomic TMB > t n Panel TMB ˆy > t 53.5% 1486 30.6% 2758 ˆy ≤t 1.1% 8201 0.5% 6838 Linear Model ˆy > t 94.3% 507 93.1% 379 ˆy ≤t 4.4% 9180 5.7% 9217 > 95% certain, ˆy > t 100.0% 102 0.0% 0 > 95% certain, ˆy < t 0.5% 7733 0.6% 7075 < 95% certain, ˆy ̸= t 40.1% 1852 33.3% 2521 Mixture Model ˆy > t 87.7% 661 81.5% 653 ˆy ≤t 3.3% 9026 3.9% 8943 > 95% certain, ˆy > t 99.6% 247 94.7% 171 > 95% certain, ˆy < t 0.5% 7716 0.8% 7415 < 95% certain, ˆy ̸= t 34.8% 1724 32.5% 2010 Tumor Normal Tumor Only Exomic TMB > t n Exomic TMB > t n Panel TMB ˆy > t 53.5% 1486 30.6% 2758 ˆy ≤t 1.1% 8201 0.5% 6838 Linear Model ˆy > t 94.3% 507 93.1% 379 ˆy ≤t 4.4% 9180 5.7% 9217 > 95% certain, ˆy > t 100.0% 102 0.0% 0 > 95% certain, ˆy < t 0.5% 7733 0.6% 7075 < 95% certain, ˆy ̸= t 40.1% 1852 33.3% 2521 Mixture Model ˆy > t 87.7% 661 81.5% 653 ˆy ≤t 3.3% 9026 3.9% 8943 > 95% certain, ˆy > t 99.6% 247 94.7% 171 > 95% certain, ˆy < t 0.5% 7716 0.8% 7415 < 95% certain, ˆy ̸= t 34.8% 1724 32.5% 2010 1
https://openalex.org/W2991133171	https://hal.science/hal-02416411/document	English	null	Peer-Reviewed Literature on Grain Legume Species in the WoS (1980–2018): A Comparative Analysis of Soybean and Pulses	Sustainability	2,019	cc-by	24,411	To cite this version: Marie-Benoît Magrini, Guillaume Cabanac, Matteo Lascialfari, Gaël Plumecocq, Marie-Josèphe Amiot, et al.. Peer-Reviewed Literature on Grain Legume Species in the WoS (1980–2018): A Compar- ative Analysis of Soybean and Pulses. Sustainability, 2019, 11 (23), pp.6833. 10.3390/su11236833. hal-02416411 To cite this version: Marie-Benoît Magrini, Guillaume Cabanac, Matteo Lascialfari, Gaël Plumecocq, Marie-Josèphe Amiot, et al.. Peer-Reviewed Literature on Grain Legume Species in the WoS (1980–2018): A Compar- ative Analysis of Soybean and Pulses. Sustainability, 2019, 11 (23), pp.6833. 10.3390/su11236833. hal-02416411 Distributed under a Creative Commons Attribution 4.0 International License Peer-Reviewed Literature on Grain Legume Species in the WoS (1980–2018): A Comparative Analysis of Soybean and Pulses Marie-Benoît Magrini 1,* , Guillaume Cabanac 2 , Matteo Lascialfari 1, Gael Plumecocq 1, Marie-Josephe Amiot 3 , Marc Anton 4, Gaelle Arvisenet 5, Alain Baranger 6, Laurent Bedoussac 7 , Jean-Michel Chardigny 8 , Gérard Duc 9, Marie-Hélène Jeuﬀroy 10, Etienne-Pascal Journet 1,11, Hervé Juin 12, Colette Larré 4, Hugues Leiser 13, Valérie Micard 14, Dominique Millot 9, Marie-Laure Pilet-Nayel 6, Christophe Nguyen-Thé 15, Tristan Salord 1, Anne-Sophie Voisin 9, Stéphane Walrand 16 and Jacques Wery 17 Marie-Benoît Magrini 1,* , Guillaume Cabanac 2 , Matteo Lascialfari 1, Gael Plumecocq 1, Marie-Josephe Amiot 3 , Marc Anton 4, Gaelle Arvisenet 5, Alain Baranger 6, Marie-Benoît Magrini 1,* , Guillaume Cabanac 2 , Matteo Lascialfari 1, Gael Plumecocq 1, Marie-Josephe Amiot 3 , Marc Anton 4, Gaelle Arvisenet 5, Alain Baranger 6, Laurent Bedoussac 7 , Jean-Michel Chardigny 8 , Gérard Duc 9, Marie-Hélène Jeuﬀroy 10, Etienne-Pascal Journet 1,11, Hervé Juin 12, Colette Larré 4, Hugues Leiser 13, Valérie Micard 14, Dominique Millot 9, Marie-Laure Pilet-Nayel 6, Christophe Nguyen-Thé 15, Tristan Salord 1, Anne-Sophie Voisin 9, Stéphane Walrand 16 and Jacques Wery 17 Laurent Bedoussac 7 , Jean-Michel Chardigny 8 , Gérard Duc 9, Marie-Hélène Jeuﬀroy 10, Etienne-Pascal Journet 1,11, Hervé Juin 12, Colette Larré 4, Hugues Leiser 13, Valérie Micard 14, Dominique Millot 9, Marie-Laure Pilet-Nayel 6, Christophe Nguyen-Thé 15, Tristan Salord 1, Anne-Sophie Voisin 9, Stéphane Walrand 16 and Jacques Wery 17 1 AGIR, INRA, Université de Toulouse, 31326 Castanet-Tolosan, France; matteo.lascialfari@inra.fr (M.L.); gael.plumecocq@inra.fr (G.P.); etienne-pascal.journet@inra.fr (E.-P.J.); tristan.salord@inra.fr (T.S.) g p q ( ) p j ( J ) ( 2 IRIT, Université de Toulouse, 31062 Toulouse, France; guillaume.cabanac@univ-tlse3.fr g 3 MOISA, CIRAD, CIHEAM-IAAM, INRA, Montpellier SupAgro, Université Montpellier, 34060 Montpellier, France; marie-josephe.amiot-carlin@inra.fr j p 4 BIA, INRA, 44000 Nantes, France; marc.anton@inra.fr (M.A.); colette.larre@inra.fr (C.L.) 5 CSGA, AgroSup Dijon, CNRS, INRA, Université Bourgogne-Franche-Comté, 21000 Dijon, France; gaelle.arvisenet@agrosupdijon.fr 6 IGEPP, INRA, Agrocampus-Ouest, Université Rennes 1, 35653 Le Rheu, France; alain.baranger@inra.fr (A.B.) marie-laure.pilet-nayel@inra.fr (M.-L.P.-N.) 7 AGIR, INRA, ENSFEA, Université de Toulouse, 31326 Castanet-Tolosan, France; laurent.bedoussac 8 8 DPTI, INRA, 75338 Paris, France; jean-michel.chardigny@inra.fr 9 Agroecologie, INRA, AgroSup Dijon, Université Bourgogne France-Comté, 21000 Dijon, France; gerard.duc@inra.fr (G.D.); dominique.millot@inra.fr (D.M.); anne-sophie.voisin@inra.fr (A.-S.V.) 9 Agroecologie, INRA, AgroSup Dijon, Université Bourgogne France-Comté, 21000 Dijon, France; gerard.duc@inra.fr (G.D.); dominique.millot@inra.fr (D.M.); anne-sophie.voisin@inra.fr (A.-S.V.) 10 Agronomie, INRA, AgroparisTech, Université Paris-Saclay, 78850 Thiverval-Grignon, France; marie helene jeuﬀroy@inra fr 10 Agronomie, INRA, AgroparisTech, Université Paris-Saclay, 78850 Thiverval-Grignon, France; marie-helene.jeuﬀroy@inra.fr PM, Université de Toulouse, INRA, CNRS, 31326 Castanet-Tolosan, France 11 LIPM, Université de Toulouse, INRA, CNRS, 31326 Castanet-Tolosan, France 12 EASM, INRA, 17700 Surgères, France; herve.juin@inra.fr 13 IST, INRA, 84140 Avignon, France; hugues.leiser@inra.fr 13 IST, INRA, 84140 Avignon, France; hugues.leiser@inra.fr 14 14 IATE, INRA, Montpellier SupAgro, CIRAD, Université Montpellier, 34060 Montpellier, France; valerie.micard@supagro.fr p g 15 SQPOV, INRA, 84140 Avignon, France; christophe.nguyen-the@inra.fr RA, 84140 Avignon, France; christophe.nguyen-the@in 16 INRA, UNH, Unité de Nutrition Humaine, CRNH Auvergne, U Clermont-Ferrand, France; stephane.walrand@inra.fr 16 INRA, UNH, Unité de Nutrition Humaine, CRNH Auvergne, Université Clermont Auvergne, 6300 Clermont-Ferrand, France; stephane.walrand@inra.fr 17 ICARDA, Ismail El-Shaaer Building, Maadi 11728, Egypt; J.Wery@cgiar.org * Correspondence: marie-benoit.magrini@inra.fr; Tel.: +33-561-285-422 Received: 25 September 2019; Accepted: 22 November 2019; Published: 2 December 2019 Abstract: Grain-legume crops are important for ensuring the sustainability of agrofood systems. HAL Id: hal-02416411 https://hal.science/hal-02416411v1 Submitted on 26 May 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License sustainability sustainability www.mdpi.com/journal/sustainability Sustainability 2019, 11, 6833; doi:10.3390/su11236833 1. Introduction A major challenge for agriculture is to greatly reduce the use of synthetic inputs and to rely on more ecological processes. To foster sustainability, increasing crop diversity with legumes has been advanced as a major driver of change [1–3]. More legume cultivation will provide considerable ecological services, in addition to their nutritional advantages, as legumes enable a renewable input of nitrogen (N) into agricultural soils through symbiotic N2 ﬁxation, lowering the fertilizer N requirements and fossil energy use of farming systems, and thus reducing the net release of greenhouse gases into the atmosphere [4]. To obtain these ecosystem services, farming systems must increase crop diversity with more legumes cultivated both in quantity and in number of legume species cultivated. Moreover, crop diversity is linked to the capacity of markets to use a variety of crops for feed and food [5,6]. For soya, huge markets exist, whereas strong innovation is still needed for pulses to break out of the lock-in they face compared to global major crops [7–9]. Even though today legumes are increasingly promoted for human protein intake, enabling a reduction in animal-based consumption, pulses have had diﬃculties to develop in those markets [7]. Within the same crop family—grain-legumes—pulses and soya have developed very diﬀerently since the 1960s. Soya is the main worldwide protein crop grown mainly for feed use (with soya oil increasingly becoming a byproduct). Nowadays, the soya crop amounts to more than 300 million metric tons, while other main grain-legumes such as pulses (pea, lentils, lupins, faba beans, chickpeas, etc.) account for less than 100 million metric tons (Appendix A, Table A1). At global scale, pulse production has gained little ground, whether for food or for feed. Thus, the United Nations launched an International Year of Pulses (IYP) in 2016 to raise awareness about the considerable contributions pulses can make to the sustainability transition of agrofood systems, and to favor their development compared to major crops such as soya [10,11]. To contribute to such new trajectories for grain-legumes, there is a consensus in Science and Technology Studies (STS) that increasing scientiﬁc research is essential. Science provides a stock of knowledge for driving new developments and innovation [12–14]. Hence, the main objective of our study is to assess the shares of research activity among the grain-legumes crops in order to evaluate whether research on grain-legumes is itself locked around soya or provides knowledge on a variety of species. Peer-Reviewed Literature on Grain Legume Species in the WoS (1980–2018): A Comparative Analysis of Soybean and Pulses Among them, pulse production is subject to strong lock-in compared to soya, the leading worldwide crop. To unlock the situation and foster more grain-legume crop diversity, scientiﬁc research is essential for providing new knowledge that may lead to new development. Our study aimed to evaluate whether research activity on grain-legumes is also locked in favor of soya. Considering more than 80 names grouped into 19 main grain-legume species, we built a dataset of 107,823 scholarly publications (articles, book, and book chapters) between 1980 and 2018 retrieved from the Web of Science (Clarivate Analytics) reﬂecting the research activity on grain-legumes. We delineated 10 scientiﬁc themes of interest running the gamut of agrofood research (e.g., genetics, agronomy, and nutrition). We indexed grain-legume species, calculated the percentage of records for each one, and conducted several analyses longitudinally and by country. Globally, we found an unbalanced research Sustainability 2019, 11, 6833; doi:10.3390/su11236833 www.mdpi.com/journal/sustainability 2 of 37 Sustainability 2019, 11, 6833 output: soya remains the main crop studied, even in the promising ﬁeld of food sciences advanced by FAO as the “future of pulses”. Our results raise questions about how to align research priorities with societal demand for more crop diversity. output: soya remains the main crop studied, even in the promising ﬁeld of food sciences advanced by FAO as the “future of pulses”. Our results raise questions about how to align research priorities with societal demand for more crop diversity. Keywords: knowledge dynamics; science and technology studies (STS); research trajectories; scientometrics; bibliometric data; agricultural sciences; food sciences; sustainability 1. Introduction Measuring science has been an ambitious challenge for many authors in STS since the seminal works of the 1980s [14]. Bibliometric and scientometric methods are now well-established for analyzing scientiﬁc advancement and for orientating research policy [15–20]. The considerable development of scientiﬁc platforms [21], combined with algorithmic advances for performing bibliometric analyses, has furthered interest in exploring the dynamics of scientiﬁc knowledge [19,22]. Through knowledge and scientiﬁc analysis, gaps and opportunities in science can be identiﬁed and addressed to meet society’s needs for innovation. For sustainability transition studies, bibliometric analysis enables us to better understand the state of knowledge of the current sociotechnical regime that needs to be changed, particularly in sectors with strong path-dependency. Consequently, the number of bibliometric analyses has been growing for a variety of ﬁelds: in electronics [23], on scientiﬁc parks and the links to open innovation [24], or in pharmaceuticals [17]. However, few have been done in agriculture (e.g., on agroecology [25]), even though there are key sustainability issues challenging research. 3 of 37 Sustainability 2019, 11, 6833 To evaluate the respective shares of grain-legume species in the sciences, we used the Web of Science (WoS) collection to retrieve the scholarly publications reﬂecting research activity recognized by peers. As advanced by STS, and more speciﬁcally by scientometrics studies [19], these research papers remain the highest quality variable to reﬂect research activity on a given topic. Even though GS (Google Scholar) is growing and presented as alternative, the metadata oﬀered by GS are still very limited, reducing the practical suitability of this source for large-scale citation analyses. In addition, GS includes around 20–50% of other type of research documents (depending on the scientiﬁc ﬁelds), such as PhD dissertations, scientiﬁc reports, and non-peer-reviewed papers that create bias in comparing research advancement from more qualitative types of research documents [26]. Moreover, language harmonization rules are required to collect data on GS. Therefore, although perhaps not a perfect resource to reﬂect all the scientiﬁc knowledge stock, we chose a traditional bibliometric platform, as the WoS as better for providing more a relevant overview of peer-reviewed research activity (i.e., the scholarly documents such as articles in peer-reviewed journals, books and book chapters), 97% of which were written in English on the WoS. 1. Introduction An alternative bibliometric platform is Scopus, but we chose WoS thanks to our institution access; and Scopus use will generate additional computing methods to extract large collection as we did on the WoS, without adding so much other records as WoS and Scopus present high overlap level [26]. We chose to focus on temperate climate species; that is, the grain-legume species grown in most Western countries; but many of them are also important grain-legumes for semi-arid or tropical areas. In analyzing the scholarly publications on grain-legumes, we wanted to assess the relative occurrences of several grain-legume species and which countries had been the most involved in this research. We performed this analysis for research over the last four decades (between 1980 and 2018), by considering ten themes (i.e., scientific fields) of interest in agrofood system research and identified by experts: Genetics, Agronomy, Ecophysiology, Biotic-stress, Feeding, Processing, Nutrition, Allergy, Acceptability, and Socioeconomics. No review of the literature to date has managed to tackle so many themes of interest together for both agricultural and food sciences, whatever the crop species considered. g g p p This original and ambitious literature review involved 26 scientiﬁc experts on legumes, coupled with database and scientometrics experts to create relevant search queries on the WoS and appropriate software to process and analyze the resulting bibliographic records. Our ﬁndings are based on a core corpus totaling 107,823 scientiﬁc publications (i.e., records) retrieved by thematic search queries addressing the title, abstract, and authors’ keywords. Since soya is a major crop used for oil unlike most other grain-legumes, we excluded records referring to the subject of “soya oil”, in order to have more relevant comparisons in the corpus created. Our results show that soya is mentioned in 43% of the records, groundnut in nearly 10% and all other pulses combined in 47%. The analyses revealed a strong imbalance within grain-legume species research, with soya dominant over all other grain-legume species; and if “soya oil” had been included in keyword searches, this percentage would be even greater. This trend has grown even stronger in recent years. We also observed that the breakdown of themes researched were not the same for soya and pulses. Processing and Nutrition were much more common themes of research for soya than for pulses. For pulses, research mainly focused on “upstream” themes linked to Genetics or Ecophysiology. 1. Introduction This imbalance questions the capacity of research to develop knowledge that would enable more food outlets for pulses, as expected by the United Nations during the IYP. 2.1. Overview of the Methodology Adopted This study was based on a bibliometric dataset retrieved from the Web of Science (WoS) a product of Clarivate Analytics. As mentioned in Section 1, Scopus is the other prominent bibliographic database, which presents a high overlap with the WoS: see [26] for a comparison of these databases stressing their limitations and advantages to conduct large-scale literature analyses. The WoS is one of the most used 4 of 37 Sustainability 2019, 11, 6833 bibliometric resources in the world. It provides access to article records from more than 30,000 journals and books in various fields of science. The WoS “Core Collection” includes about 70 million records [27]. For the present study, we worked with several scientiﬁc experts to identify domain-speciﬁc keywords and retrieve the associated scientiﬁc publications (records). First, we identiﬁed keywords covering most of the cultivated grain-legume species in Western countries (Species query). Second, we determined 10 domain-speciﬁc themes covering the main research issues on grain-legumes (e.g., Allergy). Then, search queries on the WoS were designed to delineate the species and each theme (Table 1), leading to 10 thematic corpora on grain-legumes. Finally, these 10 corpora were merged into a single corpus called Fusion. All search queries and the associated publication records are available as open data from the https://data.inra.fr repository (Appendix A, Table A2). In this article, a “corpus” is a set of records retrieved from the WoS and a “record” refers to the metadata of a scholarly document (e.g., the type of publication, such as “article”, “book chapter”; the authors; etc.). Designing these queries was an iterative process requiring a combination of skills: • Theme experts We identiﬁed leading scientists in several ﬁelds (Table 1) who helped to delineate search queries for 10 key themes and to check the validity of the records retrieved. (Delineation of scientiﬁc ﬁelds or subject areas is a question under much discussion in scientometrics. Bibliometric databases developed their own classiﬁcation reﬂecting the scope of journals. As such classiﬁcation were not directly suitable to break down agricultural and food research activity in several ﬁelds on which to build our search queries, we relied on experts’ judgement to deﬁne 10 themes of main interest covering the research on grain-legumes. We used the term of “theme” as a synonym of “subject area”. See [20] to go further on this question of delineation of scientiﬁc ﬁelds). 2.1. Overview of the Methodology Adopted Scientometricians We collaborated with scientists who specialize in the quantitative study of science and database management systems to create an online platform giving access to the corpora collected. This platform was used to then incrementally reﬁne search queries to build the corpora. Since the number of records to collect from the WoS (100 k) exceeded the amount of records one can extract from the web interface (limited to 5 k), we resorted to a Web of Science Data Integration feature. Data collection was then performed with an in-house program using the Web of Science API Expanded. Table 1. Thematic corpora investigated by experts. Theme and Underlying Corpus Name Description of the Theme Number of Scientiﬁc Experts Involved Species Names used to designate the various main grain-legume species and varieties cultivated in temperate climates 2 Genetics Varieties, genes, breeding methods and objectives 2 Agronomy Ways to grow legume crops and provided services 2 Ecophysiology Plant physiology in relation to its abiotic environment 2 BioticStress Weeds, diseases, and pests’ life traits and control in crops 2 Feeding Feeding practices, animal nutrition 2 Processing Transformation and main types of food products excluding non-food uses 4 Nutrition Nutrition subjects for humans including health 4 Allergy Concerns on allergy linked to the use of legumes in food 2 Acceptability Sensorial and organoleptic analysis for consumer acceptance 2 Socioeconomics Any subject of interest using socio-economic approaches 2 Table 1. Thematic corpora investigated by experts. Regarding the 10 domain-speciﬁc themes selected, we adopted a broad-spectrum approach covering the end-to-end workﬂow of grain-legume research from production to consumers. A growing concern in Western countries is to increase legume consumption [28]; therefore, we made sure to cover allergy and consumer acceptance subjects. We also designed a query capturing the main socioeconomic 5 of 37 Sustainability 2019, 11, 6833 research theme on grain-legumes (e.g., funding of work on breeding activities, farmers’ production choices, feed practices and business, consumer behaviors, and market functioning, policies). This query led to the corpus called Socioeconomics, a research theme complementary to the others on life sciences and engineering. Performing the statistics at the meta-level required merging the 10 thematic corpora on grain-legumes into a single corpus called Fusion. We merged all records from the underlying corpora, removing duplicates that we identiﬁed thanks to the unique identiﬁer provided for each record in the WoS (UT code). 2.1. Overview of the Methodology Adopted As a result, a record in Fusion appeared only once, even if it appeared in multiple underlying corpora. 2.2.3. Indexing as a Post-Processing Step to Cleanse the WoS Results WoS queries were issued with the standard TS operator, meaning Topic Search. TS results collect records of the database that match the user’s query on the following criteria: title, abstract, author keywords, and KeyWords Plus. KeyWords Plus are additional terms automatically generated by the WoS and attached to the records. Terms appearing more than once in the titles of the cited references in a record (i.e., in its bibliography section) are called KeyWords Plus [29]. For instance, a search like TS = “protein AND pea” matches documents whose title, abstract or author’s keywords contain the term “protein” but not “pea”, because the term “pea” occurred only in the bibliography of the document retrieved (“pea” being a KeyWords Plus here) (see, for instance, query UT = “WOS:000226807600008” AND TS = (“protein” AND “pea”) yielding one journal paper on rice mutants, whose abstract contains “protein” but not “pea”). This example stresses that the records retrieved from the WoS might contain records that were not of direct interest. In our point of view, a document of direct interest (to build a core corpus) must contain both terms in the main contents provided only by the authors: title, abstract, and author’s keywords. To circumvent the biases introduced by KeyWords Plus, we designed an indexing algorithm that cleaned the WoS results by ﬁltering out non-direct interest documents. Each remaining document had to match our criteria: only author-speciﬁed contents should be matched with the query. We applied this indexing algorithm to the 10 thematic queries. 2.2.1. Documents Type The search was restricted to the main types of scientiﬁc literature documents, namely: article, book, book chapter, and review. This translated into the query: DT = (“Article” OR “Book” OR “Book Chapter” OR “Review”). 2.2. Designing Search Queries on the WoS: Main Principles 2.2. Designing Search Queries on the WoS: Main Principles This section introduces the main principles we followed to design the search queries (Table A2) (for the syntax rules of search queries, see the Web of Science Core Collection Help: http://images. webofknowledge.com/WOKRS5251R3/help/WOS/hp_search.html). 2.2.2. Time Range We selected the period 1980–2018 (PY = “1980–2018”) to observe long-term dynamics. This timeframe encompasses document records of variable completeness: abstracts were available only starting in 1990. We expected an increase in the volume per year to raise around 1990 because search queries would match more records in both the titles and abstracts. 2.2.5. Iterative Design and Validation of the Search Queries Each thematic search query was stabilized with an iterative validation process involving the experts at each iteration. One iteration involves the following tasks applied to each thematic corpus: • Checking of a random sample from the thematic corpus. • Checking of a random sample from the thematic corpus. Each expert was asked to examine the records from a random selection of the 300 documents published during the last three years and present in the thematic corpus he/she was responsible for. Documents considered irrelevant were analyzed to deduce the changes that needed to be made on the search query in order not to retrieve these in the next iteration. Conversely, experts were also asked to identify those aspects of the theme that were not caught by the query. In particular, experts expected the leading authors or topics to appear (the three last years corresponding to the current state of the art); they used this information to adjust search queries when necessary. Overall, the search operator t1 near/10 t2 (i.e., term t1 must be within 10 words away from term t2) proved the most eﬃcient for identifying relevant thematic corpora. In our case, t1 were names of the species studied while t2 were terms related to the considered theme. This ﬁrst task iterated until the percentage of irrelevant documents was less than 20% of the random sample. • Checking of the entire thematic corpus. Each expert was asked to examine the records from a random selection of the 300 documents published during the last three years and present in the thematic corpus he/she was responsible for. D t id d i l t l d t d d th h th t d d t b d This second task relied on descriptive statistics. For each thematic corpus, experts were instructed to assess the relevance of the most frequent: This second task relied on descriptive statistics. For each thematic corpus, experts were instructed to assess the relevance of the most frequent: # terms in the title, abstract, authors’ keywords of the records; and # WoS categories (wcs) reﬂecting the scope of the journal or the book that the WoS attributes to each record of the bibliographic database. # WoS categories (wcs) reﬂecting the scope of the journal or the book that the WoS attributes to each record of the bibliographic database. 2.2.5. Iterative Design and Validation of the Search Queries Irrelevant terms or wcs with high frequencies relative to the thematic corpora were identiﬁed and used to adapt, once again, the search query. In particular, this led the experts to specify excluding conditions (see below). During this phase, the experts of closely related scientiﬁc themes (for instance, between Ecophysiology and Agronomy, or between Nutrition and Processing) collaborated to better delineate each theme. Several meetings with all experts led to adaptations of the queries between themes. This ensured a reduced asymmetry of knowledge between the scientiﬁc experts for each theme, which helped to better delineate the search queries. 2.2.4. Interactive Browsing of the Bibliographic Corpora To ease team work with the thematic experts, we designed an online interactive bibliometric platform called SCIM, enabling them to explore the resulting records. The thematic corpora are shown with a clear distinction between direct interest vs. non-direct interest documents. Skimming 6 of 37 Sustainability 2019, 11, 6833 through the latter allowed experts to check that those eliminated records were clearly out of scope (i.e., validation of the aforementioned indexing step). In addition, using descriptive statistics, experts were instructed to check the relevance of the records included. They checked the most frequent terms, journals, and Web of Science categories (wcs in the WoS parlance (http://images.webofknowledge.com/ WOKRS5251R3/help/WOS/hp_subject_category_terms_tasca.html)) associated with the records of the direct interest documents. 2.2.6. Excluding Conditions In general, non-food uses are beyond the scope of this study. development for food instead for oil [28]. o The phrase “biodiesel” as a product linked to oil fraction was excluded. In general, non- food uses are beyond the scope of this study. Finally, all of these issues meant that conducting a bibliometric study requires careful, in-depth, and iterative expert coordination for performing the many checks and reﬁning the search strategy. Compared to the delineation strategy established for this Fusion corpus (Figure 1), other delineation strategies were also tested and are reported in Appendix B. Those alternative delineation strategies resulted in less relevant corpora than with the delineation strategy of Figure 1 (see Table A5 for instance). Finally, all of these issues meant that conducting a bibliometric study requires careful, in-depth, and iterative expert coordination for performing the many checks and refining the search strategy. Compared to the delineation strategy established for this FUSION corpus (Figure 1), other delineation strategies were also tested and are reported in Appendix B. Those alternative delineation strategies resulted in less relevant corpora than with the delineation strategy of Figure 1 (see Table B2 for instance). Figure 1. Main steps to build the bibliometric dataset: the delineation strategy. 1.1. Species & Themes search queries 1.2. Thematic corpora download 2.1. Species & Themes indexing procedure by corpus Doc Type + Time Range + TS (Title, Abstract, Authors keywords, KeyWords Plus) + Excluding conditions (NOT TS/WC) 3.2 Bibliometric analysis 3.1. Validated corpora merged into a single corpus Shares of species, themes countries, time evolution… 2.2. Checking by corpus (random sample and complete corpus) Corpora retrieved from the WoS Title, Abstract, Authors keywords Delineation strategy Corpora filtered Online interactive bibliometric platform SCIM Feedback loops between steps 1-2 to stabilize search queries FUSION corpus Figure 1. Main steps to build the bibliometric dataset: the delineation strategy. Delineation strategy 1.1. Species & Themes search queries Feedback loops between steps 1-2 to stabilize search queries 1.2. Thematic corpora download Corpora retrieved from the WoS Online interactive bibliometric platform SCIM FUSION corpus Figure 1. Main steps to build the bibliometric dataset: the delineation strategy. Figure 1. Main steps to build the bibliometric dataset: the delineation strategy. This figure summarizes the main steps followed to build the thematic corpora. First, queries were submitted to the Web of Science to collect bibliographic records for each theme (Steps 1.1 and 1.2). 2.2.6. Excluding Conditions Second, the indexing phase eliminated the records retrieved because of KeyWords Plus only (Step 2.1). Then, for each theme, surviving records were checked by experts: first, on a random sample, and, second, for the complete corpus (Step 2.2). Third, the experts relied on descriptive statistics to identify the remaining irrelevant documents; they modified the query accordingly and re-ran the whole process. In this way, queries were progressively refined. Finally, the 10 thematic corpora validated by experts were merged to form a single corpus of unique records called FUSION (Step 3.1), on which the bibliometric analysis relied (Step 3.2). This ﬁgure summarizes the main steps followed to build the thematic corpora. First, queries were submitted to the Web of Science to collect bibliographic records for each theme (Steps 1.1 and 1.2). Second, the indexing phase eliminated the records retrieved because of KeyWords Plus only (Step 2.1). Then, for each theme, surviving records were checked by experts: ﬁrst, on a random sample, and, second, for the complete corpus (Step 2.2). Third, the experts relied on descriptive statistics to identify the remaining irrelevant documents; they modiﬁed the query accordingly and re-ran the whole process. In this way, queries were progressively reﬁned. Finally, the 10 thematic corpora validated by experts were merged to form a single corpus of unique records called Fusion (Step 3.1), on which the bibliometric analysis relied (Step 3.2). 2.2.6. Excluding Conditions Queries feature excluding conditions on some keywords or wcs, for speciﬁc themes or for all of them. • For instance, in the Processing query, the “germination” keyword is ambiguous, as it relates to either a food subject or an agronomic subject (as regards the germination step of seeds in the soil concerning more the Ecophysiology corpus). As “germination” was a keyword that we need to keep for the Processing query, we excluded the wcs of the Processing corpus not related to “Nutrition Dietetics” and “Food Science Technology”. • For all queries: Sustainability 2019, 11, 6833 7 of 37 # The terms “coﬀee” and “cacao” where excluded because they also appear in the underlying paper under the generic term “bean”. o The terms “coffee” and “cacao” where excluded because they also appear in the underlying paper under the generic term “bean” # The terms “coﬀee” and “cacao” where excluded because they also appear in the underlying paper under the generic term “bean”. o The terms “coffee” and “cacao” where excluded because they also appear in the underlying paper under the generic term “bean” # The terms “coﬀee” and “cacao” where excluded because they also appear in the underlying paper under the generic term “bean”. o The terms “coffee” and “cacao” where excluded because they also appear in the underlying paper under the generic term “bean” # The phrase “soya oil” was excluded due to a twofold rationale. First, it is over-represented in the literature on soya. Second, comparing soya and pulses requires selecting common features of legume interests, such as the increasing interest in plant-based protein development for food instead for oil [28]. paper under the generic term bean . o The phrase “soya oil” was excluded due to a twofold rationale. First, it is over-represented in the literature on soya. Second, comparing soya and pulses requires selecting common features of legume interests, such as the increasing interest in plant-based protein development for food instead for oil [28] # The phrase “biodiesel” and “biofuel” as products linked to oil fraction was excluded. In general, non-food uses are beyond the scope of this study. development for food instead for oil [28]. o The phrase “biodiesel” as a product linked to oil fraction was excluded. In general, non- food uses are beyond the scope of this study. # The phrase “biodiesel” and “biofuel” as products linked to oil fraction was excluded. 2.3. Focus on the Design of the Species WoS Query 2.3. Focus on the Design of the Species WoS Query The 10 thematic search queries were combined with a query targeting the names of grain-legume species. Hence, delineating the various terms referring to grain-legumes was a crucial part of our bibliometric study. This was a challenging task as different names are used in different scientific fields. Either the scientific name (generally the Latin name) or various, country-specific, common The 10 thematic search queries were combined with a query targeting the names of grain-legume species. Hence, delineating the various terms referring to grain-legumes was a crucial part of our bibliometric study. This was a challenging task as diﬀerent names are used in diﬀerent scientiﬁc ﬁelds. Either the scientiﬁc name (generally the Latin name) or various, country-speciﬁc, common names were 8 of 37 Sustainability 2019, 11, 6833 used. The expertise of two senior researchers internationally recognized on those crops, combined with the consulting of websites dedicated to data collection on plants (Table 2) and books on plant taxonomy (e.g., [30]) enabled us to list more than 80 various names that we grouped into 19 main grain-legumes species (Table 3). Only main pulses, soya, and some species from the genera Lathyrus and Vicia cultivated in temperate climates (mainly of Western countries and the Mediterranean basin) were considered here. All these species belong to the family of grain-legumes. Among them, according to the United Nations, the grain-legumes not used for oil extraction are commonly called pulses, excluding soya and groundnut for their dual richness in oil and protein. Appendix C gives a brief history of grain-legumes and reveals that while pulses were common crops for centuries, interest in them has dramatically decreased in recent decades compared to soya. Table 2. Main sources consulted to check the various terms used for grain-legumes species. Name Website Tela Botanica, the French-speaking network of botanists, presenting Latin name of species https://www.tela-botanica.org/ Feedipedia, describing all the resources used for feed in the world, and among them legumes https://www.feedipedia.org/ Atlas managed by the CGIAR—Research Program on Dryland Cereals and Legumes Agri-Food Systems (DCL) http://www.eatlasdcl.cgiar.org A personal website created by a renowned retired botanist. https://www.cropsreview.com/grain-legumes.html Inventory of food resources and constituents http://foodb.ca Table 2. Main sources consulted to check the various terms used for grain-legumes species. Table 2. Main sources consulted to check the various terms used for grain-legumes species. 2.3. Focus on the Design of the Species WoS Query 2.3. Focus on the Design of the Species WoS Query Name Website Tela Botanica, the French-speaking network of botanists, presenting Latin name of species https://www.tela-botanica.org/ Feedipedia, describing all the resources used for feed in the world, and among them legumes https://www.feedipedia.org/ Atlas managed by the CGIAR—Research Program on Dryland Cereals and Legumes Agri-Food Systems (DCL) http://www.eatlasdcl.cgiar.org A personal website created by a renowned retired botanist. https://www.cropsreview.com/grain-legumes.html Inventory of food resources and constituents http://foodb.ca Table 3 presents all the species terms included in the Species search query, classiﬁed according to a single species identiﬁer and a generic one. For instance, we gathered under the identiﬁer “soya” all occurrences of various terms referring to soya by using wildcards such as in the following list: glycine max, soja, soya$, soy$, sojabean$, soybean$, and soyabean$. These species identiﬁers served for the indexing step (explained in Step 2.3) and the exploratory statistics. As for the thematic search queries, wildcards were used to catch various forms of a term: for instance, being written in plural or singular or with varying country-dependent orthotypography (e.g., soya vs. soja and faba vs. fava bean). The asterisk () represents any group of characters (including no character), the dollar sign ($) represents zero or one character. The phrases (expressions composed of several terms), such as “chick pea”, were surrounded with quotation marks in the search queries applied on the WoS. Some generic terms (such as legumes and leguminous) were also considered, but they were only added for the Socioeconomics search query. For the other themes, using these generic terms retrieved too many irrelevant records: that is, records mentioning legumes without dealing speciﬁcally with grain-legumes. One explanation for this relates to how scientists phrase their papers: some (especially life sciences) usually work on a speciﬁc legume, while others (especially social sciences) tend to consider legumes in a more comprehensive approach. 9 of 37 Sustainability 2019, 11, 6833 Table 3. Species identiﬁer and species expressions used in the species search query. Table 3. Species identiﬁer and species expressions used in the species search query. 2.3. Focus on the Design of the Species WoS Query 2.3. Focus on the Design of the Species WoS Query Species Identiﬁer (Genus or Common Name) All Species or Common Name Terms Included in the Search Query Adzuki phaseolus angularis, vigna angularis, red mung$, red bean$, red mungbean$, adzuki$, azuki$ Bambara Bean vigna subterranean , bambara bean$ Bean phaseolus coccineus, phaseolus vulgaris, phaseolus lunatus, phaseolus spp, common bean$, common ﬁeld bean$, common ﬁeldbean$, runner bean$, runnerbean$, lima bean$, common bean$, kidney bean$, pinto bean$, vigna aconitifolia, moth bean$, vigna umbellata, rice bean$ Chickpea cicer arietinum, chickpea$, chick pea$ Cowpea vigna unguiculata, cowpea$, cow pea, cow peas, blackeyed pea, blackeyed peas, black-eye pea, black-eye peas, blackeyed bean$, catjan$, long bean$ Faba bean vicia faba, fava bean$, faba bean$, broadbean$, broad bean$, horse bean$, horsebean$, fababean$, ﬁeld bean$, ﬁeldbean$ Fenugreek trigonella foenum grecum, trigonella foenum graecum, fenugreek$, fenugrec$, fenu grec$ Lathyrus lathyrus sativus, lathyrus sativa, lathyrus ochrus, lathyrus cicera, grass pea$, red pea$, cyprus vetch$, vetchling$, gesse$ Gram bean vigna mungo, gram bean$, black bean$, black lentil$, black gram, blackgram$ Groundnut arachis hypogea, arachis hypogaea, groundnut$, peanut$ Lablab lablab purpureus, hyacinth bean$, lablab bean$, lablab$ Lentil lens culinaris, lentil$ Lupin lupinus albus, lupinus angustifolius, lupinus luteus, lupinus mutabilis, lupin$ Mungbean vigna radiata, vigna mungo, mungbean$, mung bean$, moong bean$, mungo bean$, green gram$, golden gram$, maash$, moong sanskrit$ Pea pisum sativum, pea, peas Pigeon Pea cajanus cajan, pigeon pea, pigeon peas, pigeonpea$ Soya glycine max, soja, soya$, soy$, sojabean$, soybean$, soyabean$ Vicia vetch$, vetche$, vicia sativa, vicia villosa, vicia ervilia, ervil$, vicia narbonensis, narbon bean$ Winged bean psophocarpus tetragonolobus, winged bean$, asparagus pea$, goabean$, goa bean$ Generic leguminous, legume, legumes, pulse, pulses Note: $ (respectively, ) is a wildcard replacing zero or one character (respectively, no character at all or a group of characters). For instance, “legumes” matches “grain-legumes” or “dried-legumes”. 3. Results and Discussion Bibliometric analysis allowed us to mine this dataset and infer new knowledge on grain-legume research at the global scale. The main purpose of this paper is to shed light on the share of species in these corpora, reﬂecting the research activity on grain-legumes. Thus, we calculated the percentage of records on each species within the corpora, longitudinally and at diﬀerent levels of analysis, according to species, themes, and countries, especially by comparing soya and pulse frequencies. Then, we discuss the implication of these results as regards avenues for future research and policies regarding the question of crop diversity. 3.1. Proportions of Grain-Legume Species in the Scientiﬁc Literature 3.1. Proportions of Grain-Legume Species in the Scientiﬁc Literature 3.1. Proportions of Grain-Legume Species in the Scientiﬁc Literature For this subsection, we consider ﬁve groups of grain-legume species when reporting our For this subsection, we consider ﬁve groups of grain-legume species when reporting our results: • G1 is for Soya • G1 is for Soya. • G1 is for Soya. • Pulses divided into three groups: • Pulses divided into three groups: Pulses divided into three groups: • G2 groups “PFL” including Pea, Fababean, and Lupin species. This group is the European classiﬁcation of the main protein-rich crops among pulses. • G3 groups “Other pulses” for the remaining pulses but excluding “Lathyrus and Vicia” • G4 groups Lathyrus and Vicia species together as the number of records related to those species are very low and currently the least used. • G2 groups “PFL” including Pea, Fababean, and Lupin species. This group is the European classiﬁcation of the main protein-rich crops among pulses. • G3 groups “Other pulses” for the remaining pulses but excluding “Lathyrus and Vicia” • G4 groups Lathyrus and Vicia species together as the number of records related to those species are very low and currently the least used. • G5 is for Groundnut (not considered as a pulse because of its oil richness). Sustainability 2019, 11, 6833 10 of 37 Table 4 present the shares of the ﬁve groups of grain-legume species captured in the Fusion corpus: G1, G2, and G3 appear in Table 4a, while G4 and G5 appear in Table 4b. We also distinguished the counts of species co-occurring with generic terms. Indeed, in the indexing procedure (see Section 2), we indexed the records with generic names of species such as “legumes” or “pulses”, in order to appreciate how researchers broaden their studies by referring also to the family group of legumes. Therefore, some ﬁgures in this subsection present speciﬁc counts of the co-occurrence of a generic term with a speciﬁc species’ name in records. In addition, we report the average annual growth rate of records in the Fusion corpus. Table 4. (a) Number and percentage of records in the Fusion corpus related exclusively to soya or pulses, broken down by time periods. (b) Number and percentage of records in the Fusion corpus related exclusively to groundnut, Lathyrus or Vicia, broken down by time periods. (a) PERIOD (PER.) TOTAL PER. 1st PER. 1980s 1990s 2nd PER. 3.1. Proportions of Grain-Legume Species in the Scientiﬁc Literature 2000s 2010s 3 Last Years Family species index terms of records: 1980–2018 1980–1999 1980–1989 1990–1999 2000–2018 2000–2009 2010–2018 2016–2018 Soya (alone *) 45,615 13,565 4824 8741 32,050 13,141 18,909 7076 Soya and a generic term 1440 286 7 279 1154 429 725 277 Other Pulses than PFL (alone) 20,780 7394 2719 4675 13,386 5549 7837 2819 Ibidem and a generic term 3175 569 38 531 2606 914 1692 745 PFL (alone) 16,760 7564 2743 4821 9196 4279 4917 1708 PFL and a generic term 2567 546 15 531 2021 789 1232 480 Subtotal (Soya, Pulses) 90,337 29,924 10,346 19,578 60,413 25,101 35,312 13,105 % Soya in subtotal 52% 46% 47% 46% 55% 54% 56% 56% % Subtotal within the per. 84% 86% 90% 84% 83% 84% 82% 82% Total records for the period 107,823 34,652 11,454 23,198 73,171 30,017 43,154 16,034 % per. in the Fusion corpus 100% 32% 11% 21% 68% 28% 40% 15% Annual average growth rate of the records 13% 21% 32% 12% 6% 6% 6% 6% (b) PERIOD (PER.) TOTAL PER. 1st PER. 1980s 1990s 2nd PER. 2000s 2010s 3 Last Years Family species index terms of records: 1980–2018 1980–1999 1980–1989 1990–1999 2000–2018 2000–2009 2010–2018 2016–2018 Groundnut (alone) 9305 2627 841 1786 6678 2445 4233 1595 % Groundnut within the per. 9% 8% 7% 8% 9% 8% 10% 10% Lathyrus or Vicia (alone) 1097 277 67 210 820 330 490 173 % Lathyrus or Vicia within the per. 1% 1% 1% 1% 1% 1% 1% 1% (a) Note: Each row in the table is exclusive of that group. PFL: pea, faba bean, or lupin. * For instance, this row shows the number of records containing only a generic term (a generic term such as legumes) AND a term linked to soya in the Title, Abstract or Authors’ keywords. ** The various combinations of species index (associating pulses, soya or groundnut in records) are not included here. For instance, over the entire period, we counted 838 records co-indexed with groundnut and soya; 298 records co-indexed with Lathyrus–Vicia and PFL, etc. These various co-indexations records over the entire period represent 6% of the Fusion corpus. (b) Note: Each row in the table is exclusive of that group. Table 4. (a) Number and percentage of records in the Fusion corpus related exclusively to soya or pulses, broken down by time periods. 3.1. Proportions of Grain-Legume Species in the Scientiﬁc Literature Therefore, measuring any impact from IYP 2016 on the number of publications can take at least 10 years, given that, before an increase in research activity, public and private decisions must be taken to increase funding for that research. Overall, as in the entire Core Collection, the second period (post-2000s) represents nearly two-thirds of the corpus, with a net increase in research activity (as in the WoS Core collection) since 2010: the 2010–2018 period accounts for 40% of the records in the Fusion corpus. This point is important to stress: as there has been a rapid and strong increase in scientiﬁc knowledge, it is crucial to adopt tools to analyze the ways that new knowledge is created. We need to assess the risk of over-investigating some themes or species and under-investigating others. 3.1.2. Generic Terms Referring to Legume or Pulse Family Are More Used with Pulse Species than with Soya Species Second, concerning the frequency of generic term co-occurrence with species indexes, we observed diﬀerent tendencies for soya and pulses. Table 4a shows that pulse species were more frequently co-indexed with a generic term than was soya. For instance, for the most recent period (2010–2018), 19% of pulse records are co-indexed with a generic term compared to 4% for soya records. Soya is a dominant crop having developed its own identity, making it distinct from other legumes. In other words, it seems that the identity of belonging to a larger family such as legumes is stronger for pulses species than for soya. It reﬂects also the fact that research studies focus on one species and rarely relate to the broader context of legumes or pulses. In addition, the results show a low rate of co-indexation between species (6%) (Table 5 and Figure 2). This means that the links between species are rarely stressed by the authors. In particular, only 2% of records were co-indexed both with soya and a pulse species. Thus, researchers rarely considered two (or more) species when investigating an issue, or, at least, the extension or impacts of the results for other species of the same family were rarely mentioned. This ﬁnding fundamentally questions the diﬀusion of knowledge between species and calls for future research using semantic networks to analyze which concepts (i.e., knowledge) are used when establishing connections between species. 3.1. Proportions of Grain-Legume Species in the Scientiﬁc Literature (b) Number and percentage of records in the Fusion corpus related exclusively to groundnut, Lathyrus or Vicia, broken down by time periods. Table 4. (a) Number and percentage of records in the Fusion corpus related exclusively to soya or pulses, broken down by time periods. (b) Number and percentage of records in the Fusion corpus related exclusively to groundnut, Lathyrus or Vicia, broken down by time periods. (a) Note: Each row in the table is exclusive of that group. PFL: pea, faba bean, or lupin. * For instance, this row shows the number of records containing only a generic term (a generic term such as legumes) AND a term linked to soya in the Title, Abstract or Authors’ keywords. ** The various combinations of species index (associating pulses, soya or groundnut in records) are not included here. For instance, over the entire period, we counted 838 records co-indexed with groundnut and soya; 298 records co-indexed with Lathyrus–Vicia and PFL, etc. These various co-indexations records over the entire period represent 6% of the Fusion corpus. (b) Note: Each row in the table is exclusive of that group. 3.1.1. The Number of Grain-Legume Publications Grew at the Same Rate as All Records in the WoS Core Collection First, concerning changes in the corpus size over time, we observed that, even though today there is greater awareness of legumes, the growth of scientiﬁc publications on grain-legumes is similar to that within the whole WoS Core Collection; the annual growth rate in scholarly peer-reviewed English-language journals of the WoS increased 5–6% in recent decades [27] (p. 25). The speciﬁc higher rate in the years 1980 and 1990 is due to the increase in research activity and publications observed in the entire WoS Core collection (due also to the WoS index rule changes since 1990 including both abstracts and keywords), and not to a special interest in legumes. Therefore, these ﬁgures ﬁrstly show that there has been no particular increase in legumes research, even after the United Nations’ communication about the beneﬁts of pulses for sustainability in the 1980s [31] and more recently in the 2010s (IYP in 2016). Nevertheless, researchers are aware that most publications observed in a given 11 of 37 Sustainability 2019, 11, 6833 year are the results of research conducted over the three or more previous years. 3.1. Proportions of Grain-Legume Species in the Scientiﬁc Literature Moreover, here we considered only the titles, abstracts and keywords; it is possible that the main text of the article mentioned such impacts for species other than the main one under investigation. However, titles and abstracts express the core message of an article, and considering application of results for other species does not seem to be part of this core message for most research work. 3.1.3. Soya Strongly Dominates within Grain-Legume Publications Third, these frequencies show the predominance of soya among grain-legumes (Figure 2). It is important to note that this soya predominance is underestimated, as all records referring to “soya oil” were eliminated. When such a similar exclusion is performed on groundnut (i.e., excluding “oil” theme), its ranking is lower: among 11, 612 records indexed with “groundnut”, 50% refer to oil thematic. Within the subtotal formed by soya and pulse groups (the two main species families of current interest in developed countries, notably for increasing plant-based protein), soya accounts for more than half of all records. This tendency has grown even stronger in recent years, with soya reaching 56% of the records. 12 of 37 . Sustainability 2019, 11, 6833 Table 5. Number Table 5. Number and percentage of the various grain-legume species quoted in Fusion corpus. Species Index Number of Records Share of Species Index Soya 51,395 42.6% Pea 14,175 11.7% Groundnut 11,612 9.6% Bean 8976 7.4% Cowpea 5954 4.9% Chickpea 5373 4.5% Faba bean 4640 3.8% Mungbean 3785 3.1% Lupin 3420 2.8% Lentil 2724 2.3% Pigeon Pea 2302 1.9% Vicia 2036 1.7% Gram bean 1466 1.2% Adzuki 771 0.6% Fenugreek 762 0.6% Lathyrus 491 0.4% Lablab 390 0.3% Winged bean 232 0.2% Bambara Bean 173 0.1% Total species quotes 120,677 100% Nb of records indexed with only one species (%) 100,739 (94%) Nb of records co-indexed with several species (%) 7084 (6%) Nb of records in FUSION corpus 107,823 Species Index Number of Records Share of Species Index Soya 51,395 42.6% Pea 14,175 11.7% Groundnut 11,612 9.6% Bean 8976 7.4% Cowpea 5954 4.9% Chickpea 5373 4.5% Faba bean 4640 3.8% Mungbean 3785 3.1% Lupin 3420 2.8% Lentil 2724 2.3% Pigeon Pea 2302 1.9% Vicia 2036 1.7% Gram bean 1466 1.2% Adzuki 771 0.6% Fenugreek 762 0.6% Lathyrus 491 0.4% Lablab 390 0.3% Winged bean 232 0.2% Bambara Bean 173 0.1% Total species quotes 120,677 100% Nb of records indexed with only one species (%) 100,739 (94%) Nb of records co-indexed with several species (%) 7084 (6%) Nb of records in FUSION corpus 107,823 Figure 2. Species distribution quotes in FUSION corpus. 3.1.3. Soya Strongly Dominates within Grain-Legume Publications 0% 10% 20% 30% 40% 50% Soya (51,395) Pea (14,175) Groundnut (11,612) Bean (8,976) Cowpea (5,954) Chickpea (5,373) Fababean (4,640) Mungbean (3,785) Lupin (3,420) Lentil (2,724) Pigeon Pea (2,302) Vicia (2,036) Gram bean (1,466) Adzuki (771) Fenugreek (762) Lathyrus (491) Lablab (390) Winged bean (232) Bambara Bean (173) Figure 2. Species distribution quotes in Fusion corpus. Fourth, when looking for the distribution of all the grain-legume species under study (Table 5), the predominance of soya is more accurate as the second main species, pea, accounts for nearly 12% of the mentions, compared to nearly 43% for soya. Among pulses, the ﬁve most mentioned species were, respectively: pea bean cowpea chickpea and faba bean Table 5. Number and percentage of the various grain-legume species quoted in Fusion corpus. Species Index Number of Records Share of Species Index Soya 51,395 42.6% Pea 14,175 11.7% Groundnut 11,612 9.6% Bean 8976 7.4% Cowpea 5954 4.9% Chickpea 5373 4.5% Faba bean 4640 3.8% Mungbean 3785 3.1% Lupin 3420 2.8% Lentil 2724 2.3% Pigeon Pea 2302 1.9% Vicia 2036 1.7% Gram bean 1466 1.2% Adzuki 771 0.6% Fenugreek 762 0.6% Lathyrus 491 0.4% Lablab 390 0.3% Winged bean 232 0.2% Bambara Bean 173 0.1% Total species quotes 120,677 100% Nb of records indexed with only one species (%) 100,739 (94%) Nb of records co-indexed with several species (%) 7084 (6%) Nb of records in FUSION corpus 107,823 p p Soya 51,395 42.6% Pea 14,175 11.7% Groundnut 11,612 9.6% Bean 8976 7.4% Cowpea 5954 4.9% Chickpea 5373 4.5% Faba bean 4640 3.8% Mungbean 3785 3.1% Lupin 3420 2.8% Lentil 2724 2.3% Pigeon Pea 2302 1.9% Vicia 2036 1.7% Gram bean 1466 1.2% Adzuki 771 0.6% Fenugreek 762 0.6% Lathyrus 491 0.4% Lablab 390 0.3% Winged bean 232 0.2% Bambara Bean 173 0.1% Total species quotes 120,677 100% Nb of records indexed with only one species (%) 100,739 (94%) Nb of records co-indexed with several species (%) 7084 (6%) Nb of records in FUSION corpus 107,823 Figure 2. Species distribution quotes in FUSION corpus. 0% 10% 20% 30% 40% 50% Soya (51,395) Pea (14,175) Groundnut (11,612) Bean (8,976) Cowpea (5,954) Chickpea (5,373) Fababean (4,640) Mungbean (3,785) Lupin (3,420) Lentil (2,724) Pigeon Pea (2,302) Vicia (2,036) Gram bean (1,466) Adzuki (771) Fenugreek (762) Lathyrus (491) Lablab (390) Winged bean (232) Bambara Bean (173) Figure 2. Species distribution quotes in Fusion corpus. 3.1.3. Soya Strongly Dominates within Grain-Legume Publications Fourth, when looking for the distribution of all the grain-legume species under study (Table 5), the predominance of soya is more accurate as the second main species, pea, accounts for nearly 12% of the mentions, compared to nearly 43% for soya. Among pulses, the ﬁve most mentioned species were, respectively: pea bean cowpea chickpea and faba bean 5. Number and percentage of the various grain-legume species quoted in Fusion corpus. Soya 51,395 42.6% Pea 14 175 11 7% Species Index Number of Records Share of Species Index Pea 14,175 11.7% Groundnut 11,612 9.6% Figure 2. Species distribution quotes in FUSION corpus. 0% 10% 20% 30% 40% 50% Soya (51,395) Pea (14,175) Groundnut (11,612) Bean (8,976) Cowpea (5,954) Chickpea (5,373) Fababean (4,640) Mungbean (3,785) Lupin (3,420) Lentil (2,724) Pigeon Pea (2,302) Vicia (2,036) Gram bean (1,466) Adzuki (771) Fenugreek (762) Lathyrus (491) Lablab (390) Winged bean (232) Bambara Bean (173) Figure 2. Species distribution quotes in Fusion corpus. Figure 2. Species distribution quotes in FUSION corpus. Figure 2. Species distribution quotes in Fusion corpus. Fourth, when looking for the distribution of all the grain-legume species under study (Table 5), the predominance of soya is more accurate as the second main species, pea, accounts for nearly 12% of the mentions, compared to nearly 43% for soya. Among pulses, the ﬁve most mentioned species were, respectively: pea, bean, cowpea, chickpea, and faba bean. 13 of 37 rly 12% species rly 12% species Sustainability 2019, 11, 6833 the predominance of so of the mentions comp the predominance of s of the mentions, comp As explained in Appendix B, we created alternative bibliometric corpora (Species1, Species2, Species3). Although in these alternative corpora the themes are less well delineated, it was worthwhile to check whether the species frequencies were similar, since, with the Species3 and Fusion corpora, nearly one-third of the records were not common to both. Finally, we observed the same statistics for these other corpora and the percentage of species was similar (Table A6). Overall, soya accounts for more than half of the scientiﬁc publications within the soya and pulse records, with the share of soya increasing in recent years. We also observed in these alternative corpora that soya and pulses represented around 90% of the records on grain-legumes. p y p p p As explained in Appendix B, we created alternative bibliometric corpora (SPECIES1, SPECIES2, SPECIES3). 3.1.4. Changes in the Mentions of Species over Time 3.1.4. Changes in the Mentions of Species over Time 3.1.4. Changes in the Mentions of Species over Time Figures 3 and 4 present the longitudinal evolution of the ten most mentioned species. Soya is the species with the highest number of publications: 45,615 records co-indexed with soya (Table 4a). The increase in records indexed with groundnut, chickpea, lentil, or mungbean was greater in recent years compared to other pulse species. Figures 3 and 4 present the longitudinal evolution of the ten most mentioned species. Soya is the species with the highest number of publications: 45,615 records co-indexed with soya (Table 4a). The increase in records indexed with groundnut, chickpea, lentil, or mungbean was greater in recent years compared to other pulse species. Figures 3 and 4 present the longitudinal evolution of the ten most mentioned species. Soya is the species with the highest number of publications: 45,615 records co-indexed with soya (Table 4a). The increase in records indexed with groundnut, chickpea, lentil, or mungbean was greater in recent years compared to other pulse species. Figure 3. Longitudinal evolution of main pulses species quoted in FUSION corpus. Figure 4. Longitudinal evolution of Soya and Groundnut species quoted in the FUSION corpus. Figure 3. Longitudinal evolution of main pulses species quoted in Fusion corpus. Figure 3. Longitudinal evolution of main pulses species quoted in FUSION corpus. Figure 4. Longitudinal evolution of Soya and Groundnut species quoted in the FUSION corpus. 2 Percentage of Grain-Legume Species in Literature across Countries Figure 4. Longitudinal evolution of Soya and Groundnut species quoted in the Fusion corpus. .2. Percentage of Grain-Legume Species in Literature across Countries Figure 3. Longitudinal evolution of main pulses species quoted in FUSION corpus. Figure 3. Longitudinal evolution of main pulses species quoted in Fusion corpus. Figure 3. Longitudinal evolution of main pulses species quoted in FUSION corpus. Figure 3. Longitudinal evolution of main pulses species quoted in FUSION corpus. Figure 3. Longitudinal evolution of main pulses species quoted in Fusion corpus. Figure 3. Longitudinal evolution of main pulses species quoted in FUSION corpus. Figure 3. Longitudinal evolution of main pulses species quoted in FUSION corpus. Figure 3. Longitudinal evolution of main pulses species quoted in Fusion corpus. Figure 3. Longitudinal evolution of main pulses species quoted in FUSION corpus. Figure 4 Longitudinal evolution of Soya and Groundnut species quoted in the FUSION corpus Figure 4 Longitudinal evolution of Soya and Groundnut species quoted in the Fusion corpus Figure 4. 3.1.3. Soya Strongly Dominates within Grain-Legume Publications Although in these alternative corpora the themes are less well delineated, it was worthwhile to check whether the species frequencies were similar, since, with the SPECIES3 and FUSION corpora, nearly one-third of the records were not common to both. Finally, we observed the same statistics for these other corpora and the percentage of species was similar (Table A6). Overall, soya accounts for more than half of the scientific publications within the soya and pulse records, with the share of soya increasing in recent years. We also observed in these alternative corpora that soya and pulses represented around 90% of the records on grain-legumes. As explained in Appendix B, we created alternative bibliometric corpora (SPECIES1, SPECIES2, SPECIES3). Although in these alternative corpora the themes are less well delineated, it was worthwhile to check whether the species frequencies were similar, since, with the SPECIES3 and FUSION corpora, nearly one-third of the records were not common to both. Finally, we observed the same statistics for these other corpora and the percentage of species was similar (Table A6). Overall, soya accounts for more than half of the scientific publications within the soya and pulse records, with the share of soya increasing in recent years. We also observed in these alternative corpora that soya and pulses represented around 90% of the records on grain-legumes. 3.1.4. Changes in the Mentions of Species over Time 3.1.4. Changes in the Mentions of Species over Time 3.1.4. Changes in the Mentions of Species over Time Longitudinal evolution of Soya and Groundnut species quoted in the FUSION corpus. Figure 4. Longitudinal evolution of Soya and Groundnut species quoted in the FUSION corpus. Figure 4. Longitudinal evolution of Soya and Groundnut species quoted in the Fusion corpus. g g y p 3 2 Pe e ta e of G ai Le u e S e ie i Lite atu e a o Cou t ie 3.2. Percentage of Grain-Legume Species in Literature across Countries 3.2. Percentage of Grain-Legume Species in Literature across Countries 3.2. Percentage of Grain-Legume Species in Literature across Countries g f g p The metadata of most records retrieved from the WoS identiﬁed the countries of the authors. We studied the share of soya and pulses research by country. Figures 5 and 6 and Table 6 report a proportional count for international collaboration records (that is associating several countries): 14 of 37 Sustainability 2019, 11, 6833 each country accounts for 1/n where n is the number of countries associated for the record. As most international collaborations involved few countries, those publishing the most on grain-legumes, this rule avoids overcounting them as when one uses full counting (i.e., 1 point to each country). We observed that with or without proportional counting, the ranking of countries did not change (Figure 5a,b). We also created a speciﬁc geographical index of the current 28 European Union countries (whatever the period considered), while computing the count for individual European countries. For the following data, we considered only the records indexed either with soya or with pulses and for which authors’ countries were identiﬁed (around 15,000 records in the Fusion corpus did not have this information in the WoS; these are mostly papers with multiple authors and only one reprint address, see UT = WOS:A1997XJ61100010, for instance). 3.2.1. The Ranking of Countries Was Rather Stable over Time, with China, Brazil, and India Rising First, Figures 5 and 6 show that the ranking of countries was diﬀerent for soya and pulses, but quite stable over time. Considering both soya and pulses, the two ﬁrst publishing geographical areas are the USA and the EU28, considering either the whole period or the current decade. The USA and the EU28 account for more than half of the records in the previous period, but less than one-third in the current decade (Table 6). 3.1.4. Changes in the Mentions of Species over Time 3.1.4. Changes in the Mentions of Species over Time 3.1.4. Changes in the Mentions of Species over Time More recently, China has been rising and currently represents 13% of the records in the current decade, followed by India, Brazil, Japan, Canada, and Australia. These seven countries and the EU28 account for two-thirds of the records on soya and pulses over the current decade (versus 84% in the previous period). This reveals a progression of other countries in legume research, such as South Korea and Argentina. 3.2.2. The Percentage of Soya and Pulses Records per Country Is Quite Stable over Time 3.2.2. The Percentage of Soya and Pulses Records per Country Is Quite Stable over Time Second, a clear opposition appears with some countries that focus more on pulses than on soya. This is particularly the case for the EU28 and India (and for Australia but with fewer records) and for the countries geographically close to them. Over the whole period, the EU28 accounted for more than a quarter of the studies on pulses, but less in the current decade because of India’s increase. For instance, while the EU28 has two times fewer records in the current decade than during the previous period, it is the opposite for India which has doubled records in the current decade (Table 6). Canada is the only country publishing as much on soya as on pulses, whatever the period. Other countries work more on soya, increasing the imbalance with pulses: this is particularly the case for China which currently publishes nearly four times more on soya than on pulses, with the USA three times more, and Brazil twice more. Among the 20 most publishing countries, there are six European countries in the current decade: Spain, Germany, France, Italy, England, and Poland. They also represent two-thirds of the records in the EU28. Figure 6a,b gives more details on the share of each European country in the EU28. One emerging trend is the increase of soya records, becoming more important than pulse records for the Netherlands and Romania, and near equal for Belgium, Denmark, and Austria. Poland, France, and the UK are countries with the highest proportion of pulse records compared to soya records. Overall, the ranking of European countries is stable over time, apart from the UK whose number of records is smaller in the current decade. Currently, Spain is the top European country on pulses regarding the number of publications, and it is also the top European country for pulse cultivation and consumption (Eurostats). ustainability 2019, 11, 6833 15 of 37 ustainability 2019, 11, x FOR PEER REVIEW 15 of 3 (a) (b) Figure 5. Soya and Pulses records across countries in the FUSION corpus. (a) 1980–2018. (b) 2010–2018. Note: the 20 highest frequencies are based on total records by country, a group count done for the EU28. Only records indexed with soya or with pulses were included (i.e., records co-indexed with several groups of grain-legumes were excluded). Proportional count for international collaboration records is applied. The country ranking is based on the total records number by country. Figure 5. 3.2.2. The Percentage of Soya and Pulses Records per Country Is Quite Stable over Time Soya and Pulses records across countries in the Fusion corpus. (a) 1980–2018. (b) 2010–2018. Note: the 20 highest frequencies are based on total records by country, a group count done for the EU28. Only records indexed with soya or with pulses were included (i.e., records co-indexed with several groups of grain-legumes were excluded). Proportional count for international collaboration records is applied. The country ranking is based on the total records number by country. 15 of 37 15 of 38 Sustainability 2019, 11, 6833 Su tai ability 2019 11 FOR ustainability 2019, 11, x FOR PEER REVIEW 15 of 38 (a) (a) (b) Figure 5. Soya and Pulses records across countries in the FUSION corpus. (a) 1980–2018. (b) 2010–2018. Figure 5. Soya and Pulses records across countries in the Fusion corpus. (a) 1980–2018. (b) 2010–2018. Note: the 20 highest frequencies are based on total records by country, a group count done for the EU28. (a) (b) Figure 5. Soya and Pulses records across countries in the FUSION corpus. (a) 1980–2018. (b) 2010–2018. Figure 5. Soya and Pulses records across countries in the Fusion corpus. (a) 1980–2018. (b) 2010–2018. Figure 5. Soya and Pulses records across countries in the FUSION corpus. (a) 1980–2018. (b) 2010–2018. Note: the 20 highest frequencies are based on total records by country, a group count done for the EU28. Only records indexed with soya or with pulses were included (i.e., records co-indexed with several groups of grain-legumes were excluded). Proportional count for international collaboration records is applied The country ranking is based on the total records number by country Figure 5. Soya and Pulses records across countries in the Fusion corpus. (a) 1980–2018. (b) 2010–2018. Note: the 20 highest frequencies are based on total records by country, a group count done for the EU28. Only records indexed with soya or with pulses were included (i.e., records co-indexed with several groups of grain-legumes were excluded). Proportional count for international collaboration records is applied. The country ranking is based on the total records number by country. Figure 5. Soya and Pulses records across countries in the FUSION corpus. (a) 1980–2018. (b) 2010–2018. Note: the 20 highest frequencies are based on total records by country, a group count done for the EU28. Only records indexed with soya or with pulses were included (i.e., records co-indexed with several groups of grain-legumes were excluded). 3.2.2. The Percentage of Soya and Pulses Records per Country Is Quite Stable over Time Proportional count for international collaboration records is applied The country ranking is based on the total records number by country Figure 5. Soya and Pulses records across countries in the Fusion corpus. (a) 1980–2018. (b) 2010–2018. Note: the 20 highest frequencies are based on total records by country, a group count done for the EU28. Only records indexed with soya or with pulses were included (i.e., records co-indexed with several groups of grain-legumes were excluded). Proportional count for international collaboration records is applied. The country ranking is based on the total records number by country. Sustainability 2019, 11, 6833 16 of 37 Sustainability 2019, 11, x FOR PEER REVIEW 16 of 38 (a) (b) Figure 6. Soya and Pulses records across the EU28 countries in the FUSION corpus. (a) 1980–2018. (b) 2010–2018. Note: only records indexed with soya or with pulses were included (i.e., records co-indexed with several groups of grain-legumes were excluded). Proportional count for international collaboration records is applied. The country ranking is based on the total records number. Malta had no records. Figure 6. Soya and Pulses records across the EU28 countries in the Fusion corpus. (a) 1980–2018. (b) 2010–2018. Note: only records indexed with soya or with pulses were included (i.e., records co-indexed with several groups of grain-legumes were excluded). Proportional count for international collaboration records is applied. The country ranking is based on the total records number. Malta had no records. 16 of 37 16 f 38 Sustainability 2019, 11, 6833 b l O (a) (a) (a) (b) (b) Figure 6. Soya and Pulses records across the EU28 countries in the FUSION corpus. (a) 1980–2018. (b) 2010–2018. Note: only records indexed with soya or with pulses were included (i.e., records co-indexed with several groups of grain-legumes were excluded). Proportional count for international collaboration Figure 6. Soya and Pulses records across the EU28 countries in the Fusion corpus. (a) 1980–2018. (b) 2010–2018. Note: only records indexed with soya or with pulses were included (i.e., records co-indexed with several groups of grain-legumes were excluded). Proportional count for international collaboration records is applied. The country ranking is based on the total records number. Malta had no records. 17 of 37 Sustainability 2019, 11, 6833 Table 6. The eight main areas/countries publishing the most on pulses or soya. Table 6. The eight main areas/countries publishing the most on pulses or soya. 3.2.3. International Collaboration Research Is Increasing, but Unevenly on Soya or Pulses 3.2.3. International Collaboration Research Is Increasing, but Unevenly on Soya or Pulses 3.2.3. International Collaboration Research Is Increasing, but Unevenly on Soya or Pulses 3.2.3. International Collaboration Research Is Increasing, but Unevenly on Soya or Pulses The records involving several countries (international collaboration records) amount to 19% of the records indexed with soya or pulses (16% before 2010 and 23% in the current decade). We identiﬁed 3495 combinations of countries among those collaborations and most of those involved two countries. Figure 7a,b shows the most frequent collaborations on soya or pulses globally. We observed a clear leadership of the USA in those collaborations both for soya and pulses. Although there were fewer international records on pulses than on soya among the 20 most frequent collaborations, over the current decade 26% of records on pulses involved international collaboration compared with 22% for soya. This diﬀerence at a global scale is due to the increasing collaboration among the EU28 countries, which focus more on pulses than on soya. Over the current decade, this ranking of the most frequent collaborations has remained stable. The records involving several countries (international collaboration records) amount to 19% of the records indexed with soya or pulses (16% before 2010 and 23% in the current decade). We identified 3495 combinations of countries among those collaborations and most of those involved two countries. Figure 7a,b shows the most frequent collaborations on soya or pulses globally. We observed a clear leadership of the USA in those collaborations both for soya and pulses. Although there were fewer international records on pulses than on soya among the 20 most frequent collaborations, over the current decade 26% of records on pulses involved international collaboration compared with 22% for soya. This difference at a global scale is due to the increasing collaboration among the EU28 countries, which focus more on pulses than on soya. Over the current decade, this ranking of the most frequent collaborations has remained stable. (a) (b) Figure 7. The 20 most frequent international collaborations 1980–2018 in the FUSION corpus. (a) Most frequent international collaborations on soya. (b) Most frequent international collaborations on pulses. Figure 7. The 20 most frequent international collaborations 1980–2018 in the Fusion corpus. (a) Most frequent international collaborations on soya. (b) Most frequent international collaborations on pulses. (b) (b) Figure 7. The 20 most frequent international collaborations 1980–2018 in the FUSION corpus. (a) Most frequent international collaborations on soya. (b) Most frequent international collaborations on pulses. Figure 7. 3.2.2. The Percentage of Soya and Pulses Records per Country Is Quite Stable over Time Area Rank * Area or Country 1980–2018 Period 1980–2009 Period 2010–2018 Period S / P % S % P % S % S % P % S / P % S % P % 1 USA 18,238 24% 13,487 33% 4751 13% 12,524 30% 9148 42% 3376 17% 5714 16% 4339 22% 1375 9% 2 UE28 14,854 19% 5260 13% 9594 27% 9401 22% 3078 14% 6323 31% 5453 16% 2182 11% 3271 21% 4 INDIA 7043 9% 1634 4% 5409 15% 3198 8% 741 3% 2457 12% 3845 11% 893 5% 2952 19% 3 CHINA 5926 8% 4564 11% 1362 4% 1323 3% 908 4% 415 2% 4603 13% 3656 19% 947 6% 5 BRAZIL 5712 7% 3645 9% 2067 6% 2088 5% 1284 6% 804 4% 3624 10% 2361 12% 1263 8% 6 JAPAN 4286 6% 3147 8% 1139 3% 2926 7% 2053 9% 873 4% 1360 4% 1094 6% 266 2% 7 CANADA 3524 5% 1722 4% 1802 5% 2097 5% 1027 5% 1070 5% 1427 4% 695 4% 732 5% 8 AUSTRALIA 2530 3% 747 2% 1783 5% 1702 4% 502 2% 1200 6% 828 2% 245 1% 583 4% SUBTOTAL 62,113 80% 34,206 83% 27,907 78% 35,259 84% 18,741 86% 16,518 82% 26,854 76% 15,465 79% 11,389 73% TOTAL * 77,170 100% 41,413 100% 35,757 100% 42,014 100% 21,856 100% 20,158 100% 35,156 100% 19,557 100% 15,599 100% Note: S means Soya; P means Pulses; S/P considers both. * Area ranking over 1980–2018 for Soya and Pulses records. subtotal corresponds to the sum of the eight main areas/countries. * total corresponds to all countries. Number of records indexed either with soya (only) or with pulses (only), with proportional count for international collaboration records. These counts were applied on a subset of the Fusion corpus as authors’ countries were not identiﬁed for nearly 13,000 records indexed with soya or pulses. Note: S means Soya; P means Pulses; S/P considers both. * Area ranking over 1980–2018 for Soya and Pulses records. subtotal corresponds to the sum of the eight main areas/countries. * total corresponds to all countries. Number of records indexed either with soya (only) or with pulses (only), with proportional count for international collaboration records. These counts were applied on a subset of the Fusion corpus as authors’ countries were not identiﬁed for nearly 13,000 records indexed with soya or pulses. 3.2.2. The Percentage of Soya and Pulses Records per Country Is Quite Stable over Time Sustainability 2019, 11, 6833 Sustainability 2019, 11, x FOR 18 of 37 18 of 38 3.2.3. International Collaboration Research Is Increasing, but Unevenly on Soya or Pulses 3.2.3. International Collaboration Research Is Increasing, but Unevenly on Soya or Pulses The 20 most frequent international collaborations 1980–2018 in the Fusion corpus. (a) Most frequent international collaborations on soya. (b) Most frequent international collaborations on pulses. Sustainability 2019, 11, 6833 with pulses (only), res the horizontal axis. I 19 of 37 cified in oes not The counts in Figure 7a,b correspond to the number of records indexed with soya (only) and with pulses (only), respectively, and for which the authors were only from the countries speciﬁed in the horizontal axis. In the WoS, the country indexing is alphabetically ordered and does not correspond to the authors’ order in records. 3.3. Percentage of Themes in the Soya and Pulses Literature Figure 8 presents the shares of the 10 themes considered in the FUSION corpus for soya and pulses. First, the breakdown of themes differed for soya and pulses, even in recent years: PROCESSING 3.3. Percentage of Themes in the Soya and Pulses Literature and NUTRITION were more frequent themes for soya tha for soya are slightly greater than for all pulses (see Figure 8 presents the shares of the 10 themes considered in the Fusion corpus for soya and pulses. First, the breakdown of themes diﬀered for soya and pulses, even in recent years: Processing and Nutrition were more frequent themes for soya than for pulses. In addition, while total records for soya are slightly greater than for all pulses (see Section 3.1), “upstream” themes—such as Genetics, Ecophysiology, BioticStress and Agronomy—had a few more records for pulses than soya. These comparative ﬁgures clearly highlight the fact that “downstream” themes are less invested for pulses compared to soya. Moreover, as mentioned above, while feeding is a major outlet for soya, this theme did not represent a large share of the research on soya. However, records on feeding for soya double the number of records for pulses on the same theme. The remaining minor themes, such as Acceptability and Allergy, were more developed for soya than for pulses, even in the recent period. GENETICS, ECOPHYSIOLOGY, BIOTICSTRESS and AGRONOMY—had a few more records for pulses than soya. These comparative figures clearly highlight the fact that “downstream” themes are less invested for pulses compared to soya. Moreover, as mentioned above, while feeding is a major outlet for soya, this theme did not represent a large share of the research on soya. However, records on feeding for soya double the number of records for pulses on the same theme. The remaining minor themes, such as ACCEPTABILITY and ALLERGY, were more developed for soya than for pulses, even in the recent period. (a) Figure 8. Cont. (a) Figure 8. Cont. Figure 8. Cont. Sustainability 2019, 11, 6833 20 of 37 (b) Figure 8. Shares of themes within records in the FUSION corpus over 1980–2018 and 2010–2018. (a) Records on soya. (b) Records on pulses. Figure 8. Shares of themes within records in the Fusion corpus over 1980–2018 and 2010–2018. (a) Records on soya. (b) Records on pulses. (b) (b) Figure 8. Shares of themes within records in the FUSION corpus over 1980–2018 and 2010–2018. (a) Records on soya. (b) Records on pulses. Figure 8. Shares of themes within records in the Fusion corpus over 1980–2018 and 2010–2018. (a) Records on soya. (b) Records on pulses. The results show that the USA, the EU28, India, and China are the four main countries/areas publishing on soya or pulses (Table 6). 3.3. Percentage of Themes in the Soya and Pulses Literature and NUTRITION were more frequent themes for soya tha for soya are slightly greater than for all pulses (see We calculated the thematic shares for those main countries/areas compared to all other countries to illustrate the variation of themes investigated for pulses and soya (Figure A2). On average, while compared to the USA the EU28 globally focused more on pulses than on soya, for some themes, the gap between them is smaller. For instance, in GENETICS and BIOTICSTRESS, the USA worked on these themes for pulses as much as the EU28 did, while for soya the EU28 published little on these subjects. The results show that the USA, the EU28, India, and China are the four main countries/areas publishing on soya or pulses (Table 6). We calculated the thematic shares for those main countries/areas compared to all other countries to illustrate the variation of themes investigated for pulses and soya (Figure A2). On average, while compared to the USA the EU28 globally focused more on pulses than on soya, for some themes, the gap between them is smaller. For instance, in Genetics and BioticStress, the USA worked on these themes for pulses as much as the EU28 did, while for soya the EU28 published little on these subjects. Some themes have varying interest by countries. For instance, considering China’s strong and recent focus on soya, the most investigated themes are, respectively: PROCESSING, GENETICS, ECOPHYSIOLOGY, NUTRITION, and BIOTICSTRESS. For the USA, these come in a different order: GENETICS, ECOPHYSIOLOGY, BIOTICSTRESS, PROCESSING, and AGRONOMY. Some themes have varying interest by countries. For instance, considering China’s strong and recent focus on soya, the most investigated themes are, respectively: Processing, Genetics, Ecophysiology, Nutrition, and BioticStress. For the USA, these come in a diﬀerent order: Genetics, Ecophysiology, BioticStress, Processing, and Agronomy. We previously observed that “downstream” themes were less studied for pulses, yet this difference is less pronounced for the EU28 and India. For instance, they focused almost as much on soya as on pulses for NUTRITION and ACCEPTABILITY themes. Therefore, if spatial correlation between the scholarly records on a crop and its level of production in the country seems to be an explanation at first view, further investigation is needed to better understand the variation of themes between soya and pulses according to countries. This could be linked to the research strategies of a specific research team, which would need a socio-semantic network study to uncover fully. 3.4. Implications for Future Research Policy on Grain-Legumes 3.4. Implications for Future Research Policy on Grain-Legumes The shares of crops in research are strongly path-dependent, especially as regards soya and pulses. Soya dominates research on legumes at the global scale whatever the theme, being also the crop with a dominant market size compared to pulses (Appendix A, Table A1). In addition, we observed that the gap between soya and pulses has strongly grown over time for some countries such as China. For instance, when counting the records before 2010 for China, the gap between pulses and soya was not so great, but it strongly increased afterwards. In contrast, for other countries, the gap between soya and pulses has been stable: that is to say, no opposite trend appears between the two periods for any country or theme considered here. Globally, pulses beneﬁt from less research activity than major crops such as soy, as advanced by other works [32]. As regards global plant-based protein markets for food, soya is mainly used in current product innovations compared to pulses [33]. Therefore, since markets do not seem to drive agrofood systems towards more agricultural diversity, this raises questions about how to shift research to provide knowledge that will make a transition towards more grain-legumes crop diversity possible. The present study did not examine research trends according to private or public funding. One possible avenue for future research would be to determine whether public funding favors diversity in agricultural research. Such an investigation would require analyzing the “Funding Acknowledgment Table” indexed by the WoS since 2008, through a list of institution names that need to be standardized for analysis. Finally, the shares of grain-legumes in research activity seems not to be totally dependent on the crop production of the countries and further investigation must be conducted to better analyze for which countries this spatial correlation is stronger. As the share of research between soya and pulses is highly uneven, it is essential to increase research on pulses including a wide variety of species, compared to soya as a single species, as advocated during the IYP in 2016. One main challenge remains to create links between species, what some researchers call “translational” research. This would require important changes in research. 3.3. Percentage of Themes in the Soya and Pulses Literature and NUTRITION were more frequent themes for soya tha for soya are slightly greater than for all pulses (see We previously observed that “downstream” themes were less studied for pulses, yet this diﬀerence is less pronounced for the EU28 and India. For instance, they focused almost as much on soya as on pulses for Nutrition and Acceptability themes. Therefore, if spatial correlation between the scholarly records on a crop and its level of production in the country seems to be an explanation at ﬁrst view, further investigation is needed to better understand the variation of themes between soya and pulses according to countries. This could be linked to the research strategies of a speciﬁc research team, which would need a socio-semantic network study to uncover fully. As regards the FEEDING theme, while increasing pulses in feed is an important goal for European public authorities that has led to millions in investments, there is still more research on soya by the As regards the Feeding theme, while increasing pulses in feed is an important goal for European public authorities that has led to millions in investments, there is still more research on soya by the EU28 than on pulses, representing about as many records as for the USA. Other countries are much Sustainability 2019, 11, 6833 21 of 37 involved in soya research for the Feeding theme, such as the considerable focus in South America (especially Brazil and Argentina). 3.4. Implications for Future Research Policy on Grain-Legumes For instance, if the large body of research on agronomy and ecophysiology were more systematically associated with crop modeling, their hypotheses and results would be easier to use for research protocols in other species. As shown by the relatively low number of studies combining at least two pulse species, there is also a need for more comparative analysis across species and for all the themes identiﬁed in this study. Research planning and policy should also consider that the model used for oil-legumes, focused on one species (soybean), will not work with pulses, which have strong speciﬁcities with regards to consumer preferences and food processing. Public investments and public–private partnerships will be essential to ensure that an increase in pulse research funding does not erode this pulse species diversity. It was outside the scope of this study to analyze ﬁnancial investments in research on pulses, but the number of publications on these crops, their increase during the last two decades, and the identiﬁcation of leading countries could support an international approach to the research on these crops. They would be essential components of nutrition sensitive agriculture in the Earth’s margins, recently termed “environmental nutrition” [34]. Moreover, as future pulse development would be for food and nutrition, more investment in food sciences, including processing, is crucial for providing diverse pulse-based products that ensure food security and good health. While for upstream themes required to increase production (such as Genetics and Ecophysiology) pulses have received a good deal of attention, more research in food sciences is still needed to develop markets as argued in other studies. The results here show that both Nutrition and Processing themes were strong for soya, while the food outlet for whole-grain soya represents only around 5% of its production [9]. That is to say, around 15 million tons of soya production is entirely used for food, while the food outlet for pulses is considered to be (at least) around 50% of the global production, that is, accounting for a higher food outlet of around 50 million tons according to the volumes of those crops (Table A1). However, our study found less research for pulses in 22 of 37 Sustainability 2019, 11, 6833 themes related to food sciences. 4. Conclusions Understanding the dynamics of research is an epistemic project that concerns all sciences and is one primary focus of STS. Analyzing bibliometric datasets help both decision-makers for science policies and scholars to orient science and, in the end, innovation. By using scientometric indicators on the metadata of scholarly documents retrieved from the WoS, we gave an overview of the research output on grain-legumes since 1980 at the global scale. We quantiﬁed the shares of species, analyzed by main academic ﬁelds (i.e., themes) and countries. To establish these results, this work developed a rigorous methodology for building a bibliometric dataset and a processing software, which can be used for further bibliometric research on other crops to bring a larger analysis of the dynamics of research activity on agricultural and food systems. We ﬁrst discuss main implications of this work, and then propose further works to be conducted to improve our analyses. First, these results show that research on grain-legumes is path-dependent and is strongly linked to the size of their agricultural production, as the main species researched in the past continue to be the main focus of research today. These ﬁndings should foster further discussion and reﬂection among the scientiﬁc community about grain-legumes, especially regarding the challenge of greater crop diversity. Above all, researchers must be encouraged to create links among species to enlarge the application of their results, as we found few records mentioning several species together. In addition, while upstream themes have received considerable attention (such as Genetics and Ecophysiology), downstream themes have received unequal interest among crops; particularly, compared to soya, pulses have been less researched in relation to downstream themes of food sciences. This imbalance questions the capacity of research to develop knowledge that would enable more food outlets for pulses, as expected by the United Nations during the IYP. Finally, the results show that most research on grain-legumes has been conducted by eight countries/geographical areas (including the European Union). While some geographical areas have done more research on pulses than on soya, such as the EU28 and India, others specialized on soya, such as many American countries. Newcomers such as China clearly made the choice to invest more in research on soya, establishing important collaborations with the USA. Second, there is a strategic interest in bibliometric studies on the way research is conducted on agricultural and agrofood systems, in order to deﬁne new research priorities. 3.4. Implications for Future Research Policy on Grain-Legumes By creating products meeting food habits and preferences, improving nutritional values, and increasing their usage by renowned food brands, research in processing could be a way to increase pulse consumption, to value under-used pulses species, and ﬁnally to build a value chain by making pulses more than a low-cost substitute for animal proteins [35]. A compromise between marketing opportunities and aﬀordable healthy food for consumers should more often be an objective of research in food sciences. Agricultural and food sciences have to work hand in hand for sustainable agriculture, designing coupled innovations between the upstream and downstream of supply chains [36]. Therefore, more publications co-indexed with agricultural and food sciences are expected. 4. Conclusions This type of work is not an easy task, and collaboration between scientiﬁc experts on the themes investigated and experts in scientometrics is essential. Given that we are faced with an ever increasing amount of published data, involving researchers on the measurement of sciences is essential. Moreover, those researchers could advise other researchers on the way to communicate about their work. Indeed, the way bibliometric datasets are collected and how researchers describe their works through the title, abstract, and keywords of records impact the ways we can analyze research patterns through bibliometric data. Title, abstract, and keywords are the main data on which scientometric studies relies, and thus their contents strongly determine the results obtained. Our ﬁndings raise another question about the way species are mentioned: a common dictionary of species names in scientiﬁc platforms, such as the WoS, would help to better follow species, and to conduct longitudinal analysis on the percentage of any crop in research and their links. Sustainability 2019, 11, 6833 23 of 37 Thirdly, we used English-language scholarly publications, such as peer-reviewed articles, books, and book chapters, to reﬂect research activity and to give an overview of the majority of the scientiﬁc knowledge on grain-legumes. Other data collections could enrich our dataset to have a more complete overview. The ﬁrst improvement will be to add records retrieved from the Scopus Collection (the other most used bibliometric platform), even though the WoS and Scopus overlap considerably for certain disciplines [26]. This could lead to some adjustments in the shares of themes, but probably not in their ranking. Other enrichments such as using alternative search engines, e.g., Google Scholar (GS), will require methodological solutions. GS provides access to larger collection of research such as PhD theses, scientiﬁc reports, and non-peer-reviewed articles, but no software exists to retrieve large collections from GS. In addition, no rigorous metadata are associated to enable relevant analysis among the various documents once they are collected. Last, while English dominates scientiﬁc publications, other languages are still used and including them would complicate larger record harvesting and analyses from GS. “The second most frequent language of unique GS citations was Chinese (4–12%), and all other languages have a share of 4% or lower across all subject areas. A few (5–10%) unique GS citations were published in languages outside the top 11 most frequently used languages overall” [26] (p. 14). 4. Conclusions To have a better overview of the evolution of this scholarly scientiﬁc knowledge on grain-legumes, further studies can be done (based on this dataset or an enriched dataset) such as semantic or socio-semantic network analysis. Co-words and institution mapping would provide overviews of the main and minor concepts and knowledge that characterize research according to species, themes, and countries. By identifying the main relationships between terms, bridges of knowledge and new research areas can be identiﬁed within and among species. In particular, as grain-legumes are recognized to have a high potential for more sustainable agriculture, it would be interesting to analyze the development of speciﬁc targets and wordings aiming at increasing the role of these species in a sustainable agricultural production, for both food and feed. Lastly, understanding the relationships between markets trends and research directions remains a main challenge of the STS. Future works could analyze the spatial correlation between scholarly documents on crops and the crops production/consumption levels within countries to analyze links between research activity and societal demands, as recently conducted on rice species [37]. Considering also the metadata associated to funding in notices (indexed in the WoS since 2008) will allow evaluating the shares of independent research (i.e., non-commercial funding) compared to other private-based funding, and could reveal diﬀerent research trajectories regarding the specialization vs. diversiﬁcation of crops under research. Author Contributions: Conceptualization, M.-B.M. and G.C.; Data curation, M.-B.M., G.C., M.L., H.L. and D.M.; Formal analysis, M.-B.M., G.C., M.L., M.-J.A., M.A., G.A., A.B., L.B., J.-M.C., G.D., M.-H.J., E.-P.J., H.J., C.L., H.L., V.M., D.M., M.-L.P.-N., C.N.-T., T.S., A.-S.V., S.W. and J.W.; Funding acquisition, M.-B.M.; Investigation, M.-B.M., G.C. and M.L.; Methodology, M.-B.M., G.C., M.L., M.-J.A., M.A., G.A., A.B., L.B., J.-M.C., G.D., M.-H.J., E.-P.J., H.J., C.L., H.L., V.M., D.M., M.-L.P.-N., C.N.-T., T.S., A.-S.V., S.W. and J.W.; Project administration, M.-B.M.; Resources, M.-B.M. and G.C.; Software, G.C.; Supervision, M.-B.M.; Validation, M.-B.M., G.C., M.L., G.P., M.-J.A., M.A., G.A., A.B., L.B., J.-M.C., G.D., M.-H.J., E.-P.J., H.J., C.L., H.L., V.M., D.M., M.-L.P.-N., C.N.-T., A.-S.V., S.W. and J.W.; Visualization, M.-B.M., G.C., H.L. and T.S.; Writing—original draft, M.-B.M., G.C. and G.P.; and Writing—review and editing, M.-B.M., G.C., M.L., G.P., M.-J.A., M.A., G.A., A.B., L.B., J.-M.C., G.D., M.-H.J., E.-P.J., H.J., C.L., H.L., V.M., D.M., M.-L.P.-N., C.N.-T., A.-S.V., S.W. and J.W. Author Contributions: Conceptualization, M.-B.M. and G.C.; Data curation, M.-B.M., G.C., M.L., H.L. Author Contributions: Conceptualization, M.-B.M. and G.C.; Data curation, M.-B.M., G.C., M.L., H.L. and D.M.; Formal analysis, M.-B.M., G.C., M.L., M.-J.A., M.A., G.A., A.B., L.B., J.-M.C., G.D., M.-H.J., E.-P.J., H.J., C.L., H.L., V.M., D.M., M.-L.P.-N., C.N.-T., T.S., A.-S.V., S.W. and J.W.; Funding acquisition, M.-B.M.; Investigation, M.-B.M., G.C. and M.L.; Methodology, M.-B.M., G.C., M.L., M.-J.A., M.A., G.A., A.B., L.B., J.-M.C., G.D., M.-H.J., E.-P.J., H.J., C.L., H.L., V.M., D.M., M.-L.P.-N., C.N.-T., T.S., A.-S.V., S.W. and J.W.; Project administration, M.-B.M.; Resources, M.-B.M. and G.C.; Software, G.C.; Supervision, M.-B.M.; Validation, M.-B.M., G.C., M.L., G.P., M.-J.A., M.A., G.A., A.B., L.B., J.-M.C., G.D., M.-H.J., E.-P.J., H.J., C.L., H.L., V.M., D.M., M.-L.P.-N., C.N.-T., A.-S.V., S.W. and J.W.; Visualization, M.-B.M., G.C., H.L. and T.S.; Writing—original draft, M.-B.M., G.C. and G.P.; and Writing—review and editing, M.-B.M., G.C., M.L., G.P., M.-J.A., M.A., G.A., A.B., L.B., J.-M.C., G.D., M.-H.J., E.-P.J., H.J., C.L., H.L., V.M., D.M., M.-L.P.-N., C.N.-T., A.-S.V., S.W. and J.W. Appendix A Table A1. Global production of main pulses, soya and cereals, trienniums ending 1971, 1981, 1991, 2001, 2011, and 2017 (million metric tons). Source: FAOstat. Table A1. Global production of main pulses, soya and cereals, trienniums ending 1971, 1981, 1991, 2001, 2011, and 2017 (million metric tons). Source: FAOstat. Species Year 1971 1981 1991 2001 2011 2017 Bean (dry) 12 15 18 18 24 34 Chickpea 6 5 8 6 11 14 Pea (dry) 9 7 12 10 10 16 Faba/broadbean 8 8 6 8 8 8 Lentil 1 1 2 3 4 7 Pigeon pea 2 2 2 3 4 6 Cowpea 1 1 2 3 4 7 Vetches 1 1 1 1 1 1 Lupin 0.3 1 1 1 1 1 Bambara Bean 0.03 0.03 0.08 0.08 0.14 0.18 Other pulses 3 2 4 3 3 4 Total Pulses 42 41 56 56 69 95 Soya 45 88 102 177 261 352 Cereals 1229 1632 1890 2104 2588 2980 Table A2. Links to the 11 WoS queries designed to delineate the corpus under study, each query capturing thematic corpus on grain-legumes (e.g., Allergy and BioticStress). The Species query was combined to thematic queries or used isolated. Table A2. Links to the 11 WoS queries designed to delineate the corpus under study, each query capturing thematic corpus on grain-legumes (e.g., Allergy and BioticStress). The Species query was combined to thematic queries or used isolated. 4. Conclusions and D.M.; Formal analysis, M.-B.M., G.C., M.L., M.-J.A., M.A., G.A., A.B., L.B., J.-M.C., G.D., M.-H.J., E.-P.J., H.J., C.L., H.L., V.M., D.M., M.-L.P.-N., C.N.-T., T.S., A.-S.V., S.W. and J.W.; Funding acquisition, M.-B.M.; Investigation, M.-B.M., G.C. and M.L.; Methodology, M.-B.M., G.C., M.L., M.-J.A., M.A., G.A., A.B., L.B., J.-M.C., G.D., M.-H.J., E.-P.J., H.J., C.L., H.L., V.M., D.M., M.-L.P.-N., C.N.-T., T.S., A.-S.V., S.W. and J.W.; Project administration, M.-B.M.; Resources, M.-B.M. and G.C.; Software, G.C.; Supervision, M.-B.M.; Validation, M.-B.M., G.C., M.L., G.P., M.-J.A., M.A., G.A., A.B., L.B., J.-M.C., G.D., M.-H.J., E.-P.J., H.J., C.L., H.L., V.M., D.M., M.-L.P.-N., C.N.-T., A.-S.V., S.W. and J.W.; Visualization, M.-B.M., G.C., H.L. and T.S.; Writing—original draft, M.-B.M., G.C. and G.P.; and Writing—review and editing, M.-B.M., G.C., M.L., G.P., M.-J.A., M.A., G.A., A.B., L.B., J.-M.C., G.D., M.-H.J., E.-P.J., H.J., C.L., H.L., V.M., D.M., M.-L.P.-N., C.N.-T., A.-S.V., S.W. and J.W. Funding: This work was supported by funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No. 727672—LEGVALUE (Fostering sustainable legume-based farming systems and agri-feed and food chains in the EU); from the Agence Nationale de la Recherche (ANR) under grant number ANR-11-LABX-0066; and by the French National Institute for Agricultural Research (INRA). Acknowledgments: The authors acknowledge the two anonymous reviewers whose comments and suggestions helped sharpen the argument, and Cynthia J. Johnson for her helpful comments and English editing of the manuscript. Conﬂicts of Interest: The authors declare no conﬂict of interest. 24 of 37 Sustainability 2019, 11, 6833 Appendix A Query Name Link to the Search Query Applied on the Web of Science (WoS) Core Collection THE SPECIES SEARCH QUERY SPECIES DUC, Gérard; WERY, Jacques; MAGRINI, Marie-Benoit; CABANAC, Guillaume, 2019, “Grain-Legumes Species WoS DataSet 1980–2018”, https://doi.org/10.15454/QBQFCX, Portail Data Inra THE 10 THEMATIC SEARCH QUERIES GENETICS DUC, Gérard; WERY, Jacques; MILLOT, Dominique; CABANAC, Guillaume, 2019, “Genetics and Grain-Legumes WoS DataSet 1980–2018”, https://doi.org/10.15454/PFV9JK, Portail Data Inra AGRONOMY JEUFFROY, Marie-Hélène; BEDOUSSAC, Laurent; MILLOT, Dominique; CABANAC, Guillaume, 2019, “Agronomy and Grain-Legumes WoS DataSet 1980–2018”, https://doi.org/10.15454/W6BAUG, Portail Data Inra ECOPHYSIOLOGY VOISIN, Anne-Sophie; JOURNET, Etienne-Pascal; LEISER, Hugues; CABANAC, Guillaume, 2019, “Ecophysiology and Grain-Legumes WoS DataSet 1980–2018”, https://doi.org/10.15454/F0CNNS, Portail Data Inra BIOTICSTRESS BARANGER, Alain; PILET-NAYEL, Marie-Laure; MILLOT, Dominique; CABANAC, Guillaume, 2019, “Biotic Stress and Grain-Legumes WoS DataSet 1980–2018”, https://doi.org/10.15454/L79X2K, Portail Data Inra FEEDING JUIN, Hervé; LEISER, Hugues; CABANAC, Guillaume, 2019, “Feeding and Grain-Legumes WoS DataSet 1980–2018”, https://doi.org/10.15454/BNKFVC, Portail Data Inra PROCESSING ANTON, Marc; MICARD, Valérie; NGUYEN-THE, Christophe; LEISER, Hugues; CABANAC, Guillaume, 2019, “Processing and Grain-Legumes WoS DataSet 1980–2018”, https://doi.org/10.15454/VP7PRI, Portail Data Inra NUTRITION AMIOT-CARLIN, Marie-Josephe; CHARDIGNY, Jean-Michel; WALRAND, Stéphane; LEISER, Hugues; CABANAC, Guillaume, 2019, “Nutrition and Grain-Legumes WoS DataSet 1980–2018”, https://doi.org/10.15454/5MI04S, Portail Data Inra ALLERGY LARRE, Colette; DENERY, Sandrine; LESIER, Hugues; CABANAC, Guillaume, 2019, “Allergy and Grain-Legumes WoS DataSet 1980–2018”, https://doi.org/10.15454/BZG0R7, Portail Data Inra ACCEPTABILITY ARVISENET, Gaelle; MAGRINI, Marie-Benoit; LEISER, Hugues; CABANAC, Guillaume, 2019, “Acceptability and Grain-Legumes WoS DataSet 1980–2018”, https://doi.org/10.15454/PDXRYM, Portail Data Inra SOCIOECONOMICS Magrini, Marie-Benoit; Plumecocq, Gael; Leiser, Hugues; Cabanac, Guillaume, 2019, “Socioeconomics and Grain-Legumes WoS DataSet 1980–2018”, https://doi.org/10.15454/JNIPX5, Portail Data Inra 25 of 37 Sustainability 2019, 11, 6833 Table A3. Breakdown of the Fusion corpus in the 10 underlying themes (single or combined themes indexed). Themes Index Colum A Records Number Indexed with a Single Theme * Frequency Ranking of the Single Theme Other Themes Index Combined with the Theme in Colum A ** 1st Most Freq. Nb. Records 2nd Most Freq. Nb. Records 3nd Most Freq. Nb. Records 4th Most Freq. Nb. Records 5th Most Freq. Nb. Records 6th Most Freq. Nb. Records Total nb of Records Indexed with the Theme of Column A Share of Themes Genetics 13,336 1 Ecophy. 6845 BioticS. 4887 Agro. 2176 Agro. & Ecophy. 1250 Ecophy. & BioticS. 1119 Process. 977 34,388 22% Ecophysiology (Ecophy.) 12,841 2 Genetics 6845 Agro. 3263 BioticS.s 1411 Agro. & Genetics 1250 BioticS. & Genetics 1119 Process. 878 29,867 19% Processing (Process.) 12,049 3 Nutrition 5965 Accep. 1298 Nutrition & Accept. Note: * that is neither of the Title, Abstract or Authors’ keywords contain terms present in the query of any other theme. ** that is the Title, Abstract or authors’ keywords contain terms inked to those two themes, but not of other themes. All the various combination of frequencies presented in this table represent 99% of the records in Fusion corpus. Appendix B. Robustness Assessment of the Delineation Process: Testing an Alternative Strate Appendix B. Robustness Assessment of the Delineation Process: Testing an Alternative Strate We experimented with an alternative search strategy using the WoS to delineate a core scientiﬁc literature dataset on grain-legumes. As explained in Section 2, to obtain close links between grain-legumes species and speciﬁc thematic terms, we gave preference to the use of the Boolean character near/10 in the search query design of most themes. In that way, the search query matched records whose terms joined by the operator were within 10 words of each other. However, to appreciate the diﬀerences in the frequency of records retrieved, depending on the use of the operator near or not, we also built other bibliometric datasets on those grain-legumes, resulting from search queries without the near operator. This other strategy led to the so-called Species1, Species2, and Species3 corpora, following an alternative methodology illustrated in Figure A1. This alternative delineation strategy used the same terms in the search queries than the one exposed in Section 2. We experimented with an alternative search strategy using the WoS to delineate a core scientific literature dataset on grain-legumes. As explained in Section 2, to obtain close links between grain- legumes species and specific thematic terms, we gave preference to the use of the Boolean character NEAR/10 in the search query design of most themes. In that way, the search query matched records whose terms joined by the operator were within 10 words of each other. However, to appreciate the differences in the frequency of records retrieved, depending on the use of the operator NEAR or not, we also built other bibliometric datasets on those grain-legumes, resulting from search queries without the NEAR operator. This other strategy led to the so-called SPECIES1, SPECIES2, and SPECIES3 corpora, following an alternative methodology illustrated in Figure A1. This alternative delineation strategy used the same terms in the search queries than the one exposed in Section 2. Figure A1. Main steps to build the bibliometric dataset according to an alternative delineation strategy. Figure A1 summarizes the main steps followed to build the corpora quite similar to that of Figure 1 (Section 2), but with a change in the way to design search queries and the indexing procedure. This alternative delineation strategy led to three other corpora called SPECIES1, SPECIES2, and SPECIES3. 1.1. Species search queries 1.2. Appendix B. Robustness Assessment of the Delineation Process: Testing an Alternative Strate Appendix B. Robustness Assessment of the Delineation Process: Testing an Alternative Strate Main steps to build the bibliometric dataset according to an alternative delineation strategy. Figure A1 summarizes the main steps followed to build the corpora quite similar to that of Figure 1 (Section 2), but with a change in the way to design search queries and the indexing procedure. This alternative delineation strategy led to three other corpora called Species1, Species2, and Species3. SPECIES1, SPECIES2, and SPECIES3 are the corpora retrieved from the WoS by using the SPECIES search query only, on which various indexing step strategies were applied. The aforementioned indexing step consists in keeping the records having a term of the search query among the Title, Abstract or Author’s keywords only (i.e., filtered from the KeyWord Plus). This also led to indexing each record with the search terms found in Title, Abstract or Author’s keywords. First, each record was indexed with one or several legume species according to the SPECIES terms occurring in the record (see Table 3 for species indexation). Second, we indexed each record relatively to the 10 thematic subjects when any of the terms of the records matched the terms of the thematic query (Appendix A, Table A2). In other words, this strategy did not rely on the NEAR operator but on the indexing procedure applied on the SPECIES corpus downloaded from the WoS. The three variants of the SPECIES corpora were built, depending on the way the indexing procedure was applied: Species1, Species2, and Species3 are the corpora retrieved from the WoS by using the Species search query only, on which various indexing step strategies were applied. The aforementioned indexing step consists in keeping the records having a term of the search query among the Title, Abstract or Author’s keywords only (i.e., ﬁltered from the KeyWord Plus). This also led to indexing each record with the search terms found in Title, Abstract or Author’s keywords. First, each record was indexed with one or several legume species according to the Species terms occurring in the record (see Table 3 for species indexation). Second, we indexed each record relatively to the 10 thematic subjects when any of the terms of the records matched the terms of the thematic query (Appendix A, Table A2). In other words, this strategy did not rely on the near operator but on the indexing procedure applied on the Species corpus downloaded from the WoS. Appendix A 1046 Allergy 1015 Genetics 977 Ecophy. 878 27,180 17% BioticStress (BioticS.) 9109 4 Genetics 4887 Ecophy. 1411 Agro. 1556 Ecophy. & Genetics 1119 Accept. & Nutrition 1046 Agro. & Genetics 583 20,243 13% Agronomy (Agro.) 7982 5 Ecophy. 3263 Genetics 2176 BioticS. 1556 Ecophy. & Genetics 1250 BioticS. & Genetics 583 Process. 505 19,070 12% Nutrition 4866 9 Process. 5965 Accept. & Process. 1046 Genetics & Process. 606 Genetics 578 Ecophy. 238 Ecophy. & Process. 214 15,383 10% Feeding 2888 11 Genetics 181 Process. 147 Nutrition 113 Nutrition & Process. 80 Agro. 42 Ecophysiology 36 3654 2% Allergy 1891 13 Process. 1015 Nutrition & Process. 191 Nutrition 148 Genetics 59 Genetics & Process. 28 Genetics & Nutrition 27 3650 2% Acceptability (Accept.) 745 23 Process. 1298 Nutrition & Process. 1046 Nutrition 194 Genetics & Process. 105 Genetics 90 Genetics & Nutrition 81 3972 3% Socioeconomics (Socioeco.) 565 27 Agro. 125 Process. 44 Genetics 33 Agro. & Ecophy. 31 Agro. & Genetics 27 Accept. & Process. 15 963 1% SUBTOTAL 66,272 158,370 100% in % of corpus 61% Total Corpus 107,823 Note: * that is neither of the Title, Abstract or Authors’ keywords contain terms present in the query of any other theme. that is the Title, Abstract or authors’ keywords contain terms linked to those two themes but not of other themes All the various combination of frequencies presented in this table represent 99% of the records in Fusion corpus Table A3. Breakdown of the Fusion corpus in the 10 underlying themes (single or combined themes indexed). Other Themes Index Combined with the Theme in Colum A Note: * that is neither of the Title, Abstract or Authors’ keywords contain terms present in the query of any other theme. ** that is the Title, Abstract or authors’ keywords contain terms linked to those two themes, but not of other themes. All the various combination of frequencies presented in this table represent 99% of the records in Fusion corpus. Sustainability 2019, 11, 6833 Sustainability 2019, 11, x FOR 26 of 37 26 of 38 Appendix B. Robustness Assessment of the Delineation Process: Testing an Alternative Strate Appendix B. Robustness Assessment of the Delineation Process: Testing an Alternative Strate Species corpus download Single species and thematic indexing Alternative Delineation Strategy Doc Type + Time Range + TS (Title, Abstract, Authors keywords, KeyWords Plus) + Excluding conditions (NOT TS) Corpora retrieved from the WoS Online interactive bibliometric platform SCIM Title, Abstract, Authors keywords 2. Species & Themes indexing procedures Species3 corpus 3 Corpora filtered Species2 corpus Species1 corpus Single species and double thematic indexing Double species and thematic indexing 3. Bibliometric analysis Shares of species, themes countries, time evolution… Figure A1. Main steps to build the bibliometric dataset according to an alternative delineation strategy. Figure A1 summarizes the main steps followed to build the corpora quite similar to that of Figure 1 (Section 2), but with a change in the way to design search queries and the indexing procedure. This alternative delineation strategy led to three other corpora called Species1, Species2, and Species3. Alternative Delineation Strategy Doc Type + Time Range + TS (Title, Abstract, Authors keywords, KeyWords Plus) 1.1. Species search queries + Excluding conditions (NOT TS) 1.2. Species corpus download Corpora retrieved from the WoS 2. Species & Themes indexing procedures Online interactive bibliometric platform SCIM 3 Corpora filtered Species1 corpus Shares of species, themes countries, time evolution… Figure A1. Main steps to build the bibliometric dataset according to an alternative delineation strategy. Figure A1 summarizes the main steps followed to build the corpora quite similar to that of Figure 1 (Section 2), but with a change in the way to design search queries and the indexing procedure. This alternative delineation strategy led to three other corpora called SPECIES1, SPECIES2, and SPECIES3. Figure A1. Main steps to build the bibliometric dataset according to an alternative delineation strategy. Figure A1 summarizes the main steps followed to build the corpora quite similar to that of Figure 1 (Section 2), but with a change in the way to design search queries and the indexing procedure. This alternative delineation strategy led to three other corpora called Species1, Species2, and Species3. Figure A1. Main steps to build the bibliometric dataset according to an alternative delineation strategy. Figure A1 summarizes the main steps followed to build the corpora quite similar to that of Figure 1 (Section 2), but with a change in the way to design search queries and the indexing procedure. This alternative delineation strategy led to three other corpora called SPECIES1, SPECIES2, and SPECIES3. Figure A1. Appendix B. Robustness Assessment of the Delineation Process: Testing an Alternative Strate Appendix B. Robustness Assessment of the Delineation Process: Testing an Alternative Strate The size of these corpora varied and are reported in Table A4. We observed that, of course, the more we restricted the strategy on search query and indexing, the fewer records were included. We matched the records between Fusion and Species3 corpora: we observed a diﬀerence of 22,661 records caught by Species3 but not present in Fusion; inversely, 27,813 records were caught by Fusion and not present in Species3. However, above all, the correspondence between the wcs and the themes deﬁned by experts was less adequate for the Species1, Species2, and Species3 corpora compared to the Fusion corpus. For instance, the classiﬁcation of the records with the 10 themes investigated by experts was not really relevant in Species3, while we observed strong correspondence between the wcs and the thematic indexes in Fusion. Therefore, the forthcoming analysis by themes is more biased in Species3, as this corpus induces a biased representation of the 10 themes compared with Fusion. This was encountered, for instance, with the Nutrition theme that included a lot of records dealing with plant growth and not with human nutrition in Species3. For evidence on the stronger relevance of the delineation strategy kept for Fusion Corpus, compared to the alternative delineation strategy, we present Table A5 should the number of records according to thematic indexing, respectively, in the Fusion and Species3 corpora. We observed that in Fusion, the records indexed with a single theme were more frequent (61%) than in Species3 (25%). Hence, the Fusion corpus led to a quite clear thematic classiﬁcation of the records among the 10 themes investigated, given the fact that among the remaining co-indexed thematic records 30% were indexed with two themes, and 8% with three themes. More precisely when considering thematic ranking, in Fusion corpus, the ﬁve ﬁrst thematic indexes frequencies (concerning records indexed with only one theme) were: Genetics, Ecophysiology, Processing, BioticStress and Agronomy. The remaining single theme indexes (Nutrition, Feeding, Allergy, Acceptability, and Socioeconomics) appear with less frequency as there are many fewer records on these themes, and that Genetics, Ecophysiology, Processing, BioticStress, and Agronomy are themes whose co-indexing between themselves has a high frequency of records (on that point, see Table A3 that presents frequencies on co-indexing themes in the Fusion corpus). In all, these ﬁve themes were the most frequent, respectively, 22%, 19%, 17%, 13%, and 12%. Appendix B. Robustness Assessment of the Delineation Process: Testing an Alternative Strate Appendix B. Robustness Assessment of the Delineation Process: Testing an Alternative Strate The three variants of the Species corpora were built, depending on the way the indexing procedure was applied: • SPECIES1 has single species and thematic indexing. A record was indexed with a species term and with a thematic corpus, if at least one term of the species query and at least one term of the thematic query occurred in the record. • Species1 has single species and thematic indexing. A record was indexed with a species term and with a thematic corpus, if at least one term of the species query and at least one term of the thematic query occurred in the record. • SPECIES1 has single species and thematic indexing. A record was indexed with a species term and with a thematic corpus, if at least one term of the species query and at least one term of the thematic query occurred in the record. S 2 h i l i i d i d d bl h i i d i A d i d d i h • Species1 has single species and thematic indexing. A record was indexed with a species term and with a thematic corpus, if at least one term of the species query and at least one term of the thematic query occurred in the record. Sustainability 2019, 11, 6833 27 of 37 • Species2 has single species indexing and double thematic indexing. A record was indexed with a species term and with a thematic corpus, if at least one term of the species query and at least two terms of the thematic query occurred in the record. • Species2 has single species indexing and double thematic indexing. A record was indexed with a species term and with a thematic corpus, if at least one term of the species query and at least two terms of the thematic query occurred in the record. • Species3 has both double species and thematic indexing. A record was indexed with a species term and with a thematic corpus, if at least two terms of the species query and at least two terms of the thematic query occurred in the record. • Species3 has both double species and thematic indexing. A record was indexed with a species term and with a thematic corpus, if at least two terms of the species query and at least two terms of the thematic query occurred in the record. Appendix B. Robustness Assessment of the Delineation Process: Testing an Alternative Strate Appendix B. Robustness Assessment of the Delineation Process: Testing an Alternative Strate All these remarks show the importance of the delineation strategy in building a bibliometric corpus. Moreover, it is clear that conducting bibliometric analysis requires considerable support of experts in the scientiﬁc themes investigated, since relying on wcs alone is not suﬃcient to delineate a relevant bibliometric corpus. Consequently, for us, the methodology kept to establish Fusion corpus is the most appropriate for identifying a “core” literature dataset on grain-legumes whose records can be classiﬁed by relevant themes (see also Table A3). Notwithstanding, some statistics presented in the Section 3, were also calculated on the datasets Species1, -2, and -3, to be compared to the ones established on Fusion corpus. In particular we observed that the shares of species were similar regardless of the delineation strategy of the bibliometric corpora (Table A6). Sustainability 2019, 11, 6833 28 of 37 Table A4. Size of the bibliometric corpora depending on the delineation strategy. Delineation Strategy Kept Alternative Delineation Strategy Corpus Fusion Corpus Species1 Corpus Species2 Corpus Species3 Search query applied on the WoS For most thematic corpora, species and thematic terms combined with operator near/10. See the ten thematic search queries in Appendix A. Species terms only. Species search query in Appendix A. Excluding conditions Some terms restrictions and wcs restrictions, depending on the thematic corpus. The same term restrictions as in Fusion, but no wcs restrictions. Number of records retrieved from the WoS per corpus Genetics 34,968 202,144 Agronomy 19,427 Ecophysiology 30,365 BioticStress 20,853 Feeding 4336 Processing 35,754 Nutrition 16,863 Allergy 5435 Acceptability 5459 Socioeconomics 1431 Indexing procedure One occurrence in the species terms and one occurrence in the thematic terms. One occurrence in the species terms and one occurrence in the thematic terms. One occurrence in the species terms and two occurrences in the thematic terms. Two occurrences in the species terms and two occurrences in the thematic terms. Number of records kept after indexing (share of records kept in %) Genetics 34,388 98% 160,238 (79%) 142,763 (71%) 100,248 (50%) Agronomy 19,070 98% Ecophysiology 29,867 98% BioticStress 20,243 97% Feeding 3654 84% Processing 27,180 76% Nutrition 15,383 91% Allergy 3650 67% Acceptability 3972 73% Socioeconomics 963 67% Final number of records Thematic corpora merged without duplicates: 107,823 160,238 142,763 100,248 Table A4. Size of the bibliometric corpora depending on the delineation strategy. 29 of 37 Sustainability 2019, 11, 6833 Table A5. Note: * A term keyword not contained in the title, abstract or authors’ terms present in the query of any other theme. Note: * A term keyword not contained in the title, abstract or authors’ terms present in the query of any other theme. Appendix B. Robustness Assessment of the Delineation Process: Testing an Alternative Strate Appendix B. Robustness Assessment of the Delineation Process: Testing an Alternative Strate Breakdown of the Fusion and Species3 corpora into the 10 underlying themes. FUSION Corpus SPECIES3 Corpus Themes Index Colum A Records Number Indexed with a Single Theme * Frequency Ranking of the Single Theme Records Number Indexed with the Theme of Column A Share of Themes Themes Index Colum A Records Number Indexed with a Single Theme * Frequency Ranking of the Single Theme Records Number Indexed with the Theme of Column A Share of Themes Genetics 13,336 34,388 34,388 22% Genetics 35,281 35,281 35,281 15% Ecophy. 12,841 29,867 29,867 19% Ecophy. 48,889 48,889 48,889 20% Process. 12,049 27,180 27,180 17% Process. 30,511 30,511 30,511 13% BioticSt. 9109 20,243 20,243 13% BioticSt. 17,839 17,839 17,839 7% Agronomy 7982 19,070 19,070 12% Agronomy 16,789 16,789 16,789 7% Nutrition 4866 15,383 15,383 10% Nutrition 56,021 56,021 56,021 23% Feeding 2888 3654 3654 2% Feeding 26,296 26,296 26,296 11% Allergy 1891 3650 3650 2% Allergy 2157 2157 2157 1% Accept. 745 3972 3972 3% Accept. 2280 2280 2280 1% Socioeco. 565 963 963 1% Socioeco. 3427 3427 3427 1% SUBTOTAL 66,272 158 370 100% 158 370 24,946 239 490 100% in % 61% in % 25% Total FUSION Corpus 107 823 Total SPECIES3 Corpus 100 248 Note: * A term keyword not contained in the title, abstract or authors’ terms present in the query of any other theme. FUSION Corpus 30 of 37 Sustainability 2019, 11, 6833 Table A6. Number and share of the records in Species1, -2, and -3 corpora related to soya and pulses, groundnut and lathyrus-vicia broken down by periods. Note: Each line in the table are excluding count from each other. PFL: pea, fababean or lupin. * Lecture: For instance, this line reports the number of records containing only a generic term and a term linked to soya in title, abstract or authors’ keywords for a generic term such as legumes. Note: Each line in the table are excluding count from each other. PFL: pea, fababean or lupin. * Lecture: For instance, thi and a term linked to soya in title, abstract or authors’ keywords for a generic term such as legumes. Appendix C.1. First Signs before the Common Era The ﬁrst deﬁnite signs of domesticated plants in the Old World appeared around 10,500–10,100 years before the common era (BCE). Legumes appear frequently with cereals (wheat and barley): “several grain legumes appear as constant companions of the cereals” [30] (p.1). The most frequent pulses in the early Neolithic period in the Middle East (near the Mediterranean Basin) are lentils (Lens culinaris) and peas (Pisum sativum), and two more local legume crops are chickpeas (Cicer arietinum) and bitter vetches (Vicia ervilia). Additional legumes were cultivated later, such as grass peas (Lathyrus sativus) with some evidence in Greece and Bulgaria around 8000–7000 BCE. The origin and early spread of faba beans (Vicia faba) is less clear. These archaeological ﬁndings reveal “a rule, not a single crop but rather a combination of cereals, pulses, and ﬂax appears in these early farming villages. Moreover, the assemblage seems to be similar throughout the Fertile Crescent. In other words, a common package of grain crops characterizes the development of agriculture in this ‘core area’” [30] (p. 2–3) on the spread of those crops over Europe and Mediterranean Basin during 10,500–20,000 BCE. In the Mediterranean basin and Europe, evidence from the beginning of the Common Era has found on the cultivation of the fenugreeks and the lupins, as well as the grass pea and vicias. As regards the Mesoamerican area, an illustration of legumes development was the “three sisters” system (maize, beans, and squash intercropping). Appendix C. A Brief Overview of Grain-Legumes Domestication and Their Development Annual legumes (Papilionaceae/Fabaceae or Leguminosae) cultivated for their seeds are frequent companions of cereals in most parts of the world. They are attractive because, contrary to other ﬂower plants, legumes can ﬁx atmospheric nitrogen thanks to a symbiosis with root bacteria called Rhizobium. The cultivation of legumes helps enrich the soil with nitrogen; hence, cultivated with cereals in rotation or in association, they contribute to higher fertility in soils. Moreover, as they are rich in protein, they complement cereals in diets. This protein complementarity, allowing them to substitute for animal-based proteins, was essential for the development of traditional farming communities. As underlined in [30], each important civilization in history had their basic cereals with their companion legumes. For instance, in Western Asia and Europe, wheats and barleys were frequently cultivated with peas, lentils, chickpeas, and faba beans, while, maize with several species of beans (Phaseolus) in the Americas, more with groundnut in South America. In Africa, mil and sorgho were grown along with niebe and voandzou. Soya was added among cereals in China, lablab and mungo in India, etc. Appendix B. Robustness Assessment of the Delineation Process: Testing an Alternative Strate Appendix B. Robustness Assessment of the Delineation Process: Testing an Alternative Strate Family species index terms of records SPECIES3 SPECIES2 SPECIES1 1980–2018 1980–1999 2000–2018 1980–2018 1980–1999 2000–2018 1980–2018 1980–1999 2000–2018 Only Soya—G1 42,861 9372 33,489 62,233 15,330 46,903 68,790 19,750 49,040 Soya—G1—and a generic term * 533 108 425 1840 346 1494 1895 360 1535 Only other Pulses than PFL—G3 21,394 5401 15,993 25,268 7765 17,503 28,785 10,431 18,354 Other Pulses than PFL—G3—and a generic term 16,755 5625 11,130 22,447 8473 13,974 26,619 11,412 15,207 Only PFL—G2 1736 308 1428 3851 684 3167 3966 745 3221 PFL—G2—and a generic term 1303 253 1050 3111 656 2455 3227 689 2538 Subtotal Soya/Pulses 84,582 21,067 63,515 118,750 33,254 85,496 133,282 43,387 89,895 % Soya in Soya/Pulses subtotal 51.3% 45.0% 53.4% 54.0% 47.1% 56.6% 53.0% 46.4% 56.3% % Soya/Pulses in corpus period 84.4% 85.4% 84.0% 83.2% 84.8% 82.6% 83.2% 85.2% 82.2% Groundnut 8491 1900 6591 12,133 2883 9250 14,268 4055 10,213 % Groundnut in corpus period 8.5% 7.7% 8.7% 8.5% 7.4% 8.9% 8.9% 8.0% 9.3% Lathyrus or Vicia 1374 310 1064 1684 427 1257 1907 539 1368 % Lathyrus/Vicia in corpus period 1.4% 1.3% 1.4% 1.2% 1.1% 1.2% 1.2% 1.1% 1.3% Corpus total for the period 100,248 24,661 75,587 142,763 39,216 103,547 160,238 50,929 109,309 % period in corpus total 1980–2018 100% 25% 75% 100% 27% 73% 100% 32% 68% Generic term only 1745 364 1381 1720 393 1327 1804 412 1392 Note: Each line in the table are excluding count from each other. PFL: pea, fababean or lupin. * Lecture: For instance, this line reports the number of records containing only a generic term Table A6. Number and share of the records in Species1, -2, and -3 corpora related to soya and pulses, groundnut and lathyrus-vicia broken down by periods. Table A6. Number and share of the records in Species1, -2, and -3 corpora related to soya and pulses, groundnut and lathyrus-vicia broken down by periods. Sustainability 2019, 11, 6833 31 of 37 31 of 37 Appendix C.3. From the Middle Ages to the Modern Period Sustainability 2019 11 x FOR PEER REVIEW Appendix C.3. From the Middle Ages to the Modern Period Sustainability 2019, 11, x FOR PEER REVIEW Productivity gains in agriculture remained very low until the agrarian revolutions of the 17th and 18th centuries in Europe. At this time, historians have found that legumes were second to cereals in consumption preferences, and in opposition to animal products. In France, for instance, paintings of the Renaissance period contrasted nobles who could go hunting with peasants reduced to eating lentils and bread [38]. It was a privilege of the nobility to consume meat more frequently, strongly linked to the right to hunt. This privilege has undoubtedly marked the collective unconscious towards a preference for the consumption of meat products, which in turn is also strongly correlated with the increase in incomes during the 20th century. Therefore, the current preference of Western countries for animal-based proteins is not only related to nutritional interest. However, consequently, high consumption of animal products makes it unnecessary to associate cereals and legumes consumption to meet protein needs. Appendix C.3. From the Middle Ages to the Modern Period Productivity gains in agriculture remained very low until the agrarian revolutions of the 17th and 18th centuries in Europe. At this time, historians have found that legumes were second to cereals in consumption preferences, and in opposition to animal products. In France, for instance, paintings of the Renaissance period contrasted nobles who could go hunting with peasants reduced to eating lentils and bread [38]. It was a privilege of the nobility to consume meat more frequently, strongly linked to the right to hunt. This privilege has undoubtedly marked the collective unconscious towards a preference for the consumption of meat products, which in turn is also strongly correlated with the increase in incomes during the 20th century. Therefore, the current preference of Western countries for animal-based proteins is not only related to nutritional interest. However, consequently, high consumption of animal products makes it unnecessary to associate cereals and legumes In addition, during the succession of wars aﬀecting Europe in the 19th and 20th centuries, legumes were frequently presented as ﬁlling foods during food shortages. Combined with a traditional image of “poor man’s meat” or as a food related to famines and wars, after the Second World War consumers gave up legumes, and their consumption fell in Western countries. Appendix C.3. From the Middle Ages to the Modern Period Sustainability 2019 11 x FOR PEER REVIEW Trade agreements between Europe and the USA resulted in no development of soya in Europe during several decades, and thus to important soya imports for livestock. high consumption of animal products makes it unnecessary to associate cereals and legumes consumption to meet protein needs. In addition, during the succession of wars affecting Europe in the 19th and 20th centuries, legumes were frequently presented as filling foods during food shortages. Combined with a traditional image of “poor man’s meat” or as a food related to famines and wars, after the Second World War consumers gave up legumes, and their consumption fell in Western countries. Trade agreements between Europe and the USA resulted in no development of soya in Europe during Nowadays, Europe presents the lowest consumption with 3 kg/year per capita. In some European countries, consumption is even less, such as France (1.7 kg/year per capita in 2011, Agreste statistics). Globally, legumes are far more used for feeding animals, but with a minor position in feed formulas compared with soya. In addition to this trend, chemical fertilizers development favored a nitrogen cycle conception in cropping system without legumes (see [9] for more insights on economic trade-oﬀs on legumes uses). several decades, and thus to important soya imports for livestock. Nowadays, Europe presents the lowest consumption with 3 kg/year per capita. In some European countries, consumption is even less, such as France (1.7 kg/year per capita in 2011, Agreste statistics). Globally, legumes are far more used for feeding animals, but with a minor position in feed formulas compared with soya. In addition to this trend, chemical fertilizers development favored a nitrogen cycle conception in cropping system without legumes (see [9] for more insights on economic trade-offs on legumes uses) Appendix C.2. Antiquity Period Legumes with bigger grains such as we know today reached this size in the Antiquity period. Domestication of these plants brought several major changes in plant architecture, pod size, and lodging resistance. Through cultivation, the stems became sturdier and stiﬀer, and had a reduced propensity to climb, to make them more easily cultivable in the ﬁeld. Some wild type chemical defenses had also been counter-selected to favor their consumption. Many wild legumes contain strong toxins and antimetabolites that protect them from animal predation. Gradually, the techniques of cooking and soaking or fermentation allowed the seeds to be healthy for consumption. Roman texts report diﬀerent frequencies of legumes use for human consumption. Lentils, peas, and faba beans were the most widely consumed throughout Europe and the Middle East, as well as chickpeas further south. Fenugreek was used as a condiment, especially in the Mediterranean Basin. Lupines were more concentrated around Greece and Egypt, and seems to have been domesticated later in Antiquity. Some species within the genus Vicia, having a particular taste, were consumed only during periods of famine and were reserved for medicinal purposes. Certain preferences were established for each region, still marking our culinary traditions today. For example, beans were more heavily consumed in Egypt and Spain, lentils in France. Sustainability 2019, 11, 6833 32 of 37 Referenc References [CrossRef] p Contributions of Legumes to Reducing the Environmental Risk of Agricultural Production. In Agroecosystem Diversity; Lemaire, G., de Faccio Carvalho, P.C., Kronberg, S., Recous, S., Eds.; Academic Press: Cambridge, MA, USA, 2018; pp. 123–143. 5. Rawal, V.; Bansal, V.; Thokchom, D. Biodiversity for Food and Agriculture and Food Security. An 4. Peoples, M.B.; Hauggaard-Nielsen, H.; Huguenin-Elie, O.; Jensen, E.S.; Justes, E.; Williams, M. The Contributions of Legumes to Reducing the Environmental Risk of Agricultural Production. In Agroecosystem Diversity; Lemaire, G., de Faccio Carvalho, P.C., Kronberg, S., Recous, S., Eds.; Academic Press: Cambridge, MA, USA, 2018; pp. 123–143. Exploration of Interrelationships, Background Study Paper NO. 69-2019, FAO Commission on Genetic Resources for Food and Agriculture. Available online: http://www.networkideas.org/wp- content/uploads/2019/02/ca3218en.pdf (accessed on 25 July 2019). 6. Weiner, J. Applying plant ecological knowledge to increase agricultural sustainability. J. Ecol. 2017, 105, 865 870 5. Rawal, V.; Bansal, V.; Thokchom, D. Biodiversity for Food and Agriculture and Food Security. An Exploration of Interrelationships, Background Study Paper NO. 69-2019, FAO Commission on Genetic Resources for Food and Agriculture. Available online: http://www.networkideas.org/wp-content/uploads/2019/02/ca3218en.pdf (accessed on 25 July 2019). 865–870. 7. Magrini, M.-B.; Anton, M.; Chardigny, J.-M.; Duc, G.; Duru, M.; Jeuffroy, M.-H.; Meynard, J.-M.; Micard, V ; Walrand S Pulses for sustainability: Breaking agriculture and food sectors out of lock-in Front Sustain y Weiner, J. Applying plant ecological knowledge to increase agricultural sustainability. J. Ecol. 2017, 105 865–870. [CrossRef] V.; Walrand, S. Pulses for sustainability: Breaking agriculture and food sectors out of lock-in. Front. Sustain. Food Syst. 2018, 2, 64, doi:10.3389/fsufs.2018.00064. 8. Magrini, M.-B.; Befort, N.; Nieddu, M. Technological Lock-In and Pathways for Crop Diversification in the Bio-Economy. In Agroecosystem Diversity; Lemaire, G., de Faccio Carvalho, P.C., Kronberg, S., Recous, S., 7. Magrini, M.-B.; Anton, M.; Chardigny, J.-M.; Duc, G.; Duru, M.; Jeuﬀroy, M.-H.; Meynard, J.-M.; Micard, V.; Walrand, S. Pulses for sustainability: Breaking agriculture and food sectors out of lock-in. Front. Sustain. Food Syst. 2018, 2, 64. [CrossRef] io E o o y I Ag oe o y e i e i y; e ai e, , e a io a a o, , o e g, , e ou , , Eds.; Academic Press: Cambridge, MA, USA, 2019; pp. 375–388. 9. Magrini, M.B.; Anton, M.; Cholez, C.; Corre-Hellou, G.; Duc, G.; Jeuffroy, M.H.; Meynard, J.M.; Pelzer, E.; Voisin, A.-S.; Walrand, S. Why are grain-legumes rarely present in cropping systems despite their 8. Appendix D. Corpus Broken Down by Theme and the Four Main Publishing Countries/Geographical Areas Appendix D. Corpus Broken Down by Theme and the Four M Countries/Geographical Areas Only records indexed with soya or the ones indexed with pulses were included (i.e., records co-indexed with several groups of grain-legumes were excluded); proportional count linked to international records applied; a group count done for the EU28; “Others” are all identiﬁed countries other than the USA, China, India, and those belonging to the EU28. Each theme counts for the number of records indexed with this theme (with or without co-indexing theme), representing the importance of the theme. Graphs are ordered by the amount of records by themes. Countries/Geographical Areas Only records indexed with soya or the ones indexed with pulses were included (i.e., records co- indexed with several groups of grain-legumes were excluded); proportional count linked to international records applied; a group count done for the EU28; “Others” are all identified countries other than the USA, China, India, and those belonging to the EU28. Each theme counts for the number of records indexed with this theme (with or without co-indexing theme), representing the importance of the theme. Graphs are ordered by the amount of records by themes. (a) Figure A2. Cont. (a) Figure A2. Cont. 33 of 37 Sustainability 2019, 11, 6833 tainability 2019, 11, 6833 33 of 37 Sustainability 2019, 11, x FOR PEER REVIEW 28 of 38 (b) (c) (d) (e) Figure A2. Cont. (b) (b) (c) (d) (e) (e) Figure A2. Cont. Figure A2. Cont. 34 of 37 Sustainability 2019, 11, 6833 Sustainability 2019, 11, x FOR PEER REVIEW 29 of 38 (f) (g) (h) (i) (f) (f) (g) (h) (i) (g) (h) (i) Figure A2. Cont. Figure A2. Cont. Figure A2. Cont. 35 of 37 Sustainability 2019, 11, 6833 y , , (j) Figure A2. Records number by theme and by period for the USA, the EU28, India, China, and Others. Scale based on 3 k for the figures (a)-(f), and on 0.6 k for the figures (g)-(j). Figure A2. Records number by theme and by period for the USA, the EU28, India, China, and Others. Scale based on 3 k for the ﬁgures (a–f), and on 0.6 k for the ﬁgures (g–j). (j) Figure A2. Records number by theme and by period for the USA, the EU28, India, China, and Others. Scale based on 3 k for the figures (a)-(f) and on 0 6 k for the figures (g)-(j) Figure A2. Referenc References 1. Lemaire, G.; de Faccio Carvalho, P.C.; Kronberg, S.; Recous, S. (Eds.). Agroecosystem Diversity; Academic Press: Cambridge, MA, USA, 2018. 2 Meynard J M ; Charrier F ; Le Bail M ; Magrini M B ; Charlier A ; Messéan A Socio technical lock in 1. Lemaire, G.; de Faccio Carvalho, P.C.; Kronberg, S.; Recous, S. (Eds.) Agroecosystem Diversity; Academic Press Cambridge, MA, USA, 2018. 1. Lemaire, G.; de Faccio Carvalho, P.C.; Kronberg, S.; Recous, S. (Eds.). Agroecosystem Diversity; Academic Press: Cambridge, MA, USA, 2018. 2 Meynard J M ; Charrier F ; Le Bail M ; Magrini M B ; Charlier A ; Messéan A Socio-technical lock-in 1. Lemaire, G.; de Faccio Carvalho, P.C.; Kronberg, S.; Recous, S. (Eds.) Agroecosystem Diversity; Academic Press: Cambridge, MA, USA, 2018. 2. Meynard, J.M.; Charrier, F.; Le Bail, M.; Magrini, M.B.; Charlier, A.; Messéan, A. Socio-technical lock-in hinders crop diversification in France. Agron. Sustain. Dev. 2018, 38, 54. 3 Frison E A ; Cherfas J ; Hodgkin T Agricultural biodiversity is essential for a sustainable improvement 2. Meynard, J.M.; Charrier, F.; Le Bail, M.; Magrini, M.B.; Charlier, A.; Messéan, A. Socio-technical lock-in hinders crop diversiﬁcation in France. Agron. Sustain. Dev. 2018, 38, 54. [CrossRef] 2. Meynard, J.M.; Charrier, F.; Le Bail, M.; Magrini, M.B.; Charlier, A.; Messéan, A. Socio technical lock in hinders crop diversification in France. Agron. Sustain. Dev. 2018, 38, 54. 3. Frison, E.A.; Cherfas, J.; Hodgkin, T. Agricultural biodiversity is essential for a sustainable improvement 2. Meynard, J.M.; Charrier, F.; Le Bail, M.; Magrini, M.B.; Charlier, A.; Messéan, A. Socio-technical lock-in hinders crop diversiﬁcation in France. Agron. Sustain. Dev. 2018, 38, 54. [CrossRef] 3. Frison, E.A.; Cherfas, J.; Hodgkin, T. Agricultural biodiversity is essential for a sustainable improvement in food and nutrition security. Sustainability 2011, 3, 238–253. 4. Peoples, M.B.; Hauggaard-Nielsen, H.; Huguenin-Elie, O.; Jensen, E.S.; Justes, E.; Williams, M. The p g 3. Frison, E.A.; Cherfas, J.; Hodgkin, T. Agricultural biodiversity is essential for a sustainable improvement in food and nutrition security. Sustainability 2011, 3, 238–253. [CrossRef] 3. Frison, E.A.; Cherfas, J.; Hodgkin, T. Agricultural biodiversity is essential for a sustainable improvement in food and nutrition security. Sustainability 2011, 3, 238–253. 4. Peoples, M.B.; Hauggaard-Nielsen, H.; Huguenin-Elie, O.; Jensen, E.S.; Justes, E.; Williams, M. The 3. Frison, E.A.; Cherfas, J.; Hodgkin, T. Agricultural biodiversity is essential for a sustainable improvement in food and nutrition security. Sustainability 2011, 3, 238–253. Appendix D. Corpus Broken Down by Theme and the Four Main Publishing Countries/Geographical Areas Appendix D. Corpus Broken Down by Theme and the Four M Countries/Geographical Areas Records number by theme and by period for the USA, the EU28, India, China, and Others. S l b d k f h ﬁ ( f) d k f h ﬁ ( j) (j) Figure A2. Records number by theme and by period for the USA, the EU28, India, China, and Others. Scale based on 3 k for the figures (a)-(f), and on 0.6 k for the figures (g)-(j). Figure A2. Records number by theme and by period for the USA, the EU28, India, China, and Others. Scale based on 3 k for the ﬁgures (a–f), and on 0.6 k for the ﬁgures (g–j). UK, 1986. 13. Rogers, E.M. Diﬀusion of Innovations; Free Press: New York, NY, USA, 2003. Referenc References 16. Noyons, E. Bibliometric mapping of science in a policy context. Scientometrics 2001, 50, 83–98. [CrossRef] 17. Rafols, I.; Hopkins, M.M.; Hoekman, J.; Siepel, J.; O’Hare, A.; Perianes-Rodríguez, A.; Nightingale, P. Big Pharma, little science: A bibliometric perspective on Big Pharma’s R&D decline. Technol. Forecast. Soc. Chang. 2010, 81, 22–38. 18. Barbier, M.; Bompart, M.; Garandel-Batifol, V.; Mogoutov, A. Textual Analysis and Scientometric Mapping of the Dynamic Knowledge in and around the IFSA Community. 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Report from the Commission to the Council and the European Parliament on the Development of Plant Proteins in the European Union, COM 757. Brussels. Available online: https://ec.europa.eu/info/sites/info/ﬁles/food-farming-ﬁsheries/plants_and_plant_products/ documents/report-plant-proteins-com2018-757-ﬁnal_en.pdf (accessed on 5 December 2018). 29. Garﬁeld, E.; Sher, I.H. KeyWords Plus™—Algorithmic derivative indexing. J. Am. Soc. Inf. Sci. 1993, 44, 298–299. [CrossRef] 30. Zohary, D.; Hopf, M.; Weiss, E. Referenc References Magrini, M.-B.; Befort, N.; Nieddu, M. Technological Lock-In and Pathways for Crop Diversiﬁcation in the Bio-Economy. In Agroecosystem Diversity; Lemaire, G., de Faccio Carvalho, P.C., Kronberg, S., Recous, S., Eds.; Academic Press: Cambridge, MA, USA, 2019; pp. 375–388. environmental and nutritional benefits? Analyzing lock-in in the French agrifood system. Ecol. Econ. 2016, 126, 152–162. 10. Rawal, V.; Navarro, D.K. The Global Economy of Pulses; FAO: Rome, Italy 2017. 11. Watson, C.A.; Reckling, M.; Preissel, S.; Bachinger, J.; Bergkvist, G.; Kuhlman, T.; Lindström, K.; Nemecek, 9. Magrini, M.B.; Anton, M.; Cholez, C.; Corre-Hellou, G.; Duc, G.; Jeuﬀroy, M.H.; Meynard, J.M.; Pelzer, E.; Voisin, A.-S.; Walrand, S. Why are grain-legumes rarely present in cropping systems despite their environmental and nutritional beneﬁts? Analyzing lock-in in the French agrifood system. Ecol. Econ. 2016, 126, 152–162. [CrossRef] T.; Topp, C.F.; Vanhatalo, A.; et al. Grain legume production and use in Europ Ad A 2017 144 235 303 10. Rawal, V.; Navarro, D.K. The Global Economy of Pulses; FAO: Rome, Italy, 2017. Adv. Agron. 2017, 144, 235–303. 12. Dosi, G.; Nelson, R.R. Technical Change and Industrial Dynamics as Evolutionary Processes. In Handbook of the Economics of Innovation; B. H. Hall, N. Rosenberg (eds), Elsevier: Standford, USA 2010; Volume 1, pp. 51 127 11. Watson, C.A.; Reckling, M.; Preissel, S.; Bachinger, J.; Bergkvist, G.; Kuhlman, T.; Lindström, K.; Nemecek, T.; Topp, C.F.; Vanhatalo, A.; et al. Grain legume production and use in European agricultural systems. Adv. Agron. 2017, 144, 235–303. 51–127. 13. Rogers, E.M. Diffusion of Innovations; Free Press: New York, NY, USA, 2003. 14. Callon, M.; Law, J.; Rip, A. Mapping the Dynamics of Science and Technology; Palgrave Macmillan: London, UK, 1986 12. Dosi, G.; Nelson, R.R. Technical Change and Industrial Dynamics as Evolutionary Processes. In Handbook of the Economics of Innovation; Hall, B.H., Rosenberg, N., Eds.; Elsevier: Standford, CA, USA, 2010; Volume 1, pp. 51–127. UK, 1986. 13. Rogers, E.M. Diﬀusion of Innovations; Free Press: New York, NY, USA, 2003. Sustainability 2019, 11, 6833 36 of 37 36 of 37 14. Callon, M.; Law, J.; Rip, A. Mapping the Dynamics of Science and Technology; Palgrave Macmillan: London, UK, 1986. 15. Noyons, E.C.M. Science Maps within a Science Policy Context. In Handbook of Quantitative Science and Technology Research, the use of Publication and Patent Statistics in Studies of S&T Systems; Moed, H., Glänzel, W., Schmoch, U., Eds.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 2004; pp. 237–255. Referenc References Domestication of Plants in the Old World: The Origin and Spread of Domesticated Plants in Southwest Asia, Europe, and the Mediterranean Basin, 4th ed.; Oxford University Press: Oxford, UK, 2012. 31. Aykroyd, W.R.; Doughthy, J. Les graines de légumineuses dans l’alimentation humaine; Organisation des Nations-Unies pour l’Alimentation et l’agriculture: Rome, Italy, 2008. 32. Sonnino, A. Leguminose da Granella e Ricerca Agricola—Pulses and Agricultural Research. Atti del Seminario Leguminose da Granella—Sant’Angelo Lodigiano. 14 October 2016, pp. 45–50. 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In Connecting Health and Nutrition with Environmentally Sustainable Diets, 1st ed.; Academic Press: Cambridge, MA, USA, 2019. 37 of 37 37 of 37 Sustainability 2019, 11, 6833 35. Sivasankar, S.; Ellis, N.; Buruchara, R.; Henry, C.; Rubiales, D.; Sandhu, J.S.; Negra, C. 10-year Research Strategy for Food Crops. 2016. Available online: https://pulses.org/future-of-food/10-year-research-strategy (accessed on 9 August 2019). 36. Meynard, J.M.; Jeuﬀroy, M.H.; Le Bail, M.; Lefèvre, A.; Magrini, M.B.; Michon, C. Designing coupled innovations for the sustainability transition of agrifood systems. Agric. Syst. 2017, 157, 330–339. [CrossRef] 37. Ciarli, T.; Ràfols, I. The relation between research priorities and societal demands: The case of rice. Res. Policy 2019, 48, 949–967. [CrossRef] 38. Quellier, F. Petite et grande histoire des légumineuses en Occident, Journées de la Fondation Louis Bonduelle, Paris, 2016. Available online: http://www.fondation-louisbonduelle.org/wp-content/uploads/2016/10/ ﬂorent-quellier-histoire-des-legumineuses-rencontres-fondation-louis-bonduelle-legumes-secs-proteines- vegetales.pdf (accessed on 29 July 2019). © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). 35. Sivasankar, S.; Ellis, N.; Buruchara, R.; Henry, C.; Rubiales, D.; Sandhu, J.S.; Negra, C. 10-year Research Strategy for Food Crops. 2016. Available online: https://pulses.org/future-of-food/10-year-research-strategy (accessed on 9 August 2019). 36. Referenc References Meynard, J.M.; Jeuﬀroy, M.H.; Le Bail, M.; Lefèvre, A.; Magrini, M.B.; Michon, C. Designing coupled innovations for the sustainability transition of agrifood systems. Agric. Syst. 2017, 157, 330–339. [CrossRef] 37. Ciarli, T.; Ràfols, I. The relation between research priorities and societal demands: The case of rice. Res. Policy 2019, 48, 949–967. [CrossRef] 37. Ciarli, T.; Ràfols, I. The relation between research priorities and societal demands: The case of rice. Res. Policy 2019, 48, 949–967. [CrossRef] 38. Quellier, F. Petite et grande histoire des légumineuses en Occident, Journées de la Fondation Louis Bonduelle, Paris, 2016. Available online: http://www.fondation-louisbonduelle.org/wp-content/uploads/2016/10/ ﬂorent-quellier-histoire-des-legumineuses-rencontres-fondation-louis-bonduelle-legumes-secs-proteines- vegetales.pdf (accessed on 29 July 2019). 38. Quellier, F. Petite et grande histoire des légumineuses en Occident, Journées de la Fondation Louis Bonduelle, Paris, 2016. Available online: http://www.fondation-louisbonduelle.org/wp-content/uploads/2016/10/ ﬂorent-quellier-histoire-des-legumineuses-rencontres-fondation-louis-bonduelle-legumes-secs-proteines- vegetales.pdf (accessed on 29 July 2019). © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W3029702842	https://figshare.com/articles/journal_contribution/In-situ_crystallization_and_continuous_modification_of_chromian_spinel_in_the_sulfide-poor_platinum-group_metal_ores_of_the_Norilsk-1_intrusion_Northern_Siberia_Russia_/22996676/1/files/40746755.pdf	English	null	In-Situ Crystallization and Continuous Modification of Chromian Spinel in the “Sulfide-Poor Platinum-Group Metal Ores” of the Norilsk-1 Intrusion (Northern Siberia, Russia)	Minerals	2,020	cc-by	18,845	minerals Article In-Situ Crystallization and Continuous Modiﬁcation of Chromian Spinel in the “Sulﬁde-Poor Platinum-Group Metal Ores” of the Norilsk-1 Intrusion (Northern Siberia, Russia) Ivan F. Chayka 1,2,, Liudmila M. Zhitova 1,3, Tatiana N. Antsiferova 4,5, Adam Abersteiner 6,7, Artem Ya. Shevko 1, Andrey E. Izokh 1,3, Nadezhda D. Tolstykh 1,3, Marina P. Gora 1, Valery M. Chubarov 7 and Vadim S. Kamenetsky 2,6,7 1 V. S. Sobolev Institute of Geology and Mineralogy, Siberian Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Russia; zhitova63@mail.ru (L.M.Z.); sp@igm.nsc.ru (A.Y.S.); izokh@igm.nsc.ru (A.E.I.); tolst@igm.nsc.ru (N.D.T.); gora@igm.nsc.ru (M.P.G.) 2 Institute of Experimental Mineralogy, Russian Academy of Sciences, 142432 Chernogolovka, Russia; dima.kamenetsky@utas.edu.au 3 Department of Geology and Geophysics, Novosibirsk State University, 630090 Novosibirsk, Russia 4 Institute of Geology of Ore Deposits, Petrography, Mineralogy and Geochemistry RAS (IGEM RAS), 119017 Moscow, Russia; antsifer@yandex.ru 5 Fersman Mineralogical Museum of Russian Academy of Sciences, 115162 Moscow, Russia 6 School of Natural Sciences, University of Tasmania, Hobart, Tasmania 7001, Australia; adam.abersteiner@utas.edu.au 7 Institute of Volcanology and Seismology, Far Eastern Branch of the Russian Academy of Sciences, 683006 Petropavlovsk-Kamchatsky, Russia; zond@kscnet.ru Correspondence: ivanlab211@gmail.com; Tel.: +7-9-137-723-224 Received: 22 April 2020; Accepted: 28 May 2020; Published: 30 May 2020 Abstract: Layers rich in chromian spinel (Cr-spinel) occur in numerous diﬀerentiated and layered intrusions. These layers are often characterized by elevated and even economic concentrations of platinum-group-elements (PGEs), but only scarce sulﬁde mineralization. One particular type of such lithology occurs in the roof parts of the Norilsk-type diﬀerentiated intrusions (Russia) and is referred to as the “sulﬁde-poor PGE ores”. We investigated rocks containing variable enrichments in Cr-spinel, sulﬁdes, and platinum-group minerals (PGMs) from two sections of the upper zone of the Norilsk-1 intrusion, with a focus on Cr-spinel. The rocks are dominated by two lithological types: (1) leucogabbro/troctolitic and (2) olivine-gabbro. Fine-grained (5–100 µm) disperse disseminations with varying modal abundances of Cr-spinel are characteristic for the rocks studied. Those abundances range from scarce mineralization through to very dense (up to 30 vol. % Cr-spinel) minerals Article In-Situ Crystallization and Continuous Modiﬁcation of Chromian Spinel in the “Sulﬁde-Poor Platinum-Group Metal Ores” of the Norilsk-1 Intrusion (Northern Siberia, Russia) Ivan F. Chayka 1,2,, Liudmila M. Zhitova 1,3, Tatiana N. Antsiferova 4,5, Adam Abersteiner 6,7, Artem Ya. Shevko 1, Andrey E. Izokh 1,3, Nadezhda D. Tolstykh 1,3, Marina P. Gora 1, Valery M. Chubarov 7 and Vadim S. Kamenetsky 2,6,7 1 V. S. Sobolev Institute of Geology and Mineralogy, Siberian Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Russia; zhitova63@mail.ru (L.M.Z.); sp@igm.nsc.ru (A.Y.S.); izokh@igm.nsc.ru (A.E.I.); tolst@igm.nsc.ru (N.D.T.); gora@igm.nsc.ru (M.P.G.) 2 Institute of Experimental Mineralogy, Russian Academy of Sciences, 142432 Chernogolovka, Russia; dima.kamenetsky@utas.edu.au 3 Department of Geology and Geophysics, Novosibirsk State University, 630090 Novosibirsk, Russia 4 Institute of Geology of Ore Deposits, Petrography, Mineralogy and Geochemistry RAS (IGEM RAS), 119017 Moscow, Russia; antsifer@yandex.ru 5 Fersman Mineralogical Museum of Russian Academy of Sciences, 115162 Moscow, Russia 6 School of Natural Sciences, University of Tasmania, Hobart, Tasmania 7001, Australia; adam.abersteiner@utas.edu.au 7 Institute of Volcanology and Seismology, Far Eastern Branch of the Russian Academy of Sciences, 683006 Petropavlovsk-Kamchatsky, Russia; zond@kscnet.ru Correspondence: ivanlab211@gmail.com; Tel.: +7-9-137-723-224 Received: 22 April 2020; Accepted: 28 May 2020; Published: 30 May 2020 Abstract: Layers rich in chromian spinel (Cr-spinel) occur in numerous diﬀerentiated and layered intrusions. These layers are often characterized by elevated and even economic concentrations of platinum-group-elements (PGEs), but only scarce sulﬁde mineralization. One particular type of such lithology occurs in the roof parts of the Norilsk-type diﬀerentiated intrusions (Russia) and is referred to as the “sulﬁde-poor PGE ores”. We investigated rocks containing variable enrichments in Cr-spinel, sulﬁdes, and platinum-group minerals (PGMs) from two sections of the upper zone of the Norilsk-1 intrusion, with a focus on Cr-spinel. The rocks are dominated by two lithological types: (1) leucogabbro/troctolitic and (2) olivine-gabbro. Fine-grained (5–100 µm) disperse disseminations with varying modal abundances of Cr-spinel are characteristic for the rocks studied. Those abundances range from scarce mineralization through to very dense (up to 30 vol. % Cr-spinel) minerals minerals In-Situ Crystallization and Continuous Modiﬁcation of Chromian Spinel in the “Sulﬁde-Poor Platinum-Group Metal Ores” of the Norilsk-1 Intrusion (Northern Siberia, Russia) Ivan F. Chayka 1,2,, Liudmila M. Zhitova 1,3, Tatiana N. Antsiferova 4,5, Adam Abersteiner 6,7, Artem Ya. Shevko 1, Andrey E. Izokh 1,3, Nadezhda D. Tolstykh 1,3, Marina P. Gora 1, Valery M. Chubarov 7 and Vadim S. Kamenetsky 2,6,7 Ivan F. Chayka 1,2,, Liudmila M. Zhitova 1,3, Tatiana N. Antsiferova 4,5, Adam Abersteiner 6,7, Artem Ya. Shevko 1, Andrey E. Izokh 1,3, Nadezhda D. Tolstykh 1,3, Marina P. Gora 1, Valery M. Chubarov 7 and Vadim S. Kamenetsky 2,6,7 1 V. S. Sobolev Institute of Geology and Mineralogy, Siberian Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Russia; zhitova63@mail.ru (L.M.Z.); sp@igm.nsc.ru (A.Y.S.); izokh@igm.nsc.ru (A.E.I.) tolst@igm.nsc.ru (N.D.T.); gora@igm.nsc.ru (M.P.G.) 1 V. S. Sobolev Institute of Geology and Mineralogy, Siberian Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Russia; zhitova63@mail.ru (L.M.Z.); sp@igm.nsc.ru (A.Y.S.); izokh@igm.nsc.ru (A.E.I.); tolst@igm.nsc.ru (N.D.T.); gora@igm.nsc.ru (M.P.G.) g ( ) g g ( ) 2 Institute of Experimental Mineralogy, Russian Academy of Sciences, 142432 Chernogolovka, Russia; dima.kamenetsky@utas.edu.au 2 Institute of Experimental Mineralogy, Russian Academy of Sciences, 142432 Chernogolovka, Russia; dima.kamenetsky@utas.edu.au 3 3 Department of Geology and Geophysics, Novosibirsk State University, 630090 Novosibirsk, Russia 4 Institute of Geology of Ore Deposits, Petrography, Mineralogy and Geochemistry RAS (IGEM RAS), 119017 Moscow, Russia; antsifer@yandex.ru 3 Department of Geology and Geophysics, Novosibirsk State University, 630090 Novosibirsk, Russia 4 Institute of Geology of Ore Deposits, Petrography, Mineralogy and Geochemistry RAS (IGEM RAS), 119017 Moscow, Russia; antsifer@yandex.ru 5 Fersman Mineralogical Museum of Russian Academy of Sciences, 115162 Moscow, Russia 6 School of Natural Sciences, University of Tasmania, Hobart, Tasmania 7001, Australia; adam.abersteiner@utas.edu.au 7 Institute of Volcanology and Seismology, Far Eastern Branch of the Russian Academy of Sciences, 7 Institute of Volcanology and Seismology, Far Eastern Branch of the 683006 Petropavlovsk-Kamchatsky, Russia; zond@kscnet.ru 7 Institute of Volcanology and Seismology, Far Eastern Branch o 683006 Petropavlovsk-Kamchatsky, Russia; zond@kscnet.ru 683006 Petropavlovsk-Kamchatsky, Russia; zond@kscnet.ru 683006 Petropavlovsk-Kamchatsky, Russia; zond@kscnet.ru * Correspondence: ivanlab211@gmail.com; Tel.: +7-9-137-723-224 www.mdpi.com/journal/minerals 1. Introduction Platinum-group element (PGE)-bearing reefs enriched in chromian spinel (Cr-spinel, chromite) are found in some layered igneous complexes (e.g., Bushveld, Stillwater) and are among the main global resources of PGE [1–5]. Despite numerous attempts to unravel their origin, the underlying genetic processes responsible for their outstanding chromium and PGE enrichment are still a remarkable challenge for petrologists and economic geologists. Signiﬁcant and relevant questions include how high quantities of chromium and PGEs can concentrate in a small volume rock and which processes are responsible for the geochemical links between these elements in diﬀerent geological settings and, in particular, in maﬁc-ultramaﬁc intrusions. Diﬀerentiated intrusions of the Norilsk region (Figure 1A) are renowned for bearing one of the world’s largest reserves of PGE, which are hosted in massive and disseminated sulﬁde ores [6–10]. Importantly, in the upper endocontact zones, there are discontinuous layers that consist of gabbro-, troctolite-, and picrite-like rocks with numerous patches of multi-grained Cr-spinel disseminations and extremely high PGE contents (up to 70 ppm) [11]. The presence of these layers in most intrusions of the Norilsk ore ﬁeld, along with their potential for exploration vectoring, have been emphasized in several studies [12–14]. These Cr-spinel rich rocks are comparable with the reefs of Bushveld, Stillwater and some other layered intrusions. However, there are two principal diﬀerences between reefs typical for the layered intrusions and the studied formations of the Norilsk-type intrusions. First, chromitites in layered intrusions (Bushveld, Kemi, Monchegorsk, Stillwater and others) tend to occur in their lower parts, while in the Norilsk-type intrusions, Cr-spinel rich lithologies are spatially related to the roof parts [1,15–17]. Second, most of the chromitite seams in layered intrusions are essentially planar and relatively continuous structures, while those in Norilsk-type intrusions, although being referred to as “layers” or “horizons,” are discontinuous and have complex boundaries. y p Cr-spinel is widely used as a petrological tool that can provide important insights into the origin of chromitites [18–25]. Approaches, based on chromite chemistry and chromite-hosted inclusions, proved to be eﬃcient for Bushveld, Stillwater, and Rhum complexes. A number of implications, outlined in those studies, appeared to depart from “traditional” orthomagmatic attitudes and showed that the genesis of chromitites is much more complicated than simple accumulation from maﬁc melts and may involve assimilation of the wall rocks, percolation of evolved melts through cumulates and even sub-solidus re-crystallization and compaction of Cr-spinel grains [19,26,27]. Received: 22 April 2020; Accepted: 28 May 2020; Published: 30 May 2020 Keywords: Cr-spinel; chromite; layered intrusion; Norilsk-1 Received: 22 April 2020; Accepted: 28 May 2020; Published: 30 May 2020 Abstract: Layers rich in chromian spinel (Cr-spinel) occur in numerous diﬀerentiated and layered intrusions. These layers are often characterized by elevated and even economic concentrations of platinum-group-elements (PGEs), but only scarce sulﬁde mineralization. One particular type of such lithology occurs in the roof parts of the Norilsk-type diﬀerentiated intrusions (Russia) and is referred to as the “sulﬁde-poor PGE ores”. We investigated rocks containing variable enrichments in Cr-spinel, sulﬁdes, and platinum-group minerals (PGMs) from two sections of the upper zone of the Norilsk-1 intrusion, with a focus on Cr-spinel. The rocks are dominated by two lithological types: (1) leucogabbro/troctolitic and (2) olivine-gabbro. Fine-grained (5–100 µm) disperse disseminations with varying modal abundances of Cr-spinel are characteristic for the rocks studied. Those abundances range from scarce mineralization through to very dense (up to 30 vol. % Cr-spinel) cloud-like accumulations. However, compact-grained accumulations and cumulate-like textures, which are typical for chromitites of layered intrusions, are not characteristic for the studied rocks. Instead, the disseminations exhibit chain- and trail-like alignments of Cr-spinel grains, which cross the boundaries between enclosing silicates, and sub-circular arrangements. The study revealed millimeter-scaled patchy distribution of Cr-spinel compositions within a given dissemination with Cr-spinel chemistry being strongly correlated with a kind of the enclosing silicate. (1) In unaltered rocks, plagioclase hosts more magnesian Cr-spinel (Mg# 30–60), while Cr-spinel in maﬁc minerals is less magnesian (Mg# 18–35). (2) In altered rocks, more magnesian Cr-spinel is hosted by less altered silicates, while strongly altered silicates mainly host less magnesian Cr-spinel. Systematics of trivalent cations exhibits divergent trends, even on a scale of a thin section, and depends on a kind of hosting lithology. Leucogabbro/troctolite lithologies contain Cr-spinel with anomalously low Fe3+ and extremely high Ti contents, whereas Cr-spinel from olivine-gabbro lithologies have moderate Fe3+ and moderately-high Ti contents. It is envisaged that crystallization of Cr-spinel and their host Minerals 2020, 10, 498; doi:10.3390/min10060498 www.mdpi.com/journal/minerals 2 of 30 Minerals 2020, 10, 498 rocks occurred from viscous mingled magmas, which had diﬀerent compositions and redox state. Subsequent processes involved (1) high-temperature re-equilibration of Cr-spinel with enclosing silicates and (2) post-magmatic alteration and partial recrystallization of Cr-spinel. During these processes, Cr-spinel was losing Mg and Al and gaining Fe and Ti. These chemical trends are generally coincident with those established for other intrusions worldwide, but the upper zone of the Norilsk-1 intrusion seems to possess an exceptional variety of Cr-spinel compositions, not recorded elsewhere. 1. Introduction Our study provides a large dataset on the textural and compositional characteristics of Cr-spinel in the PGE- and chromite-enriched, sulﬁde-poor rocks of the Norilsk-1 intrusion, which were sampled at two diﬀerent locations (Figure 1B). We investigated variations of Cr-spinel compositions in a variety of rocks and their dependence on enclosing silicates. Even though the studied rocks are most renowned for their PGM contents, detailed discussion of the platinum group mineralization is provided in Tolstykh et al. [28], while this study is focused only on Cr-spinel and its implications for the petrology of these rocks. The obtained textural and chemical data provides further insights into the origin of Cr-spinel mineralization and host silicate matrix. Minerals 2020, 10, 498 3 of 30 Figure 1. The Norilsk-1 intrusion and its surroundings. (A) Geological map of the Norilsk-Talnakh ore junction and the adjacent areas; (B) projection of the Norilsk-1 intrusion on the surface (outlined using borehole data). Modified after references [13,29]; (C) schematic cross section of the Norilsk-1 intrusion before extensive exploration of the Medvezhy Ruchey open pit [6,30]. Figure 1. The Norilsk-1 intrusion and its surroundings. (A) Geological map of the Norilsk-Talnakh ore junction and the adjacent areas; (B) projection of the Norilsk-1 intrusion on the surface (outlined using borehole data). Modiﬁed after references [13,29]; (C) schematic cross section of the Norilsk-1 intrusion before extensive exploration of the Medvezhy Ruchey open pit [6,30]. Figure 1. The Norilsk-1 intrusion and its surroundings. (A) Geological map of the Norilsk-Talnakh ore junction and the adjacent areas; (B) projection of the Norilsk-1 intrusion on the surface (outlined using borehole data). Modified after references [13,29]; (C) schematic cross section of the Norilsk-1 Figure 1. The Norilsk-1 intrusion and its surroundings. (A) Geological map of the Norilsk-Talnakh ore junction and the adjacent areas; (B) projection of the Norilsk-1 intrusion on the surface (outlined using borehole data) Modiﬁed after references [13 29]; (C) schematic cross section of the Norilsk-1 intrusion Figure 1. The Norilsk-1 intrusion and its surroundings. (A) Geological map of the Norilsk-Talnakh ore junction and the adjacent areas; (B) projection of the Norilsk-1 intrusion on the surface (outlined using borehole data). Modified after references [13,29]; (C) schematic cross section of the Norilsk-1 intrusion before extensive exploration of the Medvezhy Ruchey open pit [6,30]. Figure 1. The Norilsk-1 intrusion and its surroundings. 1. Introduction (A) Geological map of the Norilsk-Talnakh ore junction and the adjacent areas; (B) projection of the Norilsk-1 intrusion on the surface (outlined using borehole data). Modiﬁed after references [13,29]; (C) schematic cross section of the Norilsk-1 intrusion before extensive exploration of the Medvezhy Ruchey open pit [6,30]. 2. Geological Background 2. Geological Background The Norilsk-Talnakh ore province, which includes the Norilsk-1 intrusion, is located in Northern Siberia at the junction of the Siberian Platform with Tunguska syncline and Yenisei-Khatanga trough. Within this province, sill-like intrusions were emplaced into the upper part (from the Devonian to the Upper Permian) of the sedimentary cover of Siberian Craton and the earliest trap basalt suites [31,32]. The sedimentary sequence is composed of a variety of different rocks: fine-grained terrigenous rocks, coal-rich schists (e.g., black shales), evaporites, including massive anhydrite, and carbonate-bearing lithologies [8,31,33–37]. There are numerous intrusions in this region, and most of them contain Cu-Ni sulfides, but only three intrusions (Talnakh, Khaeralakh, and Norilsk-1) are The Norilsk-Talnakh ore province, which includes the Norilsk-1 intrusion, is located in Northern Siberia at the junction of the Siberian Platform with Tunguska syncline and Yenisei-Khatanga trough. Within this province, sill-like intrusions were emplaced into the upper part (from the Devonian to the Upper Permian) of the sedimentary cover of Siberian Craton and the earliest trap basalt suites [31,32]. The sedimentary sequence is composed of a variety of diﬀerent rocks: ﬁne-grained terrigenous rocks, coal-rich schists (e.g., black shales), evaporites, including massive anhydrite, and carbonate-bearing lithologies [8,31,33–37]. There are numerous intrusions in this region, and most of them contain Cu-Ni sulﬁdes, but only three intrusions (Talnakh, Khaeralakh, and Norilsk-1) are known to contain economic 4 of 30 Minerals 2020, 10, 498 quantities of Cu, Ni, and PGE. Furthermore, these intrusions possess the highest concentrations of Cr and PGE in their roof parts compared with other diﬀerentiated intrusions in the area. The Kharaelakh and Talnakh intrusions are located in the southern part of the Kharaelakh Trough, whereas the Norilsk-1 intrusion is situated in the north-eastern part of the Norilsk Trough. The emplacement of these intrusions was controlled by the Norilsk-Kharaelakh fault (Figure 1A,B). According to previous studies, these intrusions are referred to as “Norilsk-type.” They consist of at least three stratigraphic and lithological zones, which have sub-planar geometry and in most cases are well distinguished from each other (Figure 1C) [32,33]. The Lower Zone (LZ) is generally composed of ophitic gabbro, texturally variable gabbro with patchy textural patterns (“taxitic gabbro”), “explosive” breccia, leucogabbro, and diﬀerent metasomatic rocks with massive sulﬁdes at the endo-contact. The Main Zone (MZ) is represented by a stratiﬁed series of rocks, in which quite thick layers range upward from picritic ophitic gabbro and olivine-bearing gabbro to gabbro-diorite. 2. Geological Background 2. Geological Background The Upper Zone (UZ) is compositionally similar to the LZ in being dominantly composed of leucogabbro “taxitic” gabbro and pseudo-magmatic breccia. A characteristic feature of the UZ is the absence of sulﬁde-rich mineralization. However, discontinuous strata and lenses of rocks that are highly enriched in Cr-spinel and platinum-group minerals (PGM) with only subordinate sulﬁde (<1 vol. %) occur sporadically throughout this zone. Due to the scarcity of sulﬁde mineralization, these PGE- and Cr-rich ores were assigned the term “low-sulﬁde” in the Russian literature [13,32]. 3.1. Samples (A) Layout of the open pit with outlined main geological bodies; (B) sampled outcrop on the 300 m horizon; (C) a geological sketch of the 270 m horizon (after [13]); (D) Leucogabbro with visible Cr-spinel-rich sites. Figure 2. Field relationships of diﬀerent rocks in the Medvezhy Ruchey open pit. (A) Layout of the open pit with outlined main geological bodies; (B) sampled outcrop on the 300 m horizon; (C) a geological sketch of the 270 m horizon (after [13]); (D) Leucogabbro with visible Cr-spinel-rich sites. Borehole MS-24 was placed at the upstream of the Yuzhniy Ugolniy creek, currently buried under mine ettles (69°16′25″ N, 88°8′1″ E) (Figure 1B). With the depth of 331 m, it penetrated (top down) mine ettles (0–36.7 m), quaternary deposits (36.7–47.9), porphyric basalts and picrobasalts of the Gudchikhinskaya suite (47.9–158.0 m), tholeiitic and poikiloophitic basalts, tuffs and tuffites of Syverminskaya suite (158–269.1), basalts of Ivakinskaya suite (269.1–316.5 m), and the Upper zone on the Norilsk-1 intrusion (316.5–331 m) (Figure 3). The rocks of the intrusion are represented (top down) by breccia-like rocks with clasts of apobasaltic hornfelses, annealed coals and ophitic gabbro (316.5–322.0 m), patchy-textured (taxitic) gabbros with mingling of leucogabbro/troctolitic and olivine-gabbro lithologies (322.0–327.4 m), and ophitic gabbro of the Main zone (327.4–331.0 m) (Figure 3). Interval of the core between 321.9 m and 325.4 m contains numerous, yet uneven, Cr- spinel-rich domains and was sampled for the entire study. Samples from this borehole are labeled according to their meterage and are the following: MS24-321.9, MS24-322.3, MS24-323.4, MS24-324.0, MS24-324.3, and MS24-325.4 (Figure 3). Since their petrographic features are unique and thus essential for petrological constraints, we present their detailed description in the Results. Borehole MS-24 was placed at the upstream of the Yuzhniy Ugolniy creek, currently buried under mine ettles (69◦16′25” N, 88◦8′1” E) (Figure 1B). With the depth of 331 m, it penetrated (top down) mine ettles (0–36.7 m), quaternary deposits (36.7–47.9), porphyric basalts and picrobasalts of the Gudchikhinskaya suite (47.9–158.0 m), tholeiitic and poikiloophitic basalts, tuﬀs and tuﬃtes of Syverminskaya suite (158–269.1), basalts of Ivakinskaya suite (269.1–316.5 m), and the Upper zone on the Norilsk-1 intrusion (316.5–331 m) (Figure 3). 3.1. Samples Samples of Cr-spinel-bearing rocks of the upper zone from two distant sections of the Norilsk-1 intrusion were examined. One set of the samples was collected in 2011 from the Medvezhy Ruchey (Bear Creek) mine (Figure 2A), which is operated by Norilsk Nickel LTD. The PGE-Cr-spinel enriched layer at this mine is represented by strongly inhomogeneous gabbro-, breccia-, and skarn-like rocks [13,32,38], along with intermittent lenses of black shales, which are generally situated at the roof contact between the intrusion and Ivakinskaya suite basalts (Figure 2A–C). Although S.F. Sluzhenikin and the Geological Survey of Norilsk Nickel LTD performed extensive ﬁeld studies of this horizon during the 1990s, by 2011, most of those outcrops were inaccessible for sampling or have been buried under cliﬀdebris. However, we manage to undertake the sampling in the north-western side of the mine at the 300 m horizon (ﬂoor 274 m, top 340 m, N 69◦17′05,0” E 88◦10′13,7”) (Figure 2B). In the studied outcrop (~50 × 20 m), the PGE-Cr-spinel-bearing lithologies are represented by plagioclase-dominated rocks with diﬀerent amounts of olivine, pyroxene, and Cr-spinel. On a smaller scale, the rocks appear to be of gabbro composition and largely ophitic texture with zones of abundant Cr-spinel dissemination (Figure 2D). The PGE-Cr-spinel bearing rocks in the outcrop are overlain by a “transitional” layer, which although inaccessible, is most likely composed of leucocratic rocks with intermittent carbon-bearing pelites (black shales) and basalts, while the top of the outcrop seems to be composed mainly of metamorphosed basalts of Ivakinskaya suite (Figure 2B,C). Four samples were selected from the Cr-spinel-PGE-rich horizon in the studied outcrop (MR-14; MR-20; MR-30 and MR-31). Minerals 2020, 10, 498 5 of 30 5 of 30 Figure 2. Field relationships of different rocks in the Medvezhy Ruchey open pit. (A) Layout of the open pit with outlined main geological bodies; (B) sampled outcrop on the 300 m horizon; (C) a geological sketch of the 270 m horizon (after [13]); (D) Leucogabbro with visible Cr-spinel-rich sites. Figure 2. Field relationships of diﬀerent rocks in the Medvezhy Ruchey open pit. (A) Layout of the open pit with outlined main geological bodies; (B) sampled outcrop on the 300 m horizon; (C) a geological sketch of the 270 m horizon (after [13]); (D) Leucogabbro with visible Cr-spinel-rich sites. Figure 2. Field relationships of different rocks in the Medvezhy Ruchey open pit. 3.1. Samples The rocks of the intrusion are represented (top down) by breccia-like rocks with clasts of apobasaltic hornfelses, annealed coals and ophitic gabbro (316.5–322.0 m), patchy-textured (taxitic) gabbros with mingling of leucogabbro/troctolitic and olivine-gabbro lithologies (322.0–327.4 m), and ophitic gabbro of the Main zone (327.4–331.0 m) (Figure 3). Interval of the core between 321.9 m and 325.4 m contains numerous, yet uneven, Cr-spinel-rich domains and was sampled for the entire study. Samples from this borehole are labeled according to their meterage and are the following: MS24-321.9, MS24-322.3, MS24-323.4, MS24-324.0, MS24-324.3, and MS24-325.4 (Figure 3). Since their petrographic features are unique and thus essential for petrological constraints, we present their detailed description in the Results. 6 of 30 6 of 30 Minerals 2020, 10, 498 Mi l 2020 10 498 a , , Figure 3. MS-24 borehole with a detailed description of the studied section and a photograph of a drill core from 325.4 m depth (sample MS24-325.4), representing textural heterogeneity of Cr-spinel- bearing rocks. Methods and Data Processing Figure 3. MS-24 borehole with a detailed description of the studied section and a photograph of a drill core from 325.4 m depth (sample MS24-325.4), representing textural heterogeneity of Cr-spinel-bearing rocks. Methods and Data Processing Polished thin sections 1 × 0 5 inch were prepared for petrographic and mineralogical stu Figure 3. MS-24 borehole with a detailed description of the studied section and a photograph of a drill core from 325.4 m depth (sample MS24-325.4), representing textural heterogeneity of Cr-spinel- bearing rocks. Figure 3. MS-24 borehole with a detailed description of the studied section and a photograph of a drill core from 325.4 m depth (sample MS24-325.4), representing textural heterogeneity of Cr-spinel-bearing rocks. 3.2. Methods and Data Processing 3.2. Methods and Data Processing Polished thin sections 1 × 0.5 inch were prepared for petrographic and mineralogical studies. Grains of Cr-spinel were separated from crushed samples by gravitational concentration in water, Polished thin sections 1 × 0.5 inch were prepared for petrographic and mineralogical studies. Grains of Cr-spinel were separated from crushed samples by gravitational concentration in water, Minerals 2020, 10, 498 7 of 30 with subsequent concentration in a heavy liquid (tribromomethane) and then mounted into epoxy mounts. Petrographic studies of the thin sections were performed on a Carl Zeiss Scope A1 microscope equipped with a Canon EOS 650D camera. Scanning electron microscopy (SEM) was employed for imaging of textures and mineral assemblages. Micro-analytical studies of the Cr-spinel grains and silicates were carried out employing both electron probe microanalysis with wavelength dispersive X-ray spectroscopy (EPMA-WDS) and scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDS). Compositions of sulﬁdes and PGMs were determined using SEM-EDS. p py ( ) p g EPMA was performed on JEOL JXA-8320 and JEOL JXA-8100 microprobes at the Analytical Centre for Multi-elemental and Isotope Research of the V.S. Sobolev Institute of Geology and Mineralogy (AC IGM SB RAS), Novosibirsk, Russia. All analyses were performed at 20 kV and probe current 40 nA. Natural mineral compositions were used as standards during EPMA and were analyzed every 30–50 analyses. Imaging and quantitative analysis of mineral phases was carried out on Hitachi SU-70 Schottky ﬁeld emission SEM ﬁtted with Oxford INCA Energy XMax 80 silicon drift detector EDS system (University of Tasmania), Tescan Mira 3 LMU and JEOL JSM 6510 LV (IGM SB RAS, Novosibirsk), and Tescan Vega 3 (Institute of Volcanology and Seismology, Petropavlovsk-Kamchatsky). Probe current and acquisition time were selected individually on each instrument, while signal intensity during SEM-EDS examination was calibrated on artiﬁcial cobalt. Although applicability of SEM-EDS for quantitative analysis of minerals is debated, detailed investigations on several silicates and oxides (including Cr-spinel) have shown that this method allows to obtain numerical data on elements, which concentration is 0.3 wt. % and higher [39]. Primary EDS data was converted to element concentrations using the Aztec software package, while the quality of EDS data was controlled by real-time monitoring of σ value: element concentrations with σ more than 0.3 wt. % were considered to be inadequate. 3.2. Methods and Data Processing 3.2. Methods and Data Processing Subsequent comparison of data acquired by WDS and EDS methods substantiated the applicability of EDS for the purpose of this study, provided that all petrological implications are based on major elements in Cr-spinel, while elements with concentrations lower than 0.5% were not considered. To evaluate Fe2O3 content, we calculated mole percentage of each element and used deﬁciency of trivalent (Cr, Al, Ti, V) cations with regard to divalent ones (Fe, Mg). This method allowed to calculate Fe2O3 concentration, which was then was subtracted from total FeO to obtain a real content of FeO. Aside from routine data processing, we used graphical processing of back-scattered electron (hereinafter BSE) images, which allowed to visualize compositional features of Cr-spinel on a large area (up to a standard thin section) without employing speciﬁc equipment such as Micro-XRF technique. It was recognized that Cr-spinel with diﬀerent Mg/Fe ratio has diﬀerent brightness on grayscale BSE images. Comparison of microprobe data with BSE images of certain Cr-spinel grains revealed that this eﬀect is explained by diﬀerent atomic weights of Mg and Fe, rendering Mg-rich Cr-spinel grains darker and Fe-rich ones brighter. Although it is not usually evident from simple (low-contrast) BSE images (Figure 4A), a range of Cr-spinel grayscale hues is still signiﬁcant enough to resolve when the levels are adjusted in a raster processing software. We performed the processing in Adobe Photoshop CC, release 20.0.0. Using the “Adjustment →Curves” tool, we expanded a range of levels, corresponding to Cr-spinel grains and adjusted hues of silicates in order to better distinguish them from each other (Figure 4B). Then, we applied the “Adjustment →Gradient map” tool, assigning to the hues of Cr-spinel a rainbow stretch so that most magnesian Cr-spinel were colored by dark blue, while those with most ferrous compositions by red. Then, we attributed to plagioclase, clinopyroxene, and olivine diﬀerent hues of grey and to sulﬁdes (pure white) (Figure 4C). This algorithm allowed to obtain representative patterns, showing the compositional distribution of Cr-spinel with respect to enclosing silicate phases within a BSE-photographed site and even a whole thin section, mapped by SEM. However, it should be noted that on diﬀerent images, the same Cr-spinel compositions may be highlighted by diﬀerent colors. Thus, this method is used for qualitative visualization only and is not applicable for numeric considerations. 8 of 30 Minerals 2020, 10, 498 Figure 4. Main stages of a raster BSE (back-scattered electron) photo processing. 3.2. Methods and Data Processing 3.2. Methods and Data Processing (A) Initial image; (B) adjusted grayscale image; (C) pseudocolored image. Figure 4. Main stages of a raster BSE (back-scattered electron) photo processing. (A) Initial image; (B) adjusted grayscale image; (C) pseudocolored image. Figure 4. Main stages of a raster BSE (back-scattered electron) photo processing. (A) Initial image; (B) adjusted grayscale image; (C) pseudocolored image. Figure 4. Main stages of a raster BSE (back-scattered electron) photo processing. (A) Initial image; (B) adjusted grayscale image; (C) pseudocolored image. 4.1. Petrography and Textures of the Rocks 4.1. Petrography and Textures of the Rocks 9 of 30 Minerals 2020, 10, 498 Table 1. Summary of petrographic features of the studied rocks. Sample Rock Type Silicate Matrix 1 CrSp 2 Texture MR-14 Leucogabbro Pl +Cpx+Ol +++ Tabular Pl * grains with interstitial Cpx . Sites with >95 vol. % of Pl are present. Cr-spinel forms cloud-like abundant disseminations, which have distinct borders. Sulﬁde (pyrrhotite+chalcopyrite+pentlandite) mineralization is relatively scarce, but associated PGMs are abundant. MR-20 Ophitic gabbro Pl+Cpx +Ol + Euhedral elongated Pl with interstitial or poikilithic Cpx . Occasional sites, moderately enriched of Cr-spinel MR-30 (1) 3 Troctolite/leucogabbro Pl+Ol +Cpx +Opx ++ Euhedral tabular Pl with interstitial maﬁc minerals. Cr-spinel dissemination is relatively abundant, disperse and homogeneous. MR-30 (2) Ophitic gabbro Pl+Cpx +Ol + Euhedral elongated Pl with interstitial or poikilithic Cpx . Moderately enriched in Cr-spinel MR-30 (3) Ophitic gabbro (olivine-free) Pl +Cpx * - Elongated Pl with interstitial Cpx. No Cr-spinel. MR-31 Ophitic gabbro Pl+Cpx+Ol * + Euhedral elongated Pl with interstitial or poikilithic Cpx . Occasional sites, moderately enriched of Cr-spinel MS24-321.9 Ophitic gabbro Pl +Cpx +Ol + Euhedral elongated Pl with interstitial or poikilithic Cpx * and subhedral Ol . Thin “reef-like” band of Cr-spinel with scarce sulﬁdes (pyrrhotite + chalcopyrite + pentlandite). MS24-322.3 Ophitic gabbro Pl+Cpx+Ol Scarce Euhedral elongated Pl with interstitial or poikilithic Cpx * and subhedral Ol . Cr-spinel is only occasional. MS24-323.4 (1) Leucogabbro Pl+Cpx +Ol * +++ Tabular Pl with interstitial Cpx * and scarce Ol . Cr-spinel dissemination is dense and homogeneous with occasional Cr-spinel-free sites. Sulﬁde (pyrrhotite+chalcopyrite+pentlandite) clusters up to 5 mm. MS24-323.4 (2) Olivine-rich ophitic gabbro Pl+Cpx+Ol ++ Euhedral Pl and Ol with interstitial and poikilithic Cpx. Cr-spinel dissemination is moderately dense and homogeneous. MS24-324.0 Leucogabbro Pl +Cpx +Ol +++ Tabular Pl * with interstitial Cpx . Cr-spinel dissemination is dense and homogeneous. Sulﬁde clusters (pyrrhotite+chalcopyrite+pentlandite). up to 5 mm. MS24-324.3 Olivine-rich ophitic gabbro Pl+Cpx+Ol ++ Euhedral Pl and Ol with interstitial and poikilithic Cpx. Cr-spinel dissemination is moderately dense and homogeneous. MS24-325.4 Leucogabbro Pl+Cpx +Ol * ++ Tabular Pl * with interstitial Cpx . Cr-spinel dissemination is dense, but uneven. Sulﬁde clusters (pyrrhotite + chalcopyrite + pentlandite) are present. 1 In bold–strongly-dominating phases, in italics–subordinate phases. 2 Relative amounts from (-)–absent to (+++)–highest amount. 3 In parenthesis are given diﬀerent lithologies of a certain sample (see text for details). 4.1. Petrography and Textures of the Rocks 4.1. Petrography and Textures of the Rocks The studied samples are characterized by broadly similar mineral assemblages but highly variable textures. Petrographic features are summarized in the Table 1. In this sub-section we present general textural descriptions of the studied samples, while more detailed descriptions of Cr-spinel textures are presented in the subsequent subsection. The studied samples are characterized by broadly similar mineral assemblages but highly variable textures. Petrographic features are summarized in the Table 1. In this sub-section we present general textural descriptions of the studied samples, while more detailed descriptions of Cr-spinel textures are presented in the subsequent subsection. Mineral assemblages are dominated by plagioclase, olivine, and clinopyroxene, along with minor orthopyroxene, phlogopite and apatite. Non-silicate minerals are Cr-spinel, along with minor ilmenite, apatite, sulfides and rare baddeleyite and platinum-group minerals (PGMs). P i ili i l i ifi l l d Pl i l i i l d b Mineral assemblages are dominated by plagioclase, olivine, and clinopyroxene, along with minor orthopyroxene, phlogopite and apatite. Non-silicate minerals are Cr-spinel, along with minor ilmenite, apatite, sulﬁdes and rare baddeleyite and platinum-group minerals (PGMs). Primary silicate minerals are significantly altered. Plagioclase is to various extent replaced by albite, sericite, K-feldpar with minor epidote. Patchy alteration of pyroxenes seems to begin with fibrous amphibole (pargasite, ferro-pargasite, hornblende), while chlorite of clinochlore–chamosite series with minor Fe-hydroxides replace both pristine clinopyroxene and secondary amphibole. Olivine is extensively replaced by aggregates, composed by largely magnesian varieties of smectite, serpentine and iddingsite, the presence which is revealed by brownish colors of these aggregates. No fresh olivine was observed in the samples from the Medvezhy Ruchey mine, whereas in the samples from MS-24 core, there are sites with well-preserved euhedral olivine grains with thin rinds of the secondary aggregates. Primary silicate minerals are signiﬁcantly altered. Plagioclase is to various extent replaced by albite, sericite, K-feldpar with minor epidote. Patchy alteration of pyroxenes seems to begin with ﬁbrous amphibole (pargasite, ferro-pargasite, hornblende), while chlorite of clinochlore–chamosite series with minor Fe-hydroxides replace both pristine clinopyroxene and secondary amphibole. Olivine is extensively replaced by aggregates, composed by largely magnesian varieties of smectite, serpentine and iddingsite, the presence which is revealed by brownish colors of these aggregates. No fresh olivine was observed in the samples from the Medvezhy Ruchey mine, whereas in the samples from MS-24 core, there are sites with well-preserved euhedral olivine grains with thin rinds of the secondary aggregates. 1 In bold–strongly-dominating phases, in italics–subordinate phases. 2 Relative amounts from (-)–absent to (+++)–highest amount. 3 In parenthesis are given diﬀerent lithologies of a certain sample (see text for details). Asterisks () are used to deﬁne strongly altered/replaced silicates. 1 In bold–strongly-dominating phases, in italics–subordinate phases. 2 Relative amounts from (-)–absent to (+++)–hi certain sample (see text for details). Asterisks () are used to deﬁne strongly altered/replaced silicates. 4.1. Petrography and Textures of the Rocks 4.1. Petrography and Textures of the Rocks Asterisks () are used to deﬁne strongly altered/replaced silicates. Table 1. Summary of petrographic features of the studied rocks. Minerals 2020, 10, 498 10 of 30 A set of samples collected from the Medvezhy Ruchey open pit mine is representative of broad petrographic variations (Table 1) in a restricted volume of the upper zone of the intrusion. The sampling was performed on a small area (see higher) and although exact locations of each sample in the outcrop are missing, these petrographic varieties occur spatially close (<1–2 m) to each other. The most interesting mineralogical and textural features are observed in the sample MR-14 (Figure 5A). It has an inhomogeneous texture, which is represented by plagioclase segregations set in a matrix of smaller subhedral plagioclase and clinopyroxene crystals. Primary silicates are extensively replaced by secondary assemblages with plagioclase in segregations being totally altered. Former plagioclase grains are euhedral with an elongated and tabular shape, while former clinopyroxene is anhedral and occupies interstices between grains of plagioclase. Olivine is thought to have been present in the assemblage, but it is diﬃcult to determine its initial abundance due to extensive replacement. Cr-spinel (10–200 µm in size) is abundant and is scattered throughout the rock without showing any association with particular silicate minerals. The scattering is uneven and forms clusters without any pronounced speciﬁcation of their shapes. Distinct boundaries between Cr-spinel-rich and Cr-spinel-free sites of the rock are observed. Ore mineralization is represented by veinlets of pyrite, scarce intermittent pentlandite-pyrrhotite-chalcopyrite assemblages, and various microscopic PGMs, which are tightly associated with pentlandite-pyrrhotite-chalcopyrite clusters. Sample MR-30 exhibits three types of silicate lithologies, which have clearly deﬁned boundaries between each other (Figure 5B, Table 1) and are of signiﬁcant interest in the view of the discussion to come. The ﬁrst lithology (labeled “1”) has leucogabbro-troctolite modal composition. It is characterized by relatively fresh tabular plagioclase grains, brownish pseudomorphs after olivine and minor anhedral clinopyroxene and orthopyroxene, that are to variable extents replaced by chlorite-amphibole. Cr-spinel dissemination in this part of the sample is quite dense and homogenous, except for the former olivine, which hosts noticeably lesser amounts of Cr-spinel than plagioclase and clinopyroxene. The second lithology (labeled “2”) is used to contain less olivine and plagioclase than the previous one and can be classiﬁed as ophitic gabbro. It consists of major tabular plagioclase, interstitial clinopyroxene (both altered extensively), and a lesser amount of pseudomorphs after olivine. 4.1. Petrography and Textures of the Rocks 4.1. Petrography and Textures of the Rocks Within this lithology Cr-spinel scattering is less abundant and more uneven than within the ﬁrst one. While the ﬁrst and second lithologies of the MR-30 sample are diﬀerent only in terms of texture and modal proportions of minerals and, the third lithological type (labeled “3”) is strikingly diﬀerent from the ﬁrst and second types. It is dominated by sericitized and albitized plagioclase with minor clinopyroxene, which is replaced by chlorite and ﬁbrous amphibole, and contains no olivine and Cr-spinel, whereas elongated and spiky crystals of ilmenite are abundant. This lithology resembles “ordinary” ophitic gabbros, which compose most of the Main zone Norilsk-1 intrusions. Accessory minerals in sample MR-30 include sulﬁdes (chalcopyrite, pyrrhotite, pentlandite, and pyrite), apatite, ilmenite, and PGMs, which are usually found in association with low-Mg Cr-spinel and sulﬁdes. Samples MR-20 and MR-31 have more homogenous fabric and texture but lower abundances of Cr-spinel than samples MR-14 and MR-30. They are classiﬁed as ophitic gabbro and consist of euhedral plagioclase, subhedral to anhedral clinopyroxene, and pseudomorphs after olivine. The clinopyroxene in sample MR-31 is fresher and sometimes exhibits more euhedral shapes than the clinopyroxene in sample MR-20. However, aggregates of chlorite, amphibole, serpentine, micas, and other hydrous silicates are abundant in these samples, and it is sometimes diﬃcult to distinguish the precursor mineral. Phlogopite, sulﬁdes, apatite, ilmenite, baddeleyite, and PGMs occur as minor phases in both samples. 11 of 30 Minerals 2020, 10, 498 Figure 5. Photomicrographs of the representative thin sections of the studied Cr-spinel-rich rocks: (A) leucogabbro with Cr-spinel-rich clusters (plane polarized light); (B) three lithological domains within MR-30 thin section: troctolite (1), ophitic gabbro (2), and olivine-free ophitic gabbro without Cr-spinel (3) (crossed-polarized light); (C) abundant Cr-spinel dissemination and big sulfide aggregation in leucogabbro (reflected light), (D) leucogabbro (1) and olivine gabbro (2) lithological domains with a transitional zone (3) (cross-polarized light). Mineral abbreviations: Pl–plagioclase; Cpx–clinopyroxene, Ol– olivine; Pn–pentlandite; Po–pyrrhotite; Ccp–chalcopyrite, Py–pyrite, CrSp–chromian spinel. Samples of MS-24 core exhibit variable textures. In general, the rocks present in the considered Figure 5. 4.1. Petrography and Textures of the Rocks 4.1. Petrography and Textures of the Rocks Photomicrographs of the representative thin sections of the studied Cr-spinel-rich rocks: (A) leucogabbro with Cr-spinel-rich clusters (plane polarized light); (B) three lithological domains within MR-30 thin section: troctolite (1), ophitic gabbro (2), and olivine-free ophitic gabbro without Cr-spinel (3) (crossed-polarized light); (C) abundant Cr-spinel dissemination and big sulﬁde aggregation in leucogabbro (reﬂected light), (D) leucogabbro (1) and olivine gabbro (2) lithological domains with a transitional zone (3) (cross-polarized light). Mineral abbreviations: Pl–plagioclase; Cpx–clinopyroxene, Ol–olivine; Pn–pentlandite; Po–pyrrhotite; Ccp–chalcopyrite, Py–pyrite, CrSp–chromian spinel. section of the core change in the following sequence (top down) (Figure 3): Samples of MS-24 core exhibit variable textures. In general, the rocks present in the considered section of the core change in the following sequence (top down) (Figure 3): Hornfels-like rocks from the contact with the Ivakinskaya suite basalts. • Hornfels-like rocks from the contact with the Ivakinskaya suite basalts. • Breccia-like rocks with leucocratic ophitic gabbro matrix and abundant xenoliths of coal-bearing shales and terrigenous schists, • Mottled “taxitic” rocks with lithological domains largely represented by leucogabbro, troctolite, and olivine gabbro. Cr-spinel and sulﬁdes are abundant in this part of the core. • Gabbro- and gabbro-diorites of the main diﬀerentiated series. Minerals 2020, 10, 498 12 of 30 Sample MS24-321.9 has ophitic texture with elongated plagioclase grains, strongly altered anhedral clinopyroxene and subhedral olivine. Cr-spinel occurs as a thin reef-like (~3 mm) band with a signiﬁcant amount of sulﬁdes and sulﬁde-PGM assemblages. Sample MS24-321.9 has ophitic texture with elongated plagioclase grains, strongly altered anhedral clinopyroxene and subhedral olivine. Cr-spinel occurs as a thin reef-like (~3 mm) band with a signiﬁcant amount of sulﬁdes and sulﬁde-PGM assemblages. Sample MS24-322.5 in thin section is represented by relatively homogeneous ophitic gabbro, consisting of euhedral tabular and prismatic plagioclase grains settled within the matrix, composed of anhedral and poikilithic clinopyroxene and olivine (replaced). Cr-spinel mineralization is scarce and is represented by tiny swarms of grains, which are all hosted by silicates without any mineralogical preference. Sample MS24-323.4 is completely diﬀerent from its previous counterpart. Several studied thin sections are dominated by leucogabbro and olivine gabbro (Figure 5C,D). However, leucocratic type of rock (labeled “1” on Figure 5D) seems to be prevalent. It is composed of tabular plagioclase, which is the dominant phase and, in most cases, only slightly aﬀected by albitization and sericitization. Clinopyroxene occurs as interstitial anhedral grains, which are often replaced by chlorite and amphibole. 4.1. Petrography and Textures of the Rocks 4.1. Petrography and Textures of the Rocks Olivine is present in subordinate amounts and is largely replaced by saponite-talk-serpentine assemblages. The most important feature of this rock type is extremely high content of Cr-spinel and signiﬁcant, but lesser amounts of sulﬁdes, which form impregnated textures with clusters up to centimeter in their long dimension (Figure 5C). Olivine gabbro (labeled “2” on Figure 5D) exhibits quite homogeneous texture and contain moderate enrichments in Cr-spinel and minor sulﬁdes. Within the examined thin section, the boundary between leucocratic and olivine gabbro varieties is less distinct than in the sample MR-30 (Figure 5B,D). Instead, there is a thin transitional zone (labeled “3” in Figure 5D) where the rock fabric becomes more ﬁne-grained and plagioclase turns from tabular to elongated, olivine and clinopyroxene abundances increase, while Cr-spinel and sulﬁde abundances decrease. py p Sample MS24-323.4 is completely diﬀerent from its previous counterpart. Several studied thin sections are dominated by leucogabbro and olivine gabbro (Figure 5C,D). However, leucocratic type of rock (labeled “1” on Figure 5D) seems to be prevalent. It is composed of tabular plagioclase, which is the dominant phase and, in most cases, only slightly aﬀected by albitization and sericitization. Clinopyroxene occurs as interstitial anhedral grains, which are often replaced by chlorite and amphibole. Olivine is present in subordinate amounts and is largely replaced by saponite-talk-serpentine assemblages. The most important feature of this rock type is extremely high content of Cr-spinel and signiﬁcant, but lesser amounts of sulﬁdes, which form impregnated textures with clusters up to centimeter in their long dimension (Figure 5C). Olivine gabbro (labeled “2” on Figure 5D) exhibits quite homogeneous texture and contain moderate enrichments in Cr-spinel and minor sulﬁdes. Within the examined thin section, the boundary between leucocratic and olivine gabbro varieties is less distinct than in the sample MR-30 (Figure 5B,D). Instead, there is a thin transitional zone (labeled “3” in Figure 5D) where the rock fabric becomes more ﬁne-grained and plagioclase turns from tabular to elongated, olivine and clinopyroxene abundances increase, while Cr-spinel and sulﬁde abundances decrease. Sample MS24-324.0 is somewhat similar to the leucocratic varieties of MS24-323.4 and is composed of tabular plagioclase grains with interstitial inﬁlling of chlorite and amphibole (former clinopyroxene). It also hosts extremely dense Cr-spinel disseminations, which are scattered without any preference to the type of host silicate and substantial content of sulﬁde clusters. Sample MS24-324.3 exhibits features similar to those of olivine gabbro variety of the MS24-323.4. 4.1. Petrography and Textures of the Rocks 4.1. Petrography and Textures of the Rocks It is the most unaltered sample in the studied set and is characterized by a relatively homogeneous ophitic texture with euhedral olivine and plagioclase and interstitial clinopyroxene. Cr-spinel dissemination is dense but to a lesser extent than in leucocratic samples MS24-323.4 and MS24-324.0. Sulﬁde clusters and associated PGM mineralization are substantial but, in this study, were not examined in detail. Sample MS24-325.4 has heterogeneous fabric and texture (Figure 3). Its composition is dominated by plagioclase, which forms crystals of diﬀerent shape and size: large (>3 mm) tabular grains and smaller needle-shaped crystals. Clinopyroxene inﬁlls narrow interstices between tightly packed plagioclase and forms large poikilithic crystals that enclose the plagioclase grains. Cr-spinel distribution in the studied thin section is uneven and is not as abundant as in the leucocratic and picritic gabbro in Minerals 2020, 10, 498 13 of 30 13 of 30 the above part of the core cut. However, several embayments and trails of Cr-spinel dissemination are quite dense and have distinct boundaries (Figure 6A). Minerals 2020, 10, 498 13 of 30 Figure 6. Representative photomicrographs of specific Cr-spinel arrangements in the studied rocks. (A,B) Embayments of Cr-spinel dissemination, passing through the silicates (cross-polarized light); (C,D) Outlined by dashed lines are trails of Cr-spinel, crossing silicate borders (reflected and plane- polarized light respectively), (E) a thin chain of Cr-spinel grains, crossing the border between silicates; (F) sub-circular arrangement of Cr-spinel, surrounding a “bubble” composed by chlorite (Chl) and phlogopite (Phl) (BSE-microphoto). Mineral abbreviations: Pl–plagioclase, Ol-olivine, Cpx– clinopyroxene, Phl–phlogopite, Chl-chlorite. 4 2 C S i l T t d M h l Figure 6. Representative photomicrographs of speciﬁc Cr-spinel arrangements in the studied rocks. (A,B) Embayments of Cr-spinel dissemination, passing through the silicates (cross-polarized light); (C,D) Outlined by dashed lines are trails of Cr-spinel, crossing silicate borders (reﬂected and plane-polarized light respectively), (E) a thin chain of Cr-spinel grains, crossing the border between silicates; (F) sub-circular arrangement of Cr-spinel, surrounding a “bubble” composed by chlorite (Chl) and phlogopite (Phl) (BSE-microphoto). Mineral abbreviations: Pl–plagioclase, Ol-olivine, Cpx–clinopyroxene, Phl–phlogopite, Chl-chlorite. 4 2 Cr-Spinel: Textures and Morphology Figure 6. Representative photomicrographs of specific Cr-spinel arrangements in the studied rocks. 4.1. Petrography and Textures of the Rocks 4.1. Petrography and Textures of the Rocks (A,B) Embayments of Cr-spinel dissemination, passing through the silicates (cross-polarized light); (C,D) Outlined by dashed lines are trails of Cr-spinel, crossing silicate borders (reflected and plane- polarized light respectively), (E) a thin chain of Cr-spinel grains, crossing the border between silicates; (F) sub-circular arrangement of Cr-spinel, surrounding a “bubble” composed by chlorite (Chl) and phlogopite (Phl) (BSE-microphoto). Mineral abbreviations: Pl–plagioclase, Ol-olivine, Cpx– clinopyroxene, Phl–phlogopite, Chl-chlorite. Figure 6. Representative photomicrographs of speciﬁc Cr-spinel arrangements in the studied rocks. (A,B) Embayments of Cr-spinel dissemination, passing through the silicates (cross-polarized light); (C,D) Outlined by dashed lines are trails of Cr-spinel, crossing silicate borders (reﬂected and plane-polarized light respectively), (E) a thin chain of Cr-spinel grains, crossing the border between silicates; (F) sub-circular arrangement of Cr-spinel, surrounding a “bubble” composed by chlorite (Chl) and phlogopite (Phl) (BSE-microphoto). Mineral abbreviations: Pl–plagioclase, Ol-olivine, Cpx–clinopyroxene, Phl–phlogopite, Chl-chlorite. 4.2. Cr-Spinel: Textures and Morphology 4.2. Cr-Spinel: Textures and Morphology This is manifested in diﬀerent locations and samples, and in certain cases it is even termed as bubble- or foam-like textures [32,40]. In our studied samples, there was only one such Cr-spinel “bubble,” inﬁlled with phlogopite and chlorite, found in sample MR-14 (Figure 6F). However, well-developed sub-circular arrangements of Cr-spinel are observed on a small scale in several samples both from the Medvezhy Ruchey mine and MS-24 core. In samples MS24-323.4 and MS24-324.3, these circles are so abundant that they should be considered as a common type of Cr-spinel distribution. In contrast to the previous reports of these textures, this kind of alignment does not show aﬃliation to phase boundaries and cannot be considered as bona ﬁde agglutination of Cr-spinel on pre-existing silicates or bubbles (Figure 7). The arising problem is of importance and will be exploited in more detail below. given sample but are observed in most of the samples studied from both the Medvezhy Ruchey and MS-24 core. A remarkable textural feature, which is characteristic for the “sulfide-poor” PGE-Cr-spinel-rich lithologies in the Norilsk-type intrusions in general, is a sub-circular alignment of Cr-spinel grains. This is manifested in different locations and samples, and in certain cases it is even termed as bubble- or foam-like textures [32,40]. In our studied samples, there was only one such Cr-spinel “bubble,” infilled with phlogopite and chlorite, found in sample MR-14 (Figure 6F). However, well-developed sub-circular arrangements of Cr-spinel are observed on a small scale in several samples both from the Medvezhy Ruchey mine and MS-24 core. In samples MS24-323.4 and MS24-324.3, these circles are so abundant that they should be considered as a common type of Cr-spinel distribution. In contrast to the previous reports of these textures, this kind of alignment does not show affiliation to phase boundaries and cannot be considered as bona fide agglutination of Cr-spinel on pre-existing silicates or bubbles (Figure 7). The arising problem is of importance and will be exploited in more detail below. Figure 7. Sub-circular arrangements of Cr-spinel, hosted by unaltered silicates. (A,C–F) SEM BSE photo; (B) transmitted, cross-polarized light. Mineral abbreviations: Pl–plagioclase, Ol-olivine, Cpx– clinopyroxene, abbreviations followed by an asterisk () label strongly altered and replaced silicates. In terms of morphology, Cr-spinel varies from small (10–80 μm) rounded grains (Figure 8A) to bigger (up to 200 μm) straight-edged subhedral crystals (Figure 8B,C). 4.2. Cr-Spinel: Textures and Morphology 4.2. Cr-Spinel: Textures and Morphology The main characteristics of Cr-spinel in the studied rocks are their heterogeneity and absence of planar (or reef-like) patterns, typical for layered and differentiated intrusions. Although the The main characteristics of Cr-spinel in the studied rocks are their heterogeneity and absence of planar (or reef-like) patterns, typical for layered and diﬀerentiated intrusions. Although the abundance Minerals 2020, 10, 498 14 of 30 of Cr-spinel dissemination varies widely (from uneven accessory grains in MS24-322.3 to exceptionally abundant clusters in MR-14 and MS24-323.4), the complexity of grain distribution (e.g., swarms or clusters) is a common feature (Figures 5C and 6A,B). Borders of Cr-spinel-rich clusters often do not coincide with the margins of silicate phases and can either pass through silicate phases or be around them (Figure 6A,B). It is important that borders of Cr-spinel-rich clusters and Cr-spinel embayments passing through several silicate grains may not necessarily change their shape or show any evidence of interruption. Instead, the textures give a visual impression that Cr-spinel grains were interspersed over the pre-existing silicate matrix (Figure 6B). Upon closer inspection, thin trails of Cr-spinel grains pass through the silicates unaﬀected by the grain boundaries (Figure 6C,D). The most indicative examples of this feature are one-grain-thick chains of Cr-spinel grains, observed near the margins of the silicate phases (Figure 6E). Importantly, these kinds of alignment are not unique for a given sample but are observed in most of the samples studied from both the Medvezhy Ruchey and MS-24 core. Minerals 2020, 10, 498 14 of 30 embayments passing through several silicate grains may not necessarily change their shape or show any evidence of interruption. Instead, the textures give a visual impression that Cr-spinel grains were interspersed over the pre-existing silicate matrix (Figure 6B). Upon closer inspection, thin trails of Cr- spinel grains pass through the silicates unaffected by the grain boundaries (Figure 6C,D). The most indicative examples of this feature are one-grain-thick chains of Cr-spinel grains, observed near the margins of the silicate phases (Figure 6E). Importantly, these kinds of alignment are not unique for a given sample but are observed in most of the samples studied from both the Medvezhy Ruchey and A remarkable textural feature, which is characteristic for the “sulﬁde-poor” PGE-Cr-spinel-rich lithologies in the Norilsk-type intrusions in general, is a sub-circular alignment of Cr-spinel grains. 4.2. Cr-Spinel: Textures and Morphology 4.2. Cr-Spinel: Textures and Morphology 15 of 30 cal 15 of 30 cal Minerals 2020, 10, 498 with ilmenite ( A id f For the purpose of discussion, we address small rounded Cr-spinel grains as Cr-spinel-1 and bigger, often intergrown grains, as Cr-spinel-2. However, it should be kept in mind that these morphological types are rather end members of a continuous range than distinct populations. Chemical examination of Cr-spinel revealed that Cr-spinel-1 grains are richer in Mg and Al and poorer in Fe and Ti than Cr-spinel-2 (see the next section for the details) (Figure 8D,E). Cr-spinel-2 is often found in intergrowths with ilmenite (Figure 8C) and may contain ilmenite inclusions or lamellae. yp g y j g g g plagioclase (Figure 8F) and strongly resorbed grains in extensively altered sites of the rocks (Figure 8G). Occasionally found are Cr-spinel grains, overgrown by magnetite (Figure 8H) and a symplectite- like compositionally heterogeneous intergrowth of Cr-spinel in smectite pseudomorph after olivine (Figure 8I). In addition, up to 20% modal of Cr-spinel grains may contain rounded silicate inclusions, which mainly comprise major orthopyroxene, phlogopite, amphibole, chlorite, and silicate glass. Those inclusions were considered to be a heterogeneously trapped mixture of melt and solid phases and are studied in detail in [41]. Figure 8. A variety of Cr-spinel morphologies. (A–D)–SEM BSE photos; (E–G) colored SEM BSE photos (see Methods and data processing); (H,I) SEM BSE photos. Mineral abbreviations: Pl–plagioclase, Ol-olivine, Cpx–clinopyroxene, Sulf–sulfide, Ccp–chalcopyrite, Pn–pentlandite, Apapatite, Sm– smectite, CrSp–chromian spinel, Ilm–ilmenite, Chl–chlorite, Mag–magnetite, Amp–amphibole. Figure 8. A variety of Cr-spinel morphologies. (A–D)–SEM BSE photos; (E–G) colored SEM BSE photos (see Methods and data processing); (H,I) SEM BSE photos. Mineral abbreviations: Pl–plagioclase Ol-olivine, Cpx–clinopyroxene, Sulf–sulﬁde, Ccp–chalcopyrite, Pn–pentlandite, Apapatite, Sm–smectite CrSp–chromian spinel, Ilm–ilmenite, Chl–chlorite, Mag–magnetite, Amp–amphibole. Figure 8. A variety of Cr-spinel morphologies. (A–D)–SEM BSE photos; (E–G) colored SEM BSE photos (see Methods and data processing); (H,I) SEM BSE photos. Mineral abbreviations: Pl–plagioclase, Ol-olivine, Cpx–clinopyroxene, Sulf–sulfide, Ccp–chalcopyrite, Pn–pentlandite, Apapatite, Sm– smectite, CrSp–chromian spinel, Ilm–ilmenite, Chl–chlorite, Mag–magnetite, Amp–amphibole. Figure 8. A variety of Cr-spinel morphologies. (A–D)–SEM BSE photos; (E–G) colored SEM BSE photos (see Methods and data processing); (H,I) SEM BSE photos. Mineral abbreviations: Pl–plagioclase, Ol-olivine, Cpx–clinopyroxene, Sulf–sulﬁde, Ccp–chalcopyrite, Pn–pentlandite, Apapatite, Sm–smectite, CrSp–chromian spinel, Ilm–ilmenite, Chl–chlorite, Mag–magnetite, Amp–amphibole. Aside from these commonly present Cr-spinel morphologies, there are some rare morphological types that occur in strongly altered silicates. 4.2. Cr-Spinel: Textures and Morphology 4.2. Cr-Spinel: Textures and Morphology Importantly, the former are mostly isolated, while the latter tend to agglomerate within tightly-packed intergrowths (Figure 8C). For the purpose of discussion, we address small rounded Cr-spinel grains as Cr-spinel-1 and bigger, Figure 7. Sub-circular arrangements of Cr-spinel, hosted by unaltered silicates. (A,C–F) SEM BSE photo; (B) transmitted, cross-polarized light. Mineral abbreviations: Pl–plagioclase, Ol-olivine, Cpx–clinopyroxene, abbreviations followed by an asterisk () label strongly altered and replaced silicates. In terms of morphology, Cr-spinel varies from small (10–80 µm) rounded grains (Figure 8A) gger (up to 200 µm) straight-edged subhedral crystals (Figure 8B,C). Importantly, the former ostly isolated, while the latter tend to agglomerate within tightly-packed intergrowths (Figure 8 Figure 7. Sub-circular arrangements of Cr-spinel, hosted by unaltered silicates. (A,C–F) SEM BSE photo; (B) transmitted, cross-polarized light. Mineral abbreviations: Pl–plagioclase, Ol-olivine, Cpx– clinopyroxene, abbreviations followed by an asterisk () label strongly altered and replaced silicates. Figure 7. Sub-circular arrangements of Cr-spinel, hosted by unaltered silicates. (A,C–F) SEM BSE photo; (B) transmitted, cross-polarized light. Mineral abbreviations: Pl–plagioclase, Ol-olivine, Cpx–clinopyroxene, abbreviations followed by an asterisk () label strongly altered and replaced silicates. In terms of morphology, Cr-spinel varies from small (10–80 μm) rounded grains (Figure 8A) to bigger (up to 200 μm) straight-edged subhedral crystals (Figure 8B,C). Importantly, the former are mostly isolated, while the latter tend to agglomerate within tightly-packed intergrowths (Figure 8C). For the purpose of discussion, we address small rounded Cr-spinel grains as Cr-spinel-1 and bigger, In terms of morphology, Cr-spinel varies from small (10–80 µm) rounded grains (Figure 8A) to bigger (up to 200 µm) straight-edged subhedral crystals (Figure 8B,C). Importantly, the former are mostly isolated, while the latter tend to agglomerate within tightly-packed intergrowths (Figure 8C). In terms of morphology, Cr-spinel varies from small (10–80 μm) rounded grains (Figure 8A) to bigger (up to 200 μm) straight-edged subhedral crystals (Figure 8B,C). Importantly, the former are mostly isolated, while the latter tend to agglomerate within tightly-packed intergrowths (Figure 8C). For the purpose of discussion, we address small rounded Cr-spinel grains as Cr-spinel-1 and bigger, In terms of morphology, Cr-spinel varies from small (10–80 µm) rounded grains (Figure 8A) to bigger (up to 200 µm) straight-edged subhedral crystals (Figure 8B,C). Importantly, the former are mostly isolated, while the latter tend to agglomerate within tightly-packed intergrowths (Figure 8C). 4.2. Cr-Spinel: Textures and Morphology 4.2. Cr-Spinel: Textures and Morphology Those include jigsaw-edged grains in sericitized plagioclase (Figure 8F) and strongly resorbed grains in extensively altered sites of the rocks (Figure 8G). Occasionally found are Cr-spinel grains, overgrown by magnetite (Figure 8H) and a symplectite-like compositionally heterogeneous intergrowth of Cr-spinel in smectite pseudomorph after olivine (Figure 8I). In addition, up to 20% modal of Cr-spinel grains may contain rounded silicate inclusions, which mainly comprise major orthopyroxene, phlogopite, amphibole, chlorite, and silicate glass. Those inclusions were Minerals 2020, 10, 498 16 of 30 considered to be a heterogeneously trapped mixture of melt and solid phases and are studied in detail in [41]. Mi l 2020 10 498 16 f 30 4.3. Cr-Spinel: Chemistry 4 3 Cr Spinel: Chemistry Minerals 2020, 10, 498 17 of 30 17 of 30 In all studied samples, Cr-spinel compositions are characterized by generally low Mg# (Mg/(Mg + Fe2+) mol. %) (2 to 55 mol. %) (Figure 9A,B) and medium to high Cr# (Cr/(Cr + Al) mol. %) (48–95 mol. %). The most abundant Cr-spinel population has Mg# between 10–20 mol. %, while Cr-spinel with Mg# exceeding 45 mol. % is quite uncommon, especially for rocks from the Medvezhy Ruchey (Figure 9A). The analyzed Cr-spinel grains shows a negative correlation between Cr# and Mg# (Figure 9C) and various behavior of Cr2O3 content. The Al2O3 content in Cr-spinel varies broadly from 1 wt. % to 26 wt. % (Figure 9H), forming a positive linear correlation with Mg#. Broad ranges are observed in the Al-Cr-Fe3+ triple variations—the compositional ﬁeld of the studied Cr-spinel overlaps with signiﬁcant part of Cr-spinel compositions from intrusive rocks (Figure 9E) A characteristic feature of Cr-spinel in all the examined samples is elevated TiO2 content, which varies signiﬁcantly in grains with diﬀerent Mg# and reaches maximum of about 18 wt. % in Cr-spinel with the lowest Mg# values (Figure 9G). In general, Cr-spinel from all the samples observed exhibits anomalously broad chemical variations. The obtained chemical ranges overlap with Cr-spinel compositions from intrusive complexes worldwide, while in terms of Ti-Fe3+ systematics a large amount of the obtained compositions does not have analogues elsewhere in the world (Figure 9I). They also fully overlap with compositional ﬁelds of Cr-spinel from the Lower zone of Norilsk-type intrusions and from the eﬀusive series from the Western part of the Siberian LIP (Figure 9), being much broader than the latter ones. In addition, chemistry of the studied Cr-spinel does not correspond to any group with respect to tectonic settings, overlapping with ﬁelds of spinels from LIPs, ocean island basalts (OIBs), and even mid-oceanic ridge baslts MORBs (Figure 9H). The abundances of minor elements in the analyzed Cr-spinel, such as ZnO, NiO, and V2O3, are slightly elevated compared to typical values for magmatic Cr-spinel [42]. The compositional range for ZnO (0.05% to 0.35% wt.) shows overlap between all studied samples (Figure 9J). Vanadium contents are more variable and show a slight increase with decreasing Mg# (Figure 9K). In Cr-spinel from MS-24 drill core, V2O3 content varies in the same range (0.4–1.3 wt. %) for all studied samples, Cr-spinel from Medvezhy Ruchey exhibits diﬀerent V2O3 contents for diﬀerent samples. 4.3. Cr-Spinel: Chemistry 4 3 Cr Spinel: Chemistry We obtained a large dataset of Cr-spinel compositions (over 4000 analyses), which is listed in Table S1. Figure 9 plots the obtained compositions of Cr-spinel and reference data for layered intrusions worldwide and intrusive and eﬀusive rocks of the Norilsk region of the Siberian large igneous province (LIP) (objects are speciﬁed, and data sources are listed in the ﬁgure caption). 4.3. Cr Spinel: Chemistry We obtained a large dataset of Cr-spinel compositions (over 4000 analyses), which is listed in Table S1. Figure 9 plots the obtained compositions of Cr-spinel and reference data for layered intrusions worldwide and intrusive and effusive rocks of the Norilsk region of the Siberian large igneous province (LIP) (objects are specified, and data sources are listed in the figure caption). Figure 9. Major- (A–I) and minor-element (J,K) element variations in Cr-spinel from rocks of Medley riches open pit and drill core MS-24. Compositions for layered intrusions worldwide are from [42], Gudchikhinsky and Tuklonsky picrites [34,43–45] Cr-spinel from Norilsk-1 and Talnakh Lower zones from [46]. Fields for LIP (large igneous provinces), OIB (ocean island basalts), MORB (mid-oceanic ridge basalts) and Arc basalts on the inset (H) are as in [47]. Figure 9. Major- (A–I) and minor-element (J,K) element variations in Cr-spinel from rocks of Medley riches open pit and drill core MS-24. Compositions for layered intrusions worldwide are from [42], Gudchikhinsky and Tuklonsky picrites [34,43–45] Cr-spinel from Norilsk-1 and Talnakh Lower zones from [46]. Fields for LIP (large igneous provinces), OIB (ocean island basalts), MORB (mid-oceanic ridge basalts) and Arc basalts on the inset (H) are as in [47]. Figure 9. Major- (A–I) and minor-element (J,K) element variations in Cr-spinel from rocks of Medley riches open pit and drill core MS-24. Compositions for layered intrusions worldwide are from [42], Gudchikhinsky and Tuklonsky picrites [34,43–45] Cr-spinel from Norilsk-1 and Talnakh Lower zones from [46]. Fields for LIP (large igneous provinces), OIB (ocean island basalts), MORB (mid-oceanic ridge basalts) and Arc basalts on the inset (H) are as in [47]. Figure 9. Major- (A–I) and minor-element (J,K) element variations in Cr-spinel from rocks of Medley riches open pit and drill core MS-24. Compositions for layered intrusions worldwide are from [42], Gudchikhinsky and Tuklonsky picrites [34,43–45] Cr-spinel from Norilsk-1 and Talnakh Lower zones from [46]. Fields for LIP (large igneous provinces), OIB (ocean island basalts), MORB (mid-oceanic ridge basalts) and Arc basalts on the inset (H) are as in [47]. 4.3. Cr-Spinel: Chemistry 4 3 Cr Spinel: Chemistry Cr-spinel grains in samples MR-20 and MR-31 have the lowest V2O3 (0.4 to 0.6 wt. %) content, whereas Cr-spinel from sample MR-14 have distinctly higher concentrations (0.9–1.5 wt. % V2O3). 3 A striking feature of Cr-spinel investigated is pronouncedly variable systematics of Cr, Fe3+ and Ti. These diﬀerences are especially representative in Cr-spinel from the Medvezhy Ruchey samples and to a lesser extent in rocks from the MS-24 drill core. Cr2O3 content may either decrease with decreasing Mg# or remain stable, still reaching 40–45 wt. % when Mg# falls down below 10 mol. % (Figures 9D and 10A,B). Diﬀerence in Fe3+ systematics is reﬂected in divergent trends observed in the Fe2+ vs Fe3+ and Fe3+ vs TiO2 covariations (Figures 9F and 10C–F). Cr-spinel from samples MR-14, MR-30 and MS24-324.0, which are dominated by leucogabbro/troctolite lithologies, is characterized by weak or no change in Fe3+ with Fe2+ growth while TiO2 concentration increases sharply (“ulvospinel” trend). Meanwhile, samples with less leucocratic modal compositions show variably pronounced positive correlation between Fe2+ and Fe3+ contents and not so sharp TiO2 increase (“Ti-magnetite” trend) (Figure 10C–F). In this regard, remarkable is sample MS24-324.3, in the examined sections of which both leucogabbro and olivine gabbro are present (Figure 5D). In the same thin section of this sample Cr-spinel from leucogabbro and from olivine gabbro exhibit diverse trends in terms of Cr, Ti and Fe3+ (Figure 10B,C). Minerals 2020, 10, 498 methods) reveals 18 of 30 ure 11), 18 of 30 ure 11), Figure 10. Distinct compositional trends for Cr-spinel from different samples. (A,C,E) Medvezhy Ruchey samples; (B,D,F) drill core MS-24. Figure 10. Distinct compositional trends for Cr-spinel from diﬀerent samples. (A,C,E) Medvezhy Ruchey samples; (B,D,F) drill core MS-24. Figure 10. Distinct compositional trends for Cr-spinel from different samples. (A,C,E) Medvezhy Figure 10. Distinct compositional trends for Cr-spinel from diﬀerent samples. (A,C,E) Medvezhy Ruchey samples; (B,D,F) drill core MS-24. y p ; ( , , ) On a scale of a given silicate lithology, the composition, morphology, and spatial distribution of Cr-spinel grains are statistically related with each other. Rounded grains (Cr-spinel-1) have higher Mg#, Al2O3 and sometimes Cr2O3 values compared to intergrown subhedral straight-edged grains (Cr-spinel-2). The latter are characterized by low Mg#, high FeO (total) and high TiO2 values. This dependence is well-illustrated by transition zones between locations with high-Mg# and low-Mg# grains of Cr-spinel-1 and Cr-spinel-2, respectively (Figure 8D,E). 4.3. Cr-Spinel: Chemistry 4 3 Cr Spinel: Chemistry Minerals 2020, 10, 498 20 of 30 20 of 30 Compositional diﬀerences in Cr-spinel enclosed in fresh plagioclase, clinopyroxene, and olivine are statistically derived (Figure 12A–C). With some regard to a given sample and sampling location, Cr-spinel grains hosted by plagioclase are characterized by the highest Mg# with the main mode of about 45–50 mol. % Mg# of Cr-spinel, enclosed within clinopyroxene varies generally in range 20–40 mol. %, while those of rare Cr-spinel grains enclosed in unaltered olivine do not exceed 20 mol. % with a single grain with Mg# 31 mol. % (Figure 12A–C). In contrast, Cr-spinel hosted by altered silicates show very similar modes of Mg# (12–17 mol. %) and it is clearly seen that both in replaced plagioclase and clinopyroxene Cr-spinel loses Mg content with respect to grains, settled in fresh silicates. In a perspective of the coming discussion, it is important that despite similar main Mg# modes of Cr-spinel from replaced silicates, Cr-spinel from replaced plagioclase still appears to have a signiﬁcant population with Mg# values higher than 20, while it is not the case for Cr-spinel from replaced clinopyroxene (Figure 12D,E). Minerals 2020, 10, 498 20 of 30 Compositional differences in Cr-spinel enclosed in fresh plagioclase, clinopyroxene, and olivine are statistically derived (Figure 12A–C). With some regard to a given sample and sampling location, Cr-spinel grains hosted by plagioclase are characterized by the highest Mg# with the main mode of about 45–50 mol. % Mg# of Cr-spinel, enclosed within clinopyroxene varies generally in range 20–40 mol. %, while those of rare Cr-spinel grains enclosed in unaltered olivine do not exceed 20 mol. % with a single grain with Mg# 31 mol. % (Figure 12A–C). In contrast, Cr-spinel hosted by altered silicates show very similar modes of Mg# (12–17 mol. %) and it is clearly seen that both in replaced plagioclase and clinopyroxene Cr-spinel loses Mg content with respect to grains, settled in fresh silicates. In a perspective of the coming discussion, it is important that despite similar main Mg# modes of Cr-spinel from replaced silicates, Cr-spinel from replaced plagioclase still appears to have a significant population with Mg# values higher than 20, while it is not the case for Cr-spinel from replaced clinopyroxene (Figure 12D,E). Figure 12. Histograms, representing distribution of Mg# values in Cr-spinel enclosed in different kinds of silicates. 4.3. Cr-Spinel: Chemistry 4 3 Cr Spinel: Chemistry Host silicate phases for the histograms (A–F) are specified in the captions of each chart Figure 12. Histograms, representing distribution of Mg# values in Cr-spinel enclosed in diﬀerent kinds of silicates. Host silicate phases for the histograms (A–F) are speciﬁed in the captions of each chart. Figure 12. Histograms, representing distribution of Mg# values in Cr-spinel enclosed in different kinds of silicates. Host silicate phases for the histograms (A–F) are specified in the captions of each chart Figure 12. Histograms, representing distribution of Mg# values in Cr-spinel enclosed in diﬀerent kinds of silicates. Host silicate phases for the histograms (A–F) are speciﬁed in the captions of each chart. These numerically-derived patterns are well-illustrated by the pseudocolored images. The freshest samples, MS24-324.3 and MS24-323.4, exhibit a clear patchy pattern with the most magnesian-rich Cr-spinel being enclosed in the inner parts of plagioclase and less magnesian-rich in clinopyroxene and olivine. The least magnesian Cr-spinel is enclosed by altered clinopyroxene and olivine. (Figure 11A,B) A similar distribution is characteristic for the troctolitic assemblage in sample MR-30, where relatively fresh plagioclase hosts magnesian Cr-spinel, while extensively altered clinopyroxene contains more ferrous varieties (Figure 11C). A different pattern can be seen in the MR-31, where slightly-altered clinopyroxene hosts moderately-magnesian Cr-spinel, whilst coalescent grains of low-Mg# Cr-spinel are associated with sites composed by strongly altered former plagioclase, clinopyroxene and olivine (which actually exhibit aggregates of secondary minerals: chlorite, albite, serpentine) (Figures 8D,E and 11D,E). Although in most cases intense alteration of the silicates tends to smoothen the compositions of enclosed Cr-spinel grains (Figure 12D,E), it is not the case for the strongly altered sample MR-14, in which differences between Cr-spinel within sericite- albite-feldspar (former plagioclase) and chlorite-amphibole (former pyroxene) aggregates are obvious. While former-pyroxene-hosted Cr-spinel is ferrous and complies with the general regularity, former-plagioclase hosts jigsaw-shaped grains, which are still Mg-rich, reaching up to 50 These numerically-derived patterns are well-illustrated by the pseudocolored images. The freshest samples, MS24-324.3 and MS24-323.4, exhibit a clear patchy pattern with the most magnesian-rich Cr-spinel being enclosed in the inner parts of plagioclase and less magnesian-rich in clinopyroxene and olivine. The least magnesian Cr-spinel is enclosed by altered clinopyroxene and olivine. (Figure 11A,B) A similar distribution is characteristic for the troctolitic assemblage in sample MR-30, where relatively fresh plagioclase hosts magnesian Cr-spinel, while extensively altered clinopyroxene contains more ferrous varieties (Figure 11C). 4.3. Cr-Spinel: Chemistry 4 3 Cr Spinel: Chemistry With regard to spatial distribution of the Cr-spinel grains of diﬀerent compositions, we must emphasize that broad variation of Cr-spinel compositions may be observed at a sub-millimeter scale or even within a single intergrowth. BSE mapping of the studied thin sections with subsequent graphical processing (see Samples and methods) reveals patchy compositional distribution within Cr-spinel disseminations (Figure 11), where Cr-spinel composition is clearly dependent on the type of host silicate phase. Minerals 2020, 10, 498 Mi l 2020 10 498 19 of 30 19 f 30 Minerals 2020, 10, 498 19 of 3 Minerals 2020, 10, 498 19 of 3 Figure 11. Colored BSE photos, revealing patchy distribution of Cr-spinel compositions in poorly- altered rocks and their dependence on enclosing silicate (see Methods and data processing for the technique of coloring). (A,B)–patchy pattern in weakly-altered silicate matrix. (C,D,E)–patterns in moderately-altered rocks with more magnesian Cr-spinel associated with fresh silicates. (F)–patchy pattern in strongly altered leucogabbro. Mineral names abbreviations: Pl–plagioclase, Ol-olivine, Cpx–clinopyroxene, Sulf–sulfide, Chl–chlorite, Phl–phlogopite, abbreviations followed by an asterisk () label strongly-altered or fully-replaced silicates. Figure 11. Colored BSE photos, revealing patchy distribution of Cr-spinel compositions in poorly-altered rocks and their dependence on enclosing silicate (see Methods and data processing for the technique of coloring). (A,B)–patchy pattern in weakly-altered silicate matrix. (C–E)–patterns in moderately-altered rocks with more magnesian Cr-spinel associated with fresh silicates. (F)–patchy pattern in strongly altered leucogabbro. Mineral names abbreviations: Pl–plagioclase, Ol-olivine, Cpx–clinopyroxene, Sulf–sulﬁde, Chl–chlorite, Phl–phlogopite, abbreviations followed by an asterisk () label strongly-altered or fully-replaced silicates. Figure 11. Colored BSE photos, revealing patchy distribution of Cr-spinel compositions in poorly- altered rocks and their dependence on enclosing silicate (see Methods and data processing for the technique of coloring). (A,B)–patchy pattern in weakly-altered silicate matrix. (C,D,E)–patterns in moderately-altered rocks with more magnesian Cr-spinel associated with fresh silicates. (F)–patchy pattern in strongly altered leucogabbro. Mineral names abbreviations: Pl–plagioclase, Ol-olivine, Cpx–clinopyroxene, Sulf–sulfide, Chl–chlorite, Phl–phlogopite, abbreviations followed by an asterisk () label strongly-altered or fully-replaced silicates. Figure 11. Colored BSE photos, revealing patchy distribution of Cr-spinel compositions in poorly-altered rocks and their dependence on enclosing silicate (see Methods and data processing for the technique of coloring). (A,B)–patchy pattern in weakly-altered silicate matrix. (C–E)–patterns in moderately-altered rocks with more magnesian Cr-spinel associated with fresh silicates. (F)–patchy pattern in strongly altered leucogabbro. Mineral names abbreviations: Pl–plagioclase, Ol-olivine, Cpx–clinopyroxene, Sulf–sulﬁde, Chl–chlorite, Phl–phlogopite, abbreviations followed by an asterisk () label strongly-altered or fully-replaced silicates. 4.3. Cr-Spinel: Chemistry 4 3 Cr Spinel: Chemistry A diﬀerent pattern can be seen in the MR-31, where slightly-altered clinopyroxene hosts moderately-magnesian Cr-spinel, whilst coalescent grains of low-Mg# Cr-spinel are associated with sites composed by strongly altered former plagioclase, clinopyroxene and olivine (which actually exhibit aggregates of secondary minerals: chlorite, albite, serpentine) (Figures 8D,E and 11D,E). Although in most cases intense alteration of the silicates tends to smoothen the compositions of enclosed Cr-spinel grains (Figure 12D,E), it is not the case for the strongly altered sample MR-14, in which diﬀerences between Cr-spinel within sericite-albite-feldspar (former plagioclase) and chlorite-amphibole (former pyroxene) aggregates are obvious. While former-pyroxene-hosted Cr-spinel is ferrous and complies with the general regularity, former-plagioclase hosts jigsaw-shaped grains, which are still Mg-rich, reaching up to 50 mol. % Mg# (Figures 8F, 11F and 12D). mol. % Mg# (Figures 8F, 11F and 12D). The behavior of trivalent cations in Cr-spinel seems to not be governed by the enclosing phase as strongly as Mg-Fe2+ systematics (Figure 13). Both plagioclase and clinopyroxene enclose Cr-spinel ith affi itie fo diffe e t Fe3+ t e d (Fi u e 13B E) It ee that C i el ho ted by alte ed g g g p g g The behavior of trivalent cations in Cr-spinel seems to not be governed by the enclosing phase as strongly as Mg-Fe2+ systematics (Figure 13). Both plagioclase and clinopyroxene enclose Cr-spinel with aﬃnities for diﬀerent Fe3+ trends (Figure 13B,E). It seems that Cr-spinel hosted by altered silicates 21 of 30 21 of 30 21 of 30 21 of 30 Minerals 2020, 10, 498 Minerals 2020 10 498 have generally higher TiO2 content, than Cr-spinel enclosed in fresh silicates, while TiO2 concentration in Cr-spinel hosted by altered plagioclase is generally lower than in varieties hosted by altered clinopyroxene (Figure 13C,F). However, it should be noted that since there is no sample where a full set of enclosing phases is well represented, these subtle diﬀerences should be treated with care because the statistics can be strongly aﬀected by sample-dependent diﬀerences of trivalent cations systematics (see above). silicates have generally higher TiO2 content, than Cr-spinel enclosed in fresh silicates, while TiO2 concentration in Cr-spinel hosted by altered plagioclase is generally lower than in varieties hosted by altered clinopyroxene (Figure 13C,F). 4.3. Cr-Spinel: Chemistry 4 3 Cr Spinel: Chemistry However, it should be noted that since there is no sample where a full set of enclosing phases is well represented, these subtle differences should be treated with care because the statistics can be strongly affected by sample-dependent differences of trivalent cations systematics (see above). Figure 13. Relationships of trivalent cations and Ti in Cr-spinel hosted by fresh (A–C) and altered (D– F) silicates. On inset (B) numbered are fields from [48], which correspond to: compositions of primitive (quenched) Cr-spinel (1), Cr-spinel in olivine after ~20 years of equilibration (2) and Cr- spinel in contact with basaltic groundmass after ~20 years of equilibration (all analyses were performed on Cr-spinel inclusions in olivine from Kilauea Iki Lava Lake, Hawaii). Figure 13. Relationships of trivalent cations and Ti in Cr-spinel hosted by fresh (A–C) and altered (D–F) silicates. On inset (B) numbered are ﬁelds from [48], which correspond to: compositions of primitive (quenched) Cr-spinel (1), Cr-spinel in olivine after ~20 years of equilibration (2) and Cr-spinel in contact with basaltic groundmass after ~20 years of equilibration (all analyses were performed on Cr-spinel inclusions in olivine from Kilauea Iki Lava Lake, Hawaii). Figure 13. Relationships of trivalent cations and Ti in Cr-spinel hosted by fresh (A–C) and altered (D– F) silicates. On inset (B) numbered are fields from [48], which correspond to: compositions of primitive (quenched) Cr-spinel (1), Cr-spinel in olivine after ~20 years of equilibration (2) and Cr- spinel in contact with basaltic groundmass after ~20 years of equilibration (all analyses were performed on Cr-spinel inclusions in olivine from Kilauea Iki Lava Lake, Hawaii). Figure 13. Relationships of trivalent cations and Ti in Cr-spinel hosted by fresh (A–C) and altered (D–F) silicates. On inset (B) numbered are ﬁelds from [48], which correspond to: compositions of primitive (quenched) Cr-spinel (1), Cr-spinel in olivine after ~20 years of equilibration (2) and Cr-spinel in contact with basaltic groundmass after ~20 years of equilibration (all analyses were performed on Cr-spinel inclusions in olivine from Kilauea Iki Lava Lake, Hawaii). 5. Discussion 5. Discussion 5 1 Cr Spinel Accumulations and Their Compositional Controls: Existing Models 5.1. Cr-Spinel Accumulations and Their Compositional Controls: Existing Models 5 1 Cr Spinel Accumulations and Their Compositional Controls: Existing Models 5.1. Cr-Spinel Accumulations and Their Compositional Controls: Existing Models 5.1. Cr Spinel Accumulations and Their Compositional Controls: Existing Models There are numerous models regarding the origin of Cr-spinel-rich assemblages in mafic- ultramafic complexes. The main concern is that Cr is a minor element even in most primitive melts, which renders the notion of mass precipitation of Cr-spinel during magma evolution difficult to reconcile [1]. However, there are several models which discuss the formation of Cr-spinel under f There are numerous models regarding the origin of Cr-spinel-rich assemblages in maﬁc-ultramaﬁc complexes. The main concern is that Cr is a minor element even in most primitive melts, which renders the notion of mass precipitation of Cr-spinel during magma evolution diﬃcult to reconcile [1]. However, there are several models which discuss the formation of Cr-spinel under speciﬁc circumstances. specific circumstances. For layered and differentiated magmatic complexes, direct settling of the crystals from crystallizing magma [5], magma replenishment and emplacement of a new pulse of magma [49] or crystallization of Cr-spinel at the bottom of the magma chamber [20] were suggested as potential mechanisms of in situ crystallization. Sweeping down density currents and emplacement of crystal slurries [16,22,50,51] were proposed as gravitation-driven mechanisms for the Cr-spinel-rich “reefs” formation. Additionally, silicate-oxide liquid immiscibility was suggested to play a key role in formation of massive chromitites [52,53]. Several models imply specific physical-chemical circumstances which promote rapid and mass Cr spinel precipitation One such model favors a For layered and diﬀerentiated magmatic complexes, direct settling of the crystals from crystallizing magma [5], magma replenishment and emplacement of a new pulse of magma [49] or crystallization of Cr-spinel at the bottom of the magma chamber [20] were suggested as potential mechanisms of in situ crystallization. Sweeping down density currents and emplacement of crystal slurries [16,22,50,51] were proposed as gravitation-driven mechanisms for the Cr-spinel-rich “reefs” formation. Additionally, silicate-oxide liquid immiscibility was suggested to play a key role in formation of massive chromitites [52,53]. Several models imply speciﬁc physical-chemical circumstances which promote rapid and mass Cr-spinel precipitation. One such model favors a decompressional shift of 22 of 30 Minerals 2020, 10, 498 Cr-spinel and olivine liquidus, causing Cr-spinel crystallize alone, without olivine [18,54,55]. Some researchers [27,56,57] have suggested that it was a mixing of primitive magma with partial melts of the wall rocks, which increased the silica content and expanded the ﬁeld of Cr-spinel stability. 5 1 Cr Spinel Accumulations and Their Compositional Controls: Existing Models 5.1. Cr-Spinel Accumulations and Their Compositional Controls: Existing Models In-Situ Crystallization of Cr-Spinel and Enclosing Silicates: Textural Evidence 5 1 Cr Spinel Accumulations and Their Compositional Controls: Existing Models 5.1. Cr-Spinel Accumulations and Their Compositional Controls: Existing Models Models, implying mixing of primitive melts with fractionated residual magma [1,56] or with alkaline and reduced ﬂuids [58,59] were also proposed to be potential candidates for a number of suites. Furthermore, it was supposed that ophiolitic and alpine chromitites could have formed due to the reaction of a reduced ﬂuid with “magmatic liquid” at ~1000–1050 ◦C [60,61] or were produced by reactions between orthopyroxene and alkali-basaltic melt [62]. In support of the key role of late-magmatic process, [63] deemed that percolation and inﬁltration of evolved interstitial melts and ﬂuids through semi-consolidated cumulates may drive element re-distribution, thereby producing Cr-rich “reefs.” This theory was adopted in recent studies of [64], who proposed a similar kind of mechanism for the formation of Merensky reef and experimentally approved by [65]. Finally, there are some views that favor metasomatic, pneumatolytic, or hydrothermal processes as essential contributors to the formation of PGE-bearing chromitites in the Ural-Alaskan-type complexes [66–68], ophiolites [69,70] and Cr-spinel-rich skarns of the Talnakh intrusion (Norilsk ore junction) [71]. Thus, there is a huge variety of diﬀerent processes, which can be involved in formation of Cr-spinel accumulations. Regardless of the process responsible for the primary Cr-spinel mineralization, it is widely accepted that Cr-spinel composition is strongly aﬀected by post-crystallization modiﬁcation. Various processes are supposed to aﬀect chemistry of pre-cursor Cr-spinel. 1. Examples of Kilauea Iki and Makaopuhi lava show that even several years are suﬃcient for Cr-spinel to equilibrate with an evolving melt [48,72]. Cr-spinel entrapped within olivine represents initial compositions only in quenched scoria of the lava lakes, while in rocks sampled from other locations, Cr-spinel grains are systematically higher in Fe2+ Fe3+ and Ti values and lower in Mg, Al, and Cr [48]. 2. It is commonly accepted that processes of post-cumulus modiﬁcation of Cr-spinel occur in diﬀerentiated intrusions. The major mechanisms include both diﬀusional equilibration of Cr-spinel with interstitial melts in ‘mesocumulate’ and ‘orthocumulate’ environments [73,74] and cation interchange with the host minerals [75,76]. 3. A less likely possibility is post-magmatic, metamorphic, and hydrothermal alteration of Cr-spinel. Recent studies, however, provided clear evidence that even low-grade metamorphism may dramatically aﬀect compositions of Cr-spinel, shifting it towards more ferrous, ferric, and Zn-rich compositions [77–80]. In addition, extensive re-deposition of Cr-spinel is thought to occur during various hydrothermal and metasomatic processes [12,67–70], while experimental studies advocate signiﬁcant mobility of Cr in ﬂuids [81,82]. 5.2. 5.2. In-Situ Crystallization of Cr-Spinel and Enclosing Silicates: Textural Evidence Those would have acted as channeled passages for primitive impregnating melts, which precipitated abundant Cr-spinel due to a reaction with more evolved surrounding melts [1,56], and in this scenario, the existence of such passages would have been reﬂected in textures of silicate matrix as well. However, it actually seems like Cr-spinel textures and relationships between silicate phases weakly correlate with each other (Figure 6A–C). Instead, the observed textural oddities might be interpreted from the following perspectives: 1. Cr-spinel mineralization formed during of overprinting of already formed silicate rocks by later melts/ﬂuids; 1. Cr-spinel mineralization formed during of overprinting of already formed silicate rocks by later melts/ﬂuids; 2. Cr-spinel grains crystallized largely before the enclosing silicates but did not experience signiﬁcant displacement prior to solidiﬁcation of the silicate matrix. 2. Cr-spinel grains crystallized largely before the enclosing silicates but did not experience signiﬁcant displacement prior to solidiﬁcation of the silicate matrix. The ﬁrst scenario favors a metamorphic or metasomatic origin for either Cr-spinel mineralization or the entire rocks themselves. Even though post-magmatic/ metamorphic/ metasomatic processes were ascribed to be signiﬁcant contributors to the formation of PGE-Cr-spinel-rich rocks in the contact zones of the Norilsk-type intrusions [38,41,71,84], there is still a strong conviction that Cr-spinel mineralization and its silicate matrix are initially magmatic in origin. Thus, it appears that crystallization of Cr-spinel and enclosing silicates occurred under speciﬁc physical conditions, in which a solid phase upon crystallization became immobile until the framework of the rock solidiﬁed (e.g., viscous magma). Similar constraints are provided by ring-like (spherical in 3D [40]) alignment of Cr-spinel grains, which is a common feature in several of our studied rock samples (Figures 6F and 7). Such alignments have already been documented in Cr-spinel-rich rocks from diﬀerent Norilsk-type intrusions [32,38,40] and assigned to foaming, which was caused by volatile exsolution from a ﬂuidized magmatic mush [40]. Our observations show that ring-alignment of Cr-spinel grains has no relation to former amygdales or “bubbles” that were ultimately inﬁlled with hydrothermal minerals. Importantly, the “spheres” can be found inside unaltered silicates, where they are clearly not related to grain boundaries (Figure 7). It could be inferred that this kind of alignment was due to the agglutination of Cr-spinel on growth fronts of the silicates, which were then overgrown. However, these aggregates are essentially sub-spherical in shape and, therefore, could be hardly considered to result from this process. 5.2. In-Situ Crystallization of Cr-Spinel and Enclosing Silicates: Textural Evidence In order to place constraints on the origin of Cr-spinel accumulations, we ﬁrst exploit petrographic and textural features of the studied rocks and Cr-spinel mineralization. Despite the absence of clear-cut contact between the intrusion and Ivakinskaya suite basalts and compositional similarities between dolerites of the intrusion and high-T metamorphic rocks formed after the basalts in the contact areas [36], the ophitic textures of the studied rocks and their resemblance to dolerites (Figure 4B) strongly suggest magmatic crystallization as the initial stage of rocks’ formation. Petrographic and mineralogical heterogeneity of the studied rocks was also highlighted by previous studies as a typical feature of Upper zones of Norilsk-type intrusions [35]. Diﬀerent lithologies occurring even within a single hand sample (Figure 3) has been attributed to magma heterogeneity and immiscibility [32] or to entrapment of xenoliths of previously crystallized lithologies [83]. However, considering smooth outlines of these mingling-like textures (Figure 3) and, in certain cases, gradual transition between diﬀerent lithologies (Figure 5D), we favor the idea of magma heterogeneity and envisage existence of diﬀerent immiscible (or semi-immiscible) magmas. 23 of 30 Minerals 2020, 10, 498 In addition to these petrographic implications, textures of Cr-spinel-rich lithologies provide further insights into crystallization conditions of the rocks. An emphasis is made on features that distinguish the studied Cr-spinel mineralization from typical chromitites of layered intrusions and on speciﬁc alignment of Cr-spinel grains: 1. Heterogeneous and uneven cloud-like distribution of Cr-spinel accumulations (Figures 5A and 6A). 1. Heterogeneous and uneven cloud-like distribution of Cr-spinel accumulations (Figures 5A and 6A). 2. Absence of planar textures and dispersed character of even the most abundant Cr-spinel disseminations (Figure 5A,C). 3. Embayments, trails and chains of Cr-spinel, which cross over the boundaries between enclosing silicates (Figure 6). 4. Ring-like alignment of Cr-spinel grains, which is not controlled by neither present grain boundaries, nor bubbles and amygdales (Figure 7). Irregular and uneven shape of Cr-spinel-rich “clouds” and relatively homogeneous, dispersed scattering of Cr-spinel grains inside these clouds rule out the leading role of gravitational concentration of Cr-spinel in the studied assemblages. It has been suggested that a ﬂuid-rich dynamic or even turbulent environment was responsible for local ﬂuctuations of Cr-spinel abundance in certain parts of the “sulﬁde-poor” suite and prevented Cr-spinel from sinking and forming planar “reefs” [41]. From this perspective, embayments and trails of Cr-spinel could be explained by the existence of high-permeability zones inside crystallizing magma mush. 5.3. Cr-Spinel from Initial Crystallization to Low-T Modiﬁcation: Chemical Controls 5.3. Cr-Spinel from Initial Crystallization to Low-T Modiﬁcation: Chemical Controls Substantial compositional diﬀerences between Cr-spinel grains enclosed in diﬀerent silicates are a universal feature of intrusive rocks and are largely attributed to an extensive post-crystallization modiﬁcation [48,72–76,85]. In the entire study, the principle features to disentangle are: 1. Diﬀerent compositions of Cr-spinel enclosed in diﬀerent unaltered silicates (Figures 11A,B and 12). 2. Changing of Cr-spinel composition in altered or pseudomorph replaced silicates (Figures 8D,E, 11C E d 12) 1. Diﬀerent compositions of Cr-spinel enclosed in diﬀerent unaltered silicates (Figures 11A,B and 12). 2. Changing of Cr-spinel composition in altered or pseudomorph replaced silicates (Figures 8D,E, 11C–E and 12). 3. Contrasting ways of Cr-spinel evolution within diﬀerent lithologies (Figure 10). Although it would be logical to begin with the factors which control initial compositions of Cr-spinel, it is quite evident that the present ranges of Cr-spinel can hardly represent sensu stricto magmatic compositions [47], so the post-crystallization overprinting is most likely. Therefore, before considering primary features of Cr-spinel, these primary features should be constrained, and we ﬁrst discuss to what extent chemical ranges of Cr-spinel under study can be attributed to overprinting processes. 5.2. In-Situ Crystallization of Cr-Spinel and Enclosing Silicates: Textural Evidence Currently, no feasible mechanism(s) can be proposed to satisfactorily explain origin of these trails, chains and spherical distribution. However, these textures remained undisturbed by common “displacements” (e.g., gravitational sorting and cumulative processes) in crystallizing magmas. Therefore, we suggest that Minerals 2020, 10, 498 24 of 30 24 of 30 crystallization of Cr-spinel and enclosing silicates took place in an environment that was viscous enough to prevent not only gravitational settling of Cr-spinel but also relative displacement of Cr-spinel and silicates during solidiﬁcation. 5.3.2. Sub-Solidus Modiﬁcation Along with chemical diﬀerences in Cr-spinel enclosed within diﬀerent fresh silicates, there is a clear discrepancy between Cr-spinel hosted by fresh silicates and their altered counterparts (Figures 11 and 12). In addition, Cr-spinel-2 (big coalescent grains) seems to occur predominantly within altered sites of rock, or at close proximity to them, and contain less Mg, than Cr-spinel-1 (small rounded isolated grains) (Figure 8D,E). This implies an extensive Cr-spinel alteration driven by post-solidus processes occurring down to relatively low temperatures (stability ﬁeld of ﬁbrous amphiboles, chlorite, serpentine, albite, and sericite). Moreover, gradual transition from Cr-spinel-1 to Cr-spinel-2 (Figures 8D,E and 11E) and other morphological features of Cr-spinel such as symplectite-like Cr-spinel-saponite intergrowth (Figure 8G), fringes of Cr-magnetite overgrowing ferrochromite (Figure 8H) and strong resorption of some Cr-spinel grains settled in chlorite and smectite matrix (Figure 8I) suggest there was a complex interplay of dissolution and re-deposition processes of Cr-spinel during this stage. A large amount of chemical data on Cr-spinel with regard to an enclosing silicate, make it evident that Cr-spinel, enclosed within all altered silicates, has nearly the same main mode of Mg# at ca. 15 mol. % (Figure 11D–F). Therefore, there was a general geochemical process of modiﬁed compositions of earlier Cr-spinel. However, Cr-spinel within the alteration products of plagioclase are to a certain extent diﬀerent from their counterparts hosted by modiﬁed clinopyroxene. Firstly, Cr-spinel enclosed in former clinopyroxene has Mg# at ca. 15 mol. %, while Cr-spinel hosted by sericitized and albitized plagioclase exhibits more extended range of compositions and has higher mean Mg# values (Figure 12D,E). Second, from patterns of totally-modiﬁed sample (MR-14) it is evident that despite full replacement of plagioclase and clinopyroxene by sericite-albite-K-feldspar and amphibole-chlorite assemblages correspondingly, Cr-spinel, enclosed within former plagioclase still has pronouncedly higher Mg# value than its counterpart, enclosed within former clinopyroxene (Figure 11F). Finally, Cr-spinel-2 appears to be more common inside altered clinopyroxene than in altered plagioclase, while jigsaw-edged Cr-spinel grains are found exceptionally within products of plagioclase modiﬁcation (Figure 8F). These facts provide a robust evidence that besides a general character of the modiﬁcation processes, there were certain diﬀerences between modiﬁcation of plagioclase-hosted and clinopyroxene-hosted Cr-spinel grains. Well-described mechanisms of Cr-spinel modiﬁcation during amphibolization and chloritization of maﬁc minerals [77,86] suggest a gain of Fe and loss of Mg is a common. 5.3.1. High-T Cr-Spinel-Silicate Equilibration Strong variance of Cr-spinel chemistry enclosed within diﬀerent silicate minerals might result either from magma evolution (with more primitive Cr-spinel entrapped within earlier silicates and vice versa) or from re-equilibration with the host minerals and residual melt. Textural relationships of the silicates (Figures 5D, 6B and 7E,F) imply that the ﬁrst crystallizing phase was olivine, then plagioclase and ﬁnally–clinopyroxene. If only melt evolution controlled Cr-spinel chemistry, Mg# would be the highest in olivine-hosted Cr-spinel grains, then gradually decrease in plagioclase-hosted, reaching minimum in those hosted in clinopyroxene. However, this scenario is not applicable for the case in this study, because Cr-spinel enclosed in olivine have unexpectedly the lowest Mg# values among Cr-spinel from unaltered silicates (Figures 11 and 12). Thus, preference should be given to re-equilibration of Cr-spinel with enclosing silicates. p g Such mechanism for the Norilsk-type intrusions was previously explained by a model for layered intrusions [46] that applied these diﬀerences to post-cumulus re-equilibration of Cr-spinel. Although textural evidence rules out sensu stricto cumulative origin of the studied rocks, Cr-spinel grains within plagioclase should have less equilibrated compositions because Fe-Mg exchange between plagioclase and Cr-spinel is insigniﬁcant. Meanwhile, Cr-spinel enclosed in clinopyroxene should have been strongly aﬀected by Fe-Mg redistribution. In addition, inclusions in plagioclase are less prone to exchange of these cations than inclusions in maﬁc minerals, so the initial compositions of plagioclase-hosted spinel should better survive. Pseudocolored BSE-images provide suﬃcient support for these considerations. In fresh rocks, Cr-spinel, settled deep within plagioclase crystals, has the highest Mg#, while in clinopyroxene, olivine, and near the phase boundaries, the gain of Fe is notable (Figure 11A–C). In addition to this direct evidence, the studied Cr-spinel compositions presented as Mg# vs Cr# and Fe2+ vs Fe3+ exhibit trends that are very similar to those observed in Kilauea Iki lava lake basalts—an object, where equilibration of Cr-spinel with host silicates and evolved melt was established in real time [48]. More exactly, Cr-spinel within euhedral plagioclase can be compared with primitive Cr-spinel inclusions from Kilauea Iki, while compositions of clinopyroxene- and olivine-hosted grains are similar to Cr-spinel which experienced modiﬁcation [48] (Figure 13B). Minerals 2020, 10, 498 25 of 30 5.3.2. Sub-Solidus Modiﬁcation They also indicate that Cr-spinel can be replaced and overgrown by less magnesian varieties (ferritchromite down to pure magnetite). It appears that such scenario is well-applicable for the case under investigation. We propose that during post-solidus ﬂuid-driven re-distribution of Mg and Fe, Cr-spinel-1 grains, enclosed within replaced clinopyroxene, gained Fe and experienced recrystallization to form bigger intergrown grains of Cr-spinel-2. Undoubtedly, Cr-spinel, enclosed in plagioclase, also underwent modiﬁcation during this stage, but Fe-Mg exchange was not extensive. We attribute this diﬀerence to the assumption that even altering plagioclase grains were more reluctant to Mg and Fe diﬀusion than clinopyroxene. 5.3.3. Trivalent Cations’ Systematics Pre-Determined by Parental Media 6. Conclusions The investigation of Cr-spinel, which was performed on a set of samples of PGM-Cr-spinel-rich rocks (“sulﬁde-poor” PGE ores) from the upper zone of the Norilsk-1 intrusion, places several genetic constrains on the Cr-spinel and on rocks themselves. Initial crystallization of the rocks took place in a heterogeneous environment, where Cr-spinel-rich lithologies (leucogabbro/troctolites and olivine-rich ophitic gabbros) crystallized in situ from viscous batches of magmas with diﬀerent composition and redox state. Expansion of Cr-spinel compositional ranges was further promoted by diﬀerent extents of its re-equilibration with enclosing silicates and residual melt and, importantly, by post-solidus modiﬁcation and re-crystallization, which is thought to have lasted down to low temperatures (stability ﬁeld of chlorite, smectite and sericite). General trends with Ti and Fe contents increasing and Mg and Al contents decreasing are in accordance with trends, established for other diﬀerentiated intrusions. However, the revealed ranges of Cr-spinel compositions are unique and are not observed in any other intrusions. The study gives an example of how signiﬁcantly composition of Cr-spinel may change due to various post-crystallization processes and provides further evidence of substantial Cr mobility and Cr-spinel recrystallization during post-magmatic overprinting. Supplementary Materials: The following are available online at http://www.mdpi.com/2075-163X/10/6/498/s1, Table S1. Chemical composition of Cr-spinel grains from the studied samples with regard to enclosing silicates and aﬃnity to PGM-sulﬁde assemblages. Author Contributions: I.F.C., L.M.Z., and A.E.I. elaborated the subject and main idea of the paper, L.M.Z., A.Y.S., and M.P.G. performed sampling, I.F.C., L.M.Z., A.Y.S., N.D.T., A.A., and V.M.C. performed sample preparation and acquired analytical data, I.F.C. processed the acquired data, I.F.C. and T.N.A. prepared the illustrations, I.F.C., A.A., A.E.I., and T.N.A. produced ﬁnal manuscript, V.S.K. provided supervision of the research, administrated the project and acquired funding. All authors have read and agreed to the published version of the manuscript. Funding: The study was conducted under the State Assignment to the V.S. Sobolev Institute of Geology and Mineralogy SB RAS and was supported by the Russian Science Foundation grant 19-17-13013. Sampling and analytics were partly supported by the Russian Foundation for Basic Research grant 16-05-00945a. Acknowledgments: The authors appreciate a great eﬀort of four anonymous reviewers, which recommendations strongly improved the study. 5.3.3. Trivalent Cations’ Systematics Pre-Determined by Parental Media While controls of Mg and Fe2+ in this particular case can be understood, the control on the behavior of trivalent cations is not easily constrained. It appears that the systematics of trivalent cations in Cr-spinel does not depend on post-solidus alteration: Cr-spinel in both altered and unaltered silicates display a full range of trivalent cations’ contents (Figure 13). It follows that the main factor, governing systematics of Fe3+, Cr, and Ti, is the Cr-spinel-bearing lithology (Figure 10). This feature is well demonstrated by the samples from the Medvezhy Ruchey mine and, to a lesser extent, manifested in the samples from the MS-24 core. Moreover, Cr-spinel from diﬀerent lithologies, neighboring each other within the same thin section, exhibit considerable diﬀerence in the behavior of these elements. Titanium, Fe3+, and, to a lesser extent, Cr abundances in Cr-spinel from diﬀerent lithologies are diﬀerent for even the most magnesian Cr-spinel grains and these diﬀerences become more signiﬁcant, ultimately resulting in divergent trends Minerals 2020, 10, 498 26 of 30 26 of 30 (Figure 10). Comparing those chemical features with a kind of the host lithology, we assume that the “normal” chromite–ferritchromite–Cr-magnetite pattern is characteristic of more melanocratic, gabbro-like lithologies, while the chromite-ferritchromite-ulv˝ospinel pattern seems to dominate in leucogabbro/troctolitic lithologies. This is perhaps well-illustrated by the sample MS24-323.4, where leucogabbro and olivine-rich gabbro are in contact within a studied thin section, and Cr-spinel compositions show some kind of divergence on Mg#-Cr2O3, Fe2+-Fe3+, and Fe3+-Ti diagrams (Figure 10). Therefore, the diﬀerences in trivalent cation systematics appear to originate from conditions of initial crystallization, which were therefore contrasting for the leucogabbro/troctolite and olivine-gabbro lithological domains. Assuming that the initial crystallization took place from diﬀerent viscous magmas (see Section 5.2), we deem that magma composition largely pre-determined the trivalent cations’ systematics, while the overprinting processes signiﬁcantly extended ranges of their contents, resulting in the divergent trends. However, attention should be paid to striking diﬀerences in Fe3+/Fe2+ systematics in Cr-spinel from diﬀerent lithologies (Figure 10C,D). Likely, these diﬀerences originated not only from diﬀerent magma compositions but also from diﬀerent redox conditions. Therefore, we can suppose that leucogabbro/troctolite lithological domains were initially crystallized in unusually reduced conditions, while olivine gabbro lithological domains were in relatively oxidized circumstances, resembling those for the lower zones of the Norilsk-type intrusions (Figure 9F). References 1. Campbell, I.H.; Murck, B.W. Petrology of the G and H Chromitite Zones in the Mountain View Area of the Stillwater Complex, Montana. J. Petrol. 1993, 34, 291–316. [CrossRef] 2. Eales, H.; Cawthorn, R. The bushveld complex. In Developments in Petrology; Elsevier: Amsterdam, The Netherlands, 1996; Volume 15, pp. 181–229. 3. McCallum, I.S. The Stillwater Complex. In Developments in Petrology; Cawthorn, R.G., Ed.; Elsevier: Amsterdam, The Netherlands, 1996; Volume 15, pp. 441–483. 4. Mungall, J.E.; Naldrett, A.J. Ore deposits of the platinum-group elements. 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World-Class PGE-Cu-Ni Talnakh Deposit: New Data on the Structure and Unique Mineralization of the South-Western Branch. Minerals 2018, 8, 124. [CrossRef] Structure and Unique Mineralization of the South-Western Branch. Minerals 2018, 8, 124. [CrossRef] 8. Naldrett, A.J. Magmatic Sulﬁde Deposits: Geology, Geochemistry and Exploration; Springer Science & Business Media: Berlin/Heidelberg, Germany, 2013. 9. Sluzhenikin, S.F. Platinum-Copper-Nickel and Platinum Ores of Norilsk Region and Their Ore Minera Russ. J. Gen. Chem. 2011, 81, 1288–1301. [CrossRef] 10. Tolstykh, N.; Krivolutskaya, N.; Safonova, M.; Shapovalova, I.Y.; Zhitova, L.; Abersteiner, A. Unique Cu-rich sulphide ores of the Southern-2 orebody in the Talnakh Intrusion, Noril’sk area (Russia): Geochemistry, mineralogy and conditions of crystallization. Ore Geol. Rev. 2020, 122, 103525. [CrossRef] 11. Sluzhenikin, S.F.; Distler, V.V.; Grigoryeva, A.B. Sulﬁde-poor platinum ores of the Noril’sk region—Perspective sources of noble metals. Arctika Ekol. Ekon. 2016, 24, 32–45. 12. Ryabov, V.V.; Tsymbalist, V.G.; Yakobi, I.Y. Concentration of chromium and platinum-group elements in the roofs of Noril’sk-type layered intrusions. Dokl. Akad. Nauk Sssr 1982, 226, 466–469. 13. Sluzhenikin, S.; Distler, V.; Dyuzhikov, O.; Kravtsov, V.; Kunilov, V.; Laputina, I.; Turovtsev, D. Conﬂicts of Interest: The authors have no conﬂicts or other issues to declare. Conﬂicts of Interest: The authors have no conﬂicts or other issues to declare. 6. Conclusions We also thank many people who assisted this study: Maya Kamenetsky and Nadezhda Belkina (sample preparation); Elena Nigmatullina, Victoria Danilovskaya, Nikolay Karmanov, Mikhail Khlestov, and Karsten Goemann (EPMA assistance); and Andrey Lavrenchuk and Anastasiya Iskrina (who helped to optimize data processing in MS Excel and Adobe Photoshop respectively). 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https://openalex.org/W1483434081	https://surface.syr.edu/cgi/viewcontent.cgi?article=1047&context=istpub	English	null	Semantic Analysis for Monitoring Insider Threats	Lecture notes in computer science	2,004	cc-by	4,316	Semantic Analysis for Monitoring Insider Threats Semantic Analysis for Monitoring Insider Threats Follow this and additional works at: https://surface.syr.edu/istpub Part of the Library and Information Science Commons Part of the Library and Information Science Commons Syracuse University Syracuse University SURFACE at Syracuse University SURFACE at Syracuse University School of Information Studies - Faculty Scholarship School of Information Studies (iSchool) Recommended Citation Recommended Citation Symonenko, Svetlana; Liddy, Elizabeth D.; Yilmazel, Ozgur; Del Zoppo, Robert; and Brown, Eric, "Semantic Analysis for Monitoring Insider Threats" (2004). School of Information Studies - Faculty Scholarship. 59. https://surface.syr.edu/istpub/59 This Article is brought to you for free and open access by the School of Information Studies (iSchool) at SURFACE at Syracuse University. It has been accepted for inclusion in School of Information Studies - Faculty Scholarship by an authorized administrator of SURFACE at Syracuse University. For more information, please contact surface@syr.edu. Keywords Keywords intelligence community, IC, security, social network analysis, role-based access monitoring, insiders' communications, risk assessor, Natural Language Processing Center, NLP Description/Abstract Description/Abstract Malicious insiders’ difficult-to-detect activities pose serious threats to the intelligence community (IC) when these activities go undetected. A novel approach that integrates the results of social network analysis, role-based access monitoring, and semantic analysis of insiders ’ communications as evidence for evaluation by a risk assessor is being tested on an IC simulation. A semantic analysis, by our proven Natural Language Processing (NLP) system, of the insider’s text-based communications produces conceptual representations that are clustered and compared on the expected vs. observed scope. The determined risk level produces an input to a risk analysis algorithm that is merged with outputs from the system’s social network analysis and role-based monitoring modules. Disciplines Disciplines Library and Information Science Additional authors listed in paper. Svetlana Symonenko1, Elizabeth D. Liddy1, Ozgur Yilmazel1, Robert Del Zoppo2, Eric Brown2, Matt Downey2 1Center for Natural Language Processing, School of Information Studies, Syracuse University, Syracuse NY 13444, +1 315.443.5484 {ssymonen,liddy,oyilmaz}@mailbox.syr.edu 1Center for Natural Language Processing, School of Information Studies, Syracuse University, Syracuse NY 13444, +1 315.443.5484 {ssymonen,liddy,oyilmaz}@mailbox.syr.edu 2Information Technologies Center, Syracuse Research Corporation 6225 Running Ridge Road, North Syracuse NY 13212 +1 315.452.8000 {delzoppo,brown,downey}@syrres.com Abstract Malicious insiders’ difficult-to-detect activities pose serious threats to the intelligence community (IC) when these activities go undetected. A novel approach that integrates the results of social network analysis, role-based access monitoring, and semantic analysis of insiders’ communications as evidence for evaluation by a risk assessor is being tested on an IC simulation. A semantic analysis, by our proven Natural Language Processing (NLP) system, of the insider’s text-based communications produces conceptual representations that are clustered and compared on the expected vs. observed scope. The determined risk level produces an input to a risk analysis algorithm that is merged with outputs from the system’s social network analysis and role-based monitoring modules. Creative Commons License Creative Commons License This work is licensed under a Creative Commons Attribution 3.0 License. This article is available at SURFACE at Syracuse University: https://surface.syr.edu/istpub/59 Semantic Analysis for Monitoring Insider Threats 1 1 1 Svetlana Symonenko1, Elizabeth D. Liddy1, Ozgur Yilmazel1, Robert Del Zoppo2, Eric Brown2, Matt Downey2 Operational Scenario Intelligence analysts operate within a mission-based context, focused mainly on specific topics of interest (TOIs) and geo-political areas of interest (AOIs) that they are assigned. The role the analyst plays dictates the TOI/AOI, organizational relationships, communication patterns, intelligence products and information systems needed, and the intelligence work products created, thereby the need for monitoring Context, Role, and Semantics. The demonstration scenario we will be testing within is based on an organizational network of analysts working in various groups. Our scenario is based on a fictitious government agency with fictitious information targets. However, our SMEs will ensure that the scenario will be representative of the information assurance problem of malicious insider threats in the U.S. Intelligence Community. Introduction Natural Language Processing (NLP) has proven successful in a range of applications of significance to the intelligence community (IC). Most of these applications support the IC’s need for improved representation of, and access to, large amounts of textual information for tasks such as information retrieval, question-answering, cross-language information retrieval, cross-document summarization, and information extraction. In the research we are herein reporting, we adapt our proven NLP capabilities to provide fine-grained content representation and analysis of text-based communications in a novel application – detecting insider threats via semantic analysis of text-based artifacts either produced by or accessed by IC analysts. Our full insider threat solution integrates evidence from social network analysis and role- based access monitoring of system usage with our semantic analysis of insiders’ cyber communications as inputs to a risk analysis algorithm that assesses these inputs and produces an indication of the potential risk of an insider threat within the organization. This research is being conducted as part of ARDA’s Information Assurance for the Intelligence Community Program, and therefore, it is being modeled on and tested in a simulated IC malicious insider threat scenario developed by Subject Matter Experts (SMEs) on our project with years of experience with the community. A malicious insider is someone who, while a valid user of IC systems, decides to perform unauthorized malicious acts, including sharing of information with groups unfriendly to the US. The goal of this ARDA program is to develop solutions for efficiently detecting and monitoring such unwanted behaviors. While the IC is the main focus of our development efforts, the banking and securities industries have the same need to recognize potential insider threats and will be able to utilize this model and methodology for distinguishing between normal and abnormal cyber behavior of their employees. To accomplish our goal, we are developing and testing an Insider Threat Model that integrates Context, Role, and Semantics, here defined as: Context – the tasks the analyst is assigned; Role – the analyst’s assigned job functions within that context, and; Semantics – the content of the information produced or accessed by the analyst. Given these inputs, the model will detect levels of insider threat risk by comparing expected cyber behaviors against observed cyber behaviors. This paper reports on the Semantic Analysis module. Approach The insider threat scenario described above presents the following problem amenable to the semantic analysis module of our system. Given the set of textual data available electronically and ranging in genre from news articles to analyst reports, official documents, email messages, query logs, and so on, the system should be able to identify the TOI / AOI mentioned in the documents and compare them against the expected TOI and AOI. In other words, the task is to detect an outlier, i.e. a TOI / AOI, which is significantly different from the expected ones. Our approach is based on a number of assumptions developed in the course of our talks with members of the IC. First, we assume that analysts are assigned relatively long-term tasks and dedicate most of their work time to it.1 Next, we assume, there may be more than one analyst who is assigned the same main topic and that each would then work on particular subtopics. Finally, we assume that the analysts work with documents and engage in email communication on topics related to their assigned task. We can also expect that the analysts working on subtopics of the same main topic would access different, but topically related, documents. Given the above assumptions, we can expect that clustering documents that the analysts work with would yield a larger cluster(s) containing on-topic documents, and a few smaller clusters of off-topic documents. Further, we can train a clustering model on the dataset containing mainly on-topic documents. The topical description of a cluster will be generated from the n most frequent concepts in the clustered documents. Then, we can assess whether the documents accessed or created by the analyst fall within the scope of on-topic cluster(s) or whether they are significantly far from such topical cluster(s). We will experimentally compare and select from the range of available hierarchical clustering methods2 the most appropriate one for our task of developing a model of expected TOI/AOI for the documents that the analyst accesses/generates. Then, each new document will be assessed in terms of its semantic distance from the existing cluster(s). As a result, the document will be merged with on-topic cluster(s), or existing off-topic cluster(s), or will start a new off-topic cluster. It is important to note that not every off-topic cluster should raise an alert flag. First, clustering algorithms can generate sporadic clusters. 1 This assumption does not cover analysts working on time-critical requests that need to be turned in within a couple of hours. Such analysts are expected to change topics quickly. A different TOI / AOI model would be needed for them. Related work To the best of our knowledge, there is no account of the integrated social context, role, and semantics approach that we are taking. While some projects have addressed these dimensions individually, most research appears to be focused on cyber threat and cyber security. When semantics has been utilized, it is applied to describe the role-based access policy of an organization (RAND, 1999; Upadhyaya et al., 2001). In related work, a research project by Raskin et al. (2002) aims to use a natural language-based ontology to scan texts for indicators of possible intellectual property leakage. The 2003 NSF / NIJ Symposium on Intelligence and Security Informatics marked an increased interest in the research community in applying linguistic analysis to the problems of cyber security. Stolfo et al. (2003) mined subject lines of email messages for patterns typical for particular user groups (e.g. software developers vs. the legal department). Patman & Thompson (2003) reported on the implementation of a personal name disambiguation module that utilizes knowledge of cultural contexts. Burgoon et al. (2003) looked for linguistic indicators of deception in interview transcripts. Zhou et al. (2003) conducted a longitudinal study of linguistic cues of deception in email messages. Zheng et al. (2003) compared machine-learning algorithms on the task of recognizing the authorship of email messages, and evaluated the efficiency of using different semantic features such as style markers, structural features, and content-specific features. Sreenath et al. (2003) employed latent semantic analysis to reconstruct users’ original queries from their online browsing paths and applied this technique to detecting malicious (e.g. terrorist) trends. The novelty of our approach is in both the problem and the scope. The patterns that we are seeking to detect may not look malicious. In fact, they may look perfectly legitimate but, when considering the user’s role and context of their assignment (i.e. topics and geo- political focus), they indicate that the insider’s activities are out of range of “expected behavior”. Our task is to assess the semantic distance between the content of the documents that the insider is currently accessing and creating and the expected content, given the analyst’s assigned TOI and AOI. For this purpose, concept-based semantic analysis will be applied to the wide range of textual documents that analysts use and produce while working on a task, e.g. documents provided by other organizations or from internal collections, email communication, or database or Internet query logs. 3 Known to negatively affect computational effectiveness of clustering algorithms (Hotho et al., 2003) 2 See Ward, 1963; Zhao & Karypis, 2002 for details on methods. 3 2 See Ward, 1963; Zhao & Karypis, 2002 for details on methods. 3 Approach Also, realistically, analysts cannot be expected to work on their assigned topic 100% of their time. Finally, the emergent topic can be a legitimate development in the analyst’s work. Therefore, the system will check the semantic distance between the off-topic cluster and the on-topic cluster(s), and also the size of the off-topic cluster. When both parameters exceed thresholds, the semantic analysis module emits an indicator to the insider threat monitoring application. A human (e.g. an information assurance engineer) can then review the indicators for their relevancy. Documents assessed as being on-topic will be added to the model; thus, adjusting the semantics of the expected TOI/AOI and the on-topic cluster parameters. We intend to boost the efficacy of the clustering methods by use of multiple ontologies, which will enable mapping of individual terms and locations to appropriate categories and, thus, will reduce the high dimensionality of data3 and, more importantly, contribute to the conceptual coverage of the resulting clusters. 5 Italicizing indicates data amenable to semantic analysis 4 http://cns.miis.edu/ 5 Resources Data Data One of the challenges of this project is to develop a test collection of questions / topics and related documents for training and testing to adequately represent the spectrum of textual data accessed / generated during the analyst’s work processes. Such data collection is bound to be diverse in both, format (such as txt, html, doc, tabular) and genre (e.g. formal documents, analytic reports, online news stories, email messages). Being aware of the constraints on data procurement from operational settings, we gathered resources that would best fit the context of the IC. The resulting collection, discussed in greater detail below, is an example of collaboration and sharing among different research teams involved in ARDA and DARPA funded projects. The analysts’ tasks were modeled on scenarios developed by Center for Non-Proliferation Studies (CNS)4 experts for use in ARDA’s AQUAINT (Advanced Question and Answering for Intelligence) Program. We also make use of the scenario-based questions generated at the 2003 ARDA-NRRC workshop on Scenario-Based Question-Answering (Liddy, 2003). A scenario consists of a question (i.e. particular task that the analyst is charged with) and a set of sub-questions, thus, modeling the analyst’s decomposition of the main question into a set of contextually related sub-topics and posing them iteratively against the appropriate information resources (Figure 1). ( g ) Main Question/Topic Despite having complete access, to this day UN inspections have been unable to find any biological weapons, or remnants thereof, in Iraq. Why has it proven so difficult to discover hard information about Iraq’s biological weapons program and what are the implications of these difficulties for the international biological arms control regime? Question Decomposition / Subtopics (selected from 15) 1. What does it take to determine/find signatures of a biological weapons program? 2. What are UN capabilities and procedures for inspection? 3. Are they looking for the right thing? 4. Where are they likely to be? 5. Signature of the inspections: how predictable were they? Did they lend themselves to deception? 6. What is the Iraqi denial and deception capability? How much effort is involved in hiding it? What evidence is available? Sources to Answer the Question(s)5 • Arms control agreements • UN databases, guidelines, and procedures • UNSCOM report • CNS data for weapons info • Office of Technology Assessment reports • Foreign press reports • General search • Talk to inspectors • Geospatial sources Figure 1. Resources Sample AQUAINT scenario 4 http://cns.miis.edu/ 5 Italicizing indicates data amenable to semantic analysis Main Question/Topic Despite having complete access, to this day UN inspections have been unable to find any biological weapons, or remnants thereof, in Iraq. Why has it proven so difficult to discover hard information about Iraq’s biological weapons program and what are the implications of these difficulties for the international biological arms control regime? Question Decomposition / Subtopics (selected from 15) 1. What does it take to determine/find signatures of a biological weapons program? 2. What are UN capabilities and procedures for inspection? 3. Are they looking for the right thing? 4. Where are they likely to be? 5. Signature of the inspections: how predictable were they? Did they lend themselves to deception? 6. What is the Iraqi denial and deception capability? How much effort is involved in hiding it? What evidence is available? Sources to Answer the Question(s)5 • Arms control agreements • UN databases, guidelines, and procedures • UNSCOM report • CNS data for weapons info • Office of Technology Assessment reports • Foreign press reports • General search • Talk to inspectors • Geospatial sources Figure 1. Sample AQUAINT scenario 4 http://cns.miis.edu/ 5 Italicizing indicates data amenable to semantic analysis 8 National Geographic Intelligence Agency; former name is National Imagery and Mapping Agency 9www.alexandria.ucsb.edu/~lhill/FeatureTypes Figure 2. Example of term-mapping Figure 2. Example of term-mapping For the conceptual organization of AOI, we will utilize the SPAWAR Gazetteer, also developed under the AQUAINT Program. It combines resources of four publicly available gazetteers (NGA8; USGS; CIA World Factbook; DARPA’s TIPSTER Program), and is dynamically updated. The gazetteer uses a single comprehensive categorization scheme based on the Alexandria Digital Library thesaurus9. When tested on text annotation tasks, it was shown to cover 90% of geographic references in texts. Main Question/Topic From our conversations with intelligence analysts, we have learned that these scenarios fairly accurately represent actual analysts’ tasks. Another benefit of the AQUAINT scenarios is that they were developed under the premise that much of the needed information can be found in the CNS collection, in particular, in: datasets on nuclear weapons and missile proliferation; country profiles for North Korea and China; NIS Nuclear Profiles; a Nuclear Trafficking Database; the news archive on CBW / WMD. The resources are of various genres: news (including translations); analytic reports by various agencies, and; treaties. Our data set also includes a collection of online news topically related to the CNS data, compiled by the AQUAINT team at SUNY-Albany6. 7 http://ontolingua.stanford.edu. 8 6 http://www.hitiqa.albany.edu/index.html 7 Ontology I h In the semantic analysis approach, rather than using the literal words in texts, we develop algorithms to augment the document terms selected for clustering with appropriate concepts. Given that the focus will be on TOI and AOI, we needed an ontology for the nonproliferation domain, as well as a gazetteer. Through collaboration with ISI / SAIC / Ontolingua, we obtained access to an ontology of CNS concepts7, which also includes topics from non-CNS knowledge bases on terrorism. We will adjust this ontology to incorporate our currently employed taxonomy. Figure 2 illustrates the current semantic mapping of the terms sarin and mustard gas to a type cweap (chemical weapon) and its augmentation with CNS topics (WMD, weapons). cbw092502 .. the regime has accumulated substantial stockpiles of deadly liquid agents such as mustard gas, and ominous nerve agents, such as sarin and VX, the report said. entity = mustard_gas\|NN type = cweap Cat = WMD Top Cat = weapon entity = sarin\|NN type = cweap Cat = WMD Top Cat = weapon Fi 2 E l f t i cbw092502 .. the regime has accumulated substantial stockpiles of deadly liquid agents such as mustard gas, and ominous nerve agents, such as sarin and VX, the report said. 9www.alexandria.ucsb.edu/~lhill/FeatureTypes 8 National Geographic Intelligence Agency; former name 9 10 Extracted entities include nouns (missile) and noun phrases (biological warhead), as well as named entities, which are proper names (China, Scud). Preliminary Example To exemplify our methods, consider the following example that we developed in order to familiarize ourselves with the data collection we were assembling. We selected a small set (five) of documents from the North Korea collection compiled by CNS. All documents were of a similar genre, namely, chronology of proliferation events. Two documents came from the Missile subset, and three documents came from the Chemical subset. We ran the documents through CNLP’s text processor and analyzed the extracted entities and named entities10. The analysis led to a few important observations. First, selecting only entities to represent the conceptual scope of the document reduces it by about 3/4th, and further limiting to the named entities cut it to about 1/10th of its original size (Table 1): Tokens Doc92 Doc95 Doc47_96 Doc97_00 Doc01_02 Words 5356 4102 2736 1787 690 Entities + Named Entities 1399 1136 748 462 181 Named Entities only 420 405 252 161 81 , p p ( ) 11 Such as: launcher, gun, nuclear, Nodong (a proper name for the nuclear missile) 12 12 For Doc95, the TOI frequency would be boosted by 31.5% Table 1. Count of document terms Second, using a gazetteer to resolve individual location names to their upper level geographic concept appears beneficial for identifying important AOIs. For instance, out of 39 Russia-related place names in Doc92, 23 were literally Russia[n]. The rest (one third) constituted city names (Moscow – 11, Miass – 4) and a region name (Ural). Another example: of 13 mentions of South Korea, 8 (two thirds) referred to Seoul. Assuming that locations are almost exclusively proper names, we estimated AOI frequencies against the named entities only. Table 2 shows prevalent AOIs (in %) for the two Missile documents. AOI Doc92 Doc95 North Korea 29.05 19.01 South Korea 3.1 4.44 United States 4.29 4.94 Syria 6.19 0 Iran 8.57 4.94 Russia 9.29 .25 10 Extracted entities include nouns (missile) and noun phrases (biological warhead), as well as named entities, which are proper names (China, Scud). 11 Such as: launcher, gun, nuclear, Nodong (a proper name for the nuclear missile) 12 For Doc95 the TOI frequency would be boosted by 31 5% 2 For Doc95, the TOI frequency would be boosted by 31.5% 14 For example, in query logs, every word is assumed to be on topic, which is not true for a news story where most content-indicative terms are located in the lead sentence/paragraph. Table 2. AOI frequency for Missile documents Next, we wanted to compare the topicality of Missile vs. Chemical documents. Table 3 shows TOI frequency across all five documents. Obviously, Doc92 and Doc95 focus on the Missile topic, whereas the other three documents mainly discuss Chemical/Biological Weapons. Again, the concept-based approach seems promising. For example, out of 174 Missile-related terms in Doc92, 131 were literal missile[s]. The document also contained 40 mentions of a topically important term, Scud (a ballistic missile); including 23 cases where the term was used just as a proper name. Applying the TOI ontology would group these and other11 terms under the Missile concept, thus, increasing its frequency by 24.7%12. TOI Doc92 Doc95 Doc47_96 Doc97_00 Doc01_02 Missile 12.44 14.35 3.21 2.6 1.66 Chem/Bio .07 .7 4.95 5.19 6.63 Table 3. TOI frequency for Missile and Chemical documents TOI Doc92 Doc95 Doc47_96 Doc97_00 Doc01_02 Missile 12.44 14.35 3.21 2.6 1.66 Chem/Bio .07 .7 4.95 5.19 6.63 Table 3. TOI frequency for Missile and Chemical documents Acknowledgements g This work is supported by the Advanced Research and Development Activity (ARDA). 13 Compare, for example, the style of email communication (informal, abundant in morphologic and syntactic shortcuts) and official briefing reports. 14 Conclusion This project further extends the idea of combining NLP and machine learning (clustering) techniques to an application in the field of information security. This merging presents a few challenges, as well as potential areas of contribution, to the problem of knowledge acquisition. First, the majority of the prior research focused on a particular genre (news stories, or email messages, or query logs). Our data collection combines various genres, differing in style, syntax, and semantics13. We will, therefore, be enhancing our existing NLP tools to deal with genre specifics at the term extraction, term mapping, and term/concept-weighting stages14. Next, we will further investigate benefits and issues related to an ontology-driven approach to identifying important topical structures in large and stylistically diverse datasets. While this is a nascent project, we believe that the application area, the approach, and the model described herein should be of interest to researchers in the area of insider threats as well as other NLP teams dealing with analogous situations. References Allan, J., Lavrenko, V., Malin, D., & Swan, R. 2000. Detections, Bounds, and Timelines: UMass and TDT-3. http://citeseer.nj.nec.com/455856.html Anderson, R. 1999. Research and Development Initiatives Focused on Preventing, Detecting, and Responding to Insider Misuse of Critical Defense Information Systems: Results of a Three-Day Workshop. f y p http://www.rand.org/publications/CF/CF151/CF151.pdf Berkhin, P. 2002. Survey Of Clustering Data Mining Techniques. http://citeseer.nj.nec.com/berkhin02survey.html Burgoon, J., Blair, J., Qin, T., Nunamaker, J., Jr. Detecting Deception Through Linguistic Analysis. Presentation at 2003 NSF/NIJ Symposium on Intelligence and Security Informatics. Hotho, A., Staab, S. & Stumme, G. 2003. Text clustering based on background knowledge. University of Karlsruhe, Institute AIFB. http://citeseer.nj.nec.com/hotho03text.html Intelligence and Security Informatics: First NSF/NIJ Symposium, Tucson, AZ, USA, June 2-3, 2003. Proceedings. (Eds.). Chen, H., Miranda, R., Zeng, D.D., Demchak, C., Schroeder, J., & Madhusudan, T. Volume 2665 / 2003. Springer-Verlag Heidelberg. Lidd E D 2003 S i B d Q i A S AQUAINT 2003 PI M ti Intelligence and Security Informatics: First NSF/NIJ Symposium, Tucson, AZ, USA, June 2-3, 2003. Proceedings. (Eds.). Chen, H., Miranda, R., Zeng, D.D., Demchak, C., Schroeder, J., & Madhusudan, T. Volume 2665 / 2003. Springer-Verlag Heidelberg. Liddy, E.D. 2003. Scenario Based Question-Answer Systems. AQUAINT 2003 PI Meeting. http://cnlp.org/presentations/present.asp?show=conference Liddy, E.D. 2003. Scenario Based Question-Answer Systems. AQUAINT 2003 PI Meeting. http://cnlp.org/presentations/present.asp?show=conference lp.org/presentations/present.asp?show=conference Patman, F. & Thompson, P. 2003. A New Frontier in Text Mining. In Intelligence and Security Informatics, p. 27-38. Raskin, V., Hempelmann, C., Triezenberg, K., & Nirenburg, S. 2001. Ontology in Information Security: a Useful Theoretical Foundation and Methodological Tool. Proceedings of the 2001 Workshop on New Security Paradigms, p.53-59. Sreenath, D.V., Grosky, W.I., & Fotouhi, F. 2003. Emergent Semantics from Users' Browsing Paths. In Intelligence and Security Informatics, p. 355-357. Steinbach, M., Karypis, G., & Kumar, V. 2000. A comparison of document clustering techniques. http://citeseer.nj.nec.com/steinbach00comparison.html Stolfo, S., Hershkop, S., Wang, K., Nimeskern, O., & Hu, C. 2003. Behavior Profiling of Email. Presentation at 2003 NSF/NIJ Symposium on Intelligence and Security Informatics. Upadhyaya, S., Chinchani, R., Kwiat. K. 2001. An Analytical Framework for Reasoning About Intrusions. 20th IEEE Symposium on Reliable Distributed Systems, p. 99-108. Ward, J. H., Jr. 1963. Hierarchical grouping to optimize an objective function. Journal of the American Statistical Association, vol. 58, p.236-244. Zhao, Y. & Karypis, G. 2002. Evaluation of Hierarchical Clustering Algorithms for Document Datasets. http://citeseer.nj.nec.com/zhao02evaluation.html p j Zheng, R., Yi, O., Zan, H., & Hsinchun, C. 2003. Authorship Analysis in Cybercrime Investigation. References In Intelligence and Security Informatics, p. 59-73. Zhou, L., Burgoon, J.K., & Twitchell, D.P. 2003. A Longitudinal Analysis of Language Behavior of Deception in E-mail. In Intelligence and Security Informatics, p. 102-110.
https://openalex.org/W2153597650	https://europepmc.org/articles/pmc3201920?pdf=render	English	null	Optimal Timing of Insecticide Fogging to Minimize Dengue Cases: Modeling Dengue Transmission among Various Seasonalities and Transmission Intensities	PLoS neglected tropical diseases	2,011	cc-by	5,923	Mika Oki, Toshihiko Sunahara, Masahiro Hashizume, Taro Yamamoto International Health, Institute of Tropical Medicine, The Global Center of Excellence, Nagasaki University, Nagasaki, Japan epartment of International Health, Institute of Tropical Medicine, The Global Center of Excellence, Nagasaki University, Nagasaki, Jap Abstract This is an open-access article distributed under the terms of the Creative Commons Attribution License, use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology, and the Global Center for Excellence Program of the Ministry of Education, Culture, Sports, Science and Technology. No funding bodies had any role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. E-mail: mrs_southern_wind@yahoo.co.jp www.plosntds.org Optimal Timing of Insecticide Fogging to Minimize Dengue Cases: Modeling Dengue Transmission among Various Seasonalities and Transmission Intensities Mika Oki, Toshihiko Sunahara, Masahiro Hashizume, Taro Yamamoto Department of International Health, Institute of Tropical Medicine, The Global Center of Excellence, Nagasaki University, Nagasaki, Japan Mika Oki, Toshihiko Sunahara, Masahiro Hashizume, Taro Yamamoto Abstract Background: Dengue infection is endemic in many regions throughout the world. While insecticide fogging targeting the vector mosquito Aedes aegypti is a major control measure against dengue epidemics, the impact of this method remains controversial. A previous mathematical simulation study indicated that insecticide fogging minimized cases when conducted soon after peak disease prevalence, although the impact was minimal, possibly because seasonality and population immunity were not considered. Periodic outbreak patterns are also highly influenced by seasonal climatic conditions. Thus, these factors are important considerations when assessing the effect of vector control against dengue. We used mathematical simulations to identify the appropriate timing of insecticide fogging, considering seasonal change of vector populations, and to evaluate its impact on reducing dengue cases with various levels of transmission intensity. Methodology/Principal Findings: We created the Susceptible-Exposed-Infectious-Recovered (SEIR) model of dengue virus transmission. Mosquito lifespan was assumed to change seasonally and the optimal timing of insecticide fogging to minimize dengue incidence under various lengths of the wet season was investigated. We also assessed whether insecticide fogging was equally effective at higher and lower endemic levels by running simulations over a 500-year period with various transmission intensities to produce an endemic state. In contrast to the previous study, the optimal application of insecticide fogging was between the onset of the wet season and the prevalence peak. Although it has less impact in areas that have higher endemicity and longer wet seasons, insecticide fogging can prevent a considerable number of dengue cases if applied at the optimal time. Conclusions/Significance: The optimal timing of insecticide fogging and its impact on reducing dengue cases were greatly influenced by seasonality and the level of transmission intensity. We suggest that these factors should be considered when planning a control strategy against dengue vectors. Citation: Oki M, Sunahara T, Hashizume M, Yamamoto T (2011) Optimal Timing of Insecticide Fogging to Minimize Dengue Transmission among Various Seasonalities and Transmission Intensities. PLoS Negl Trop Dis 5(10): e1367. doi:10.1371/journal.pntd.0 Editor: Roberto Barrera, Centers for Disease Control and Prevention, Puerto Rico, United States of America Received June 6, 2011; Accepted September 2, 2011; Published October 25, 2011 Copyright: 2011 Oki 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 Oki et al. October 2011 \| Volume 5 \| Issue 10 \| e1367 www.plosntds.org The model We used the structure of the N&R model [10] and partly modified it to: 1) add seasonality and 2) produce the endemic state. Equations are presented below. Host population was divided into Sh (susceptible), Eh (exposed), Ih (infectious) and Rh (recovered). Vector population was also divided into Sv (susceptible), Ev (exposed) and Iv (infectious). Host population dSh dt ~ Nh Tlh {Sh(cvh Iv Nh z 1 Tlh ) ð1Þ dEh dt ~Shcvh Iv Nh {Eh( 1 Tiit z 1 Tlh ) ð2Þ dIh dt ~ Eh Tiit {Ih( 1 Tid z 1 Tlh ) ð3Þ dRh dt ~ Ih T { Rh T ð4Þ dSh dt ~ Nh Tlh {Sh(cvh Iv Nh z 1 Tlh ) ð1Þ ð1Þ detected dengue cases is recommended by the World Health Organization during the early phase of a disease outbreak, and is practiced in many endemic areas [7]. However, it has been suggested that fogging following case detection is not conducted early enough to prevent virus transmission occurring across a wider area [7,8]. dEh dt ~Shcvh Iv Nh {Eh( 1 Tiit z 1 Tlh ) ð2Þ ð2Þ In recent years, ‘‘in-advance’’ treatment has been proposed. Fogging is sometimes conducted very early in, or even before, the rainy season [9], however the rationale for such in-advance treatment has yet to be established. Newton and Reiter (N&R) [10] reported that based on a mathematical simulation, the strongest effect of insecticide fogging in preventing dengue cases is expected when insecticides are applied several days after the prevalence peak; however, this method had little impact on disease prevention, with only 6.8% of the cases prevented. Many other researchers have referred to this study as evidence of the ineffectiveness of insecticide fogging [11–13]. dIh dt ~ Eh Tiit {Ih( 1 Tid z 1 Tlh ) ð3Þ ð3Þ dRh dt ~ Ih Tid { Rh Tlh ð4Þ ð4Þ Vector population dSv dt ~e{Sv chv (IhzIh visit) Nh z 1 Tlv ð5Þ ð5Þ However, the basic assumptions of the N&R model were oversimplified when compared with the real situation in dengue endemic areas. For example, the N&R model did not take into account seasonal fluctuations in climatic conditions, which influence vector population dynamics and viral development within vectors. Author Summary Dengue virus infection is a serious infectious disease transmitted by Aedes mosquitoes in the tropics and sub- tropics. Disease control often involves the use of insecticide fogging against mosquito vectors. However, the effectiveness of this method for reducing dengue cases, in addition to appropriate application procedures, is still debated. The previous mathematical simulation study reported that insecticide fogging reduces dengue cases most effectively when applied soon after the epidemic peak; however, the model did not take into account seasonality and population immunity, which strongly affect the epidemic pattern of dengue infection. Consid- ering these important factors, we used a mathematical simulation model to explore the most effective time for insecticide fogging and to evaluate its impact on reducing dengue cases. Simulations were conducted with various lengths of the wet season and population immunity levels. We found that insecticide fogging substantially reduces dengue cases if conducted at an appropriate time. In contrast to the previously suggested application time during the peak of disease prevalence, the optimal timing is relatively early: between the beginning of the dengue season and the prevalence peak. Optimal Timing of Fogging to Minimize Dengue Cases Optimal Timing of Fogging to Minimize Dengue Cases When assessing the current dengue situation, seasonality and transmission intensity are critical determinants of epidemic patterns that should be taken into consideration when evaluating and optimizing the impact of insecticide fogging. Some studies suggested that the optimal timing and the impact of insecticide fogging might differ from results reported by N&R when also considering seasonality [12,17]. However, the most appropriate time for insecticide fogging to effectively prevent dengue incidence was not definitively provided in these studies. Thus, we aimed to identify the optimal timing for insecticide fogging and its impact on reducing cases of DENV infection by using a mathematical simulation model of dengue transmission dynamics that included various seasonal settings and transmission intensities. www.plosntds.org www.plosntds.org Introduction Tetravalent dengue vaccines are now under development and have the potential to effectively prevent disease [4]; however, these vaccines are not currently approved for clinical use. Tetravalent dengue vaccines are now under development and have the potential to effectively prevent disease [4]; however, these vaccines are not currently approved for clinical use. Dengue virus (DENV) infection is a mosquito-borne viral disease of serious health concern in recent decades. More than two-fifths of the global population is considered to be at risk of dengue infection, principally in the tropics and sub-tropics [1]. Increases in dengue epidemics are likely due to the rapid and broad-ranging migration of people and urbanization, which is accompanied by expanded infestation of vector mosquito: Aedes aegypti [2]. To date, vector control has been the only measure for dengue prevention. In contrast to the substantial progress observed for vaccine development, vector control strategies have shown limited improvement. The major vector control measures conducted in many dengue endemic areas include: 1) fogging ultra-low-volume insecticide particles (insecticide fogging) that target adult mosqui- toes; 2) chemical and biological controls for mosquito larvae in the key containers; and 3) larval source reduction. Among those, insecticide fogging has been commonly implemented, but its impact on reducing dengue cases is still controversial [5,6]. Clinical manifestations of dengue infection range from a mild febrile form (dengue fever: DF) to severe and sometimes to fatal forms (dengue hemorrhagic fever: DHF and dengue shock syndrome: DSS). Although the case fatality rate of DHF/DSS has been declining [3], such severe forms always require intensive care and fluid management under hospitalization. Consequently, a major outbreak represents a serious burden on medical facilities. Aedes aegypti is a highly domesticated species that tends to rest in locations hidden indoors, making it hard for insecticide to reach adult mosquitoes [6]. Appropriate timing for insecticide applica- tion is also under discussion. Fogging in and around the houses of October 2011 \| Volume 5 \| Issue 10 \| e1367 1 October 2011 \| Volume 5 \| Issue 10 \| e1367 Optimal Timing of Fogging to Minimize Dengue Cases The model In addition, the human population was assumed to be completely naı¨ve to DENV, and the magnitude of the outbreak in their simulation was very large, which resulted in 7,651 people out of 10,000 being infected in an outbreak [11]. This phenomenon might be observed in specific situations, like the first dengue outbreak in Easter Island [14], but would not apply to areas where dengue infections are already endemic. dEv dt ~Sv chv (IhzIh visit) Nh {Ev( 1 Teit z 1 Tlv ) ð6Þ ð6Þ dIv dt ~ Ev Teit { Iv Tlv ð7Þ ð7Þ Parameters and parameter values are shown in Table 1. Seasonality. We considered both wet and dry seasons. In the real-life situation, seasonal changes in temperature have a great impact on mosquito survival and viral growth, and rainfall influences the availability of larval habitat of the vector [18–20]. However, to minimize model complexity, we assumed two seasons that affect only the mosquito lifespan (Tlv). The emergence rate of adult mosquitoes (e) was set based on N&R’s assumption [10] that carrying capacity was divided by vector lifespan (4 days). When we added seasonality into the model, we assumed that mosquito Population immunity is also likely to widely vary in endemic regions. For example, 100% of Nicaraguan children at the age of 16 are seropositive for at least one of the DENVs [15], whereas only 6.5% of junior high school children in Singapore have been exposed to these viruses [16]. Although dengue is endemic in both countries, the transmission intensity appears to be much higher in Nicaragua, resulting in higher immunity levels compared with Singapore. October 2011 \| Volume 5 \| Issue 10 \| e1367 2 Optimal Timing of Fogging to Minimize Dengue Cases Table 1. Parameter values for the simulations. The model Parameter Symbol Value Source Host population Nh 10,000 10 Host life span Tlh 600,060 hours (68.5 years) 10 Intrinsic incubation period Tiit 5 days 10 Extrinsic incubation period Teit 10 days 10 Number of mosquitoes per person MPP 2–15 Emerging rate of adult mosquitoes e 5,000–37,500/day * 10 Vector life span Tlv 4 days (wet season) 3 days (dry season) 10 Visiting infectious host Ih_visit 0 in Simulation 1 and 2 0.001 in Simulation 3 and4 20 Host infection duration Tid 3 days 10 Effective contact rate, vector to host cvh 0.75/day 10 Effective contact rate, host to vector chv 0.375/day 10 *5,000 for Simulation 1–3 (MPP = 2); 7,500, 12,500, 20,000 and 37,500 for MPP = 3, 5, 8 and15 in Simulation 4. doi:10.1371/journal.pntd.0001367.t001 produce an endemic state. When we simulated dengue transmission over many years with a single initial introduction of DENV, we often found that the prevalence decreased to a very low level during the dry season and never recovered to the visible level during the successive wet season. This is not the case in actual dengue endemic areas, where infected hosts or vectors occasionally enter the system and maintain transmission. To simulate stable seasonal dynamics, the number of infected hosts that temporarily visit an area but are not included in the resident population (Ih_visit) was added into equations 5 and 6. For strict mathematical consistency, Ih_visit should be added to the denominator; however, as we set Ih_visit to be a very small value (0.001) compared with the total population (10,000), it was omitted from the denominator. Therefore, we assumed a constant rate of virus introduction (Ih_visit), which was 0.00001% of the total host population from the first day of the simulation. As the coefficient of variation for annual cases was less than 1% over 10 years (the 490th–499th year) in all settings, we considered that the endemic state had been reached in this stage. Fogging was applied in the 500th year, and the optimum day for fogging to minimize dengue cases in this year was calculated. emergence is not affected by the season and is a constant, which may be true in areas where the dengue vector breed in domestic water containers that do not receive rainwater. The density of adult mosquitoes was calculated as eTlv at the equilibrium and was higher during the wet season. Insecticide. Single pulse fogging was conducted in all simulations. The model Fogging was implemented at noon on each day of application. For each insecticide application, 60% of the total mosquitoes were assumed to be killed. No residual insecticide efficacy was included in our model so that, after application, there was no effect on surviving or newly-emerged mosquitoes [10,17]. N&R assumed a density-dependent recovery rate after insecticide fogging; however, we assumed that the fogging did not affect larval and pupal population. Density dependence was therefore not included in adult population dynamics, and the emergence rate of mosquitoes after fogging was also the same rate at e. Simulations Fogging was applied each day from the 1st to the 365th day of the year, during which time, the wet season was assumed to occur at the beginning of the year. The annual number of infected cases was calculated at each application and the day when fogging resulted in a maximum reduction of dengue cases was defined as the optimal day for fogging. The simulation was conducted numerically with a time-step of one hour using Microsoft Excel. Simulation 4: Simulation 3 + various transmission intensities. These simulations were conducted using higher transmission intensities than in Simulation 2. The number of mosquitoes per person (MPP) was increased from 2 to 3, 5, 8 and 15 for low, moderate, high and very high endemic situations, respectively. The day and proportion of most prevented cases were investigated at each MPP. Simulation 1: Identical settings to N&R’s base case. This simulation was carried out for 1 year using the same settings as N&R’s base case simulation [10]; the host population was completely naı¨ve to DENV and the initial value of Iv was set to 1 on the first day of the year. The optimal day for fogging, and the proportion of the prevented cases, were compared with the results from N&R’s simulation. Simulation 1 Our results were very similar to those obtained for N&R’s simulation (Table 2). The maximum reduction in cases was observed when fogging was conducted 6 days after the prevalence peak; however this reduction only amounted to 6.7% of the total cases (Fig. 1A). Simulation 2: Simulation 1 + seasonality. We added seasonality to the model by changing vector life span (Tlv) for 4 days in the wet season and 3 days in the dry season during the course of the simulation. Wet season duration was set to 4, 5 and 6 months (one month = 30 days). All parameters except Tlv were the same as for Simulation 1. The optimal day for fogging and maximum case reduction were investigated for each wet season in a year. Simulation 2: Simulation 1 + seasonality. We added seasonality to the model by changing vector life span (Tlv) for 4 days in the wet season and 3 days in the dry season during the course of the simulation. Wet season duration was set to 4, 5 and 6 months (one month = 30 days). All parameters except Tlv were the same as for Simulation 1. The optimal day for fogging and maximum case reduction were investigated for each wet season in a year. Discussion We successfully developed a model for predicting the most optimal time for insecticide fogging against dengue mosquitoes, which will potentially help reduce the number of dengue cases in endemic regions of the world. By including additional parameters, such as seasonality and disease transmission rates, our model more accurately depicted epidemic outbreaks when compared with the previously published model. Our simulation results for a naı¨ve population with no seasonal setting were similar to those obtained with the N&R model [10]. The greatest reduction of dengue cases was observed when fogging was conducted several days after the prevalence peak, but the impact was minimal. When climatic conditions are favorable for mosquitoes throughout the year, insecticide fogging only slows down the epidemic curve temporarily even if implemented intensively. After fogging, mosquito populations recover rapidly and transmit DENV to susceptible people. Dengue incidence subsequently continues to increase until population immunity reaches a level at which the recovery rate exceeds the new infection rate. In such a situation, fogging reduced dengue cases when conducted after the prevalence peak by accelerating the natural decline of the epidemic. Figure 1. Dengue prevalence with and without optimal insecticide fogging. A: naı¨ve population under non-seasonal condition (Simulation 1), B: naı¨ve population adding 5-month wet season (Simulation 2), and C: endemic state with 5-month wet season (Simulation 3). Black lines indicate untreated epidemics and dotted red lines show epidemics after insecticide treatment. All simulations were conducted using the number of mosquito per person (MPP) = 2. Note that prevalence in Simulation 3 differed from that in Simulation 1 and 2. doi:10.1371/journal.pntd.0001367.g001 When we considered seasonality, the results were completely different. The optimum timing for insecticide fogging shifted earlier than the prevalence peak; because it interferes with the exponential epidemic growth at a certain point and prevents the prevalence peak from reaching the original level by the end of the wet season (Fig. 1B). Furthermore, when considering both endemicity and seasonality, the optimum timing for insecticide fogging shifted to an earlier time and the proportion of prevented cases was greater. The period of greatest prevention was observed relatively early in the wet season (Fig. 2, in green). time, climatic conditions for mosquitoes are unfavorable. Our results showed that the optimal day for fogging was earlier than in Simulation 1 for all wet season durations assessed. The proportion of prevented cases was greater during a shorter wet season (Table 2). Simulation 3 In the endemic state, the yearly number of cases was much smaller than that observed in Simulation 1 and 2 (Table 2, Fig. 1C). Optimal timing of fogging shifted to a much earlier time than in Simulation 2, and more than 40% of the cases were prevented during the wet season of any length. Our model however, does have some limitations when applying simulations to actual dengue endemic areas, due to the simplification of parameters to understand the overall effects of insecticide fogging. First, our assumption of seasonal change was represented by two different values of mosquito lifespan, which Discussion DENV has four different serotypes that simultaneously circulate in most dengue endemic countries. Such co-circulation of multiple serotypes greatly influences long-term epidemic patterns. We additionally evaluated the optimal timing of insecticide fogging by including the co-circulation of four serotypes in endemic situation. The results indicated that the optimal application was also between the onset of the wet season and the prevalence peak (results are shown in Text S1). Therefore, we suggest that hyperendemicity did not affect our findings. Optimal Timing of Fogging to Minimize Dengue Cases 2, and the green area indicates the greatest proportion of cases prevented (.40%). The proportion of prevented cases at and after the prevalence peak was not optimal in any settings for an endemic situation. Optimal Timing of Fogging to Minimize Dengue Cases Figure 1. Dengue prevalence with and without optimal insecticide fogging. A: naı¨ve population under non-seasonal condition (Simulation 1), B: naı¨ve population adding 5-month wet season (Simulation 2), and C: endemic state with 5-month wet season (Simulation 3). Black lines indicate untreated epidemics and dotted red lines show epidemics after insecticide treatment. All simulations were conducted using the number of mosquito per person (MPP) = 2. Note that prevalence in Simulation 3 differed from that in Simulation 1 and 2. doi:10.1371/journal.pntd.0001367.g001 situations (MPP = 3 and 5), a maximum reduction in cases was observed between 81 and 116 days before the prevalence peak, and over 40% of cases were mostly prevented during the wet season of any length. In the higher endemic situations (MPP = 8 and 15), a maximum reduction in cases was also observed earlier than the prevalence peak. The proportion of prevented cases was 35.9–39.6%, which was slightly lower than a MPP of 2, 3 and 5. situations (MPP = 3 and 5), a maximum reduction in cases was observed between 81 and 116 days before the prevalence peak, and over 40% of cases were mostly prevented during the wet season of any length. In the higher endemic situations (MPP = 8 and 15), a maximum reduction in cases was also observed earlier than the prevalence peak. The proportion of prevented cases was 35.9–39.6%, which was slightly lower than a MPP of 2, 3 and 5. Overall, the most effective time for insecticide fogging was early in the wet season, when over 35% of the cases were prevented at any transmission intensity level. The greatest impact of fogging was observed during shorter wet seasons and for lower transmission intensities. The proportion of cases prevented by fogging on each day of the year is shown in Fig. 2, and the green area indicates the greatest proportion of cases prevented (.40%). The proportion of prevented cases at and after the prevalence peak was not optimal in any settings for an endemic situation. Overall, the most effective time for insecticide fogging was early in the wet season, when over 35% of the cases were prevented at any transmission intensity level. The greatest impact of fogging was observed during shorter wet seasons and for lower transmission intensities. The proportion of cases prevented by fogging on each day of the year is shown in Fig. www.plosntds.org Simulation 2 The epidemic magnitude was smaller when the wet season was shorter (Table 2). Dengue incidence generally increased exponen- tially during the wet season (Fig. 1B), and started to decline rapidly within the few days after the onset of the dry season, during which Simulation 3: Simulation 2 + endemic state. This simulation was run for 499 years without any intervention to Simulation 3: Simulation 2 + endemic state. This simulation was run for 499 years without any intervention to www.plosntds.org October 2011 \| Volume 5 \| Issue 10 \| e1367 October 2011 \| Volume 5 \| Issue 10 \| e1367 3 October 2011 \| Volume 5 \| Issue 10 \| e1367 Simulation 4 Population immunity level also increased with an increase in MPP and the length of wet season (Table 1). In the lower endemic October 2011 \| Volume 5 \| Issue 10 \| e1367 4 Optimal Timing of Fogging to Minimize Dengue Cases Table 2. Summary of the simulation results. Setting MPP Wet season (months) Herd immunity Day of prevalence peak No. of annual cases Prevented cases Best day of fogging Difference from the peak Without fogging With fogging N&R’s base case 2 12 0% 163 7,561.9 7,044.2 6.8% 169 +6 Simulation1 2 12 0% 163 7,616.0 7,120,0 6.7% 169 +6 Simulation2 2 4 0% 125 2,614.8 1,874.0 28.3% 60 265 2 5 0% 152 4,788.8 3,793.0 20.8% 112 240 2 6 0% 163 6,259.7 5,592.0 14.4% 139 224 Simulation3 2 4 14.7% 125 21.4 12.8 40.5% 53 272 2 5 19.8% 154 29.0 17.3 40.5% 66 288 2 6 24.6% 183 35.8 21.4 40.2% 78 2105 Simulation4 3 4 41.7% 125 61.0 35.7 41.4% 44 281 3 5 45.5% 154 66.5 39.2 41.1% 58 296 3 6 49.0% 184 71.5 42.5 40.6% 71 2113 5 4 64.8% 125 94.8 56.0 40.9% 40 285 5 5 67.1% 154 98.1 58.4 40.5% 56 298 5 6 69.2% 184 101.1 60.6 40.0% 68 2116 8 4 77.9% 125 113.9 68.8 39.6% 41 284 8 5 79.4% 154 116.0 70.4 39.3% 57 297 8 6 80.8% 184 117.9 72.1 38.8% 70 2114 15 4 88.1% 125 128.9 81.9 36.4% 45 280 15 5 89.0% 154 130.0 82.7 36.3% 63 291 15 6 89.7% 184 131.0 84.0 35.9% 79 2105 doi:10.1371/journal.pntd.0001367.t002 doi:10.1371/journal.pntd.0001367.t002 optimal strategy to reduce the first generation of mosquitoes in the season. was too simple to describe real seasonal dynamics. However, as we aimed to provide a practical strategy for determining optimal insecticide fogging in general, we prioritized the model simplicity and clearly distinguished the on and off-dengue seasons. Various biological features may fluctuate seasonally and affect dengue epidemics. However, when a year can be divided into the on and off-dengue seasons, temporary reduction of adult mosquito population by fogging in the middle of the on-dengue season would delay epidemic growth and prevent cases (Fig. 1B). Thus, we considered that our simple setting for seasonality can typically represent more complex dynamics in the real world. Simulation 4 Our study also analyzed the effect of insecticide fogging on preventing total cases in a single year, but not the effect on longer- term total cases. When insecticide fogging prevented many cases, it also reduced immunity in the host population. Consequently, the susceptible population would potentially cause even larger epidemics in subsequent years. We should therefore carefully foresee and take action between epidemics after applying insecticide fogging. Furthermore, when insecticide treatment was routinely conducted every year, we should have also considered the development of insecticide-resistance in the vector population [23], which was not included in our model. As insecticide resistance has already become a serious problem in many dengue endemic countries [24,25], it is important to carefully consider which insecticides can effectively reduce mosquitoes in the target areas on the basis of biological evidence. The spraying method used to allow the insecticides to reach mosquitoes also requires further investigation. Although our results may not show the best strategy for the long-term prevention of dengue epidemics, they should be interpreted as the optimal strategy for the non-regular emergency treatment during major epidemics. Second, since our model was derived from the N&R model [10], and because we aimed to directly compare our simulations with their conclusion, we set the mosquito lifespan assumption to be identical to this previous study. This was originally obtained from a field study carried out in Thailand (four days in the wet season) [21]. In general, the lifespan of Ae. aegypti in the field is estimated to be slightly longer than our assumption: 5.3–9.1 days [22]. However, the low vector survival rate in our model did not affect our conclusion because when we simulated with a 10 day lifespan in the wet season and 7.5 day lifespan in the dry season, the optimal timing of fogging was also between the beginning of the wet season and the prevalence peak (results not shown). Despite these limitations, our model has a clear practical significance for dengue control in regions where this disease has been endemic for a long time and its epidemic pattern is affected by seasonal climate factors. The optimal timing of insecticide fogging to reduce dengue incidence most effectively is between the onset of the wet season and the prevalence peak, rather than waiting until the peak of a major outbreak occurs. Third, our model did not consider spatial heterogeneity. www.plosntds.org Simulation 4 In our settings, the ‘‘in advance’’ treatment did not appear to be the most effective strategy if implemented too early (Fig. 2). However, incase vector populations survived the dry season in limited areas and expanded the distribution range gradually in the wet season, in-advance focal fogging targeting those areas might be the October 2011 \| Volume 5 \| Issue 10 \| e1367 October 2011 \| Volume 5 \| Issue 10 \| e1367 5 Optimal Timing of Fogging to Minimize Dengue Cases Figure 2. Proportion of cases prevented by single insecticide fogging. MPP is the number of mosquitoes per person. The wet season occurs at the beginning of the year. Blue lines represent the optimal day of fogging and red lines indicate the prevalence peak. doi:10.1371/journal.pntd.0001367.g002 Figure 2. Proportion of cases prevented by single insecticide fogging. MPP is the number of mosquitoes per person. The wet season occurs at the beginning of the year. Blue lines represent the optimal day of fogging and red lines indicate the prevalence peak. doi:10 1371/journal pntd 0001367 g002 Author Contributions Conceived and designed the experiments: TS MO. Performed the experiments: MO. Analyzed the data: MO TS MH TY. Wrote the paper: MO. Text S1 Supplementary method and results of the simulations with four DENV serotypes. (DOC) Acknowledgments The authors thank Drs. Katsuyuki Eguchi and Junko Okumura for their valuable comments on this manuscript. References Control Strategy in Iquitos, Peru and Kanchanaburi, Thailand. Am J Trop Med Hyg 84: 208–217. 1. World Health Organization. Available: http://www.who.int/csr/disease/den- gue/en. Accessed on 2011 Apr 20. 1. World Health Organization. Available: http://www.who.int/csr/disease/den- gue/en. Accessed on 2011 Apr 20. Control Strategy in Iquitos, Peru and Kanchanaburi, Thailand. Am J Trop Med Hyg 84: 208–217. 2. Gubler DJ (1998) Dengue and dengue hemorrhagic fever. Clin Microbiol Rev 11: 480–496. 10. Newton EA, Reiter P (1992) A model of the transmission of dengue fever with an evaluation of the impact of ultra-low volume (ULV) insecticide applications on dengue epidemics. Am J Trop Med Hyg 47: 709–720. 2. Gubler DJ (1998) Dengue and dengue hemorrhagic fever. Clin Microbiol Rev 11: 480–496. 3. Kuno G (2009) Emergence of the Severe Syndrome and Mortality Associated with Dengue and Dengue-Like Illness: Historical Records (1890 to 1950) and Their Compatibility with Current Hypotheses on the Shift of Disease Manifestation. Clinical Microbiology Reviews 22: 186–201. g p p yg 11. Teixeiraa M, Barretoa M, Maria da Concei o N, Ferreirab L, Moratoa V, et al. (2007) Exposure to the risk of dengue virus infection in an urban setting: ecological versus individual heterogeneity. Dengue Bulletin 31: 36–46. 4. Whitehead SS, Blaney JE, Durbin AP, Murphy BR (2007) Prospects for a dengue virus vaccine. Nature Reviews Microbiology 5: 518–528. 4. Whitehead SS, Blaney JE, Durbin AP, Murphy BR (2007) Prospects for a dengue virus vaccine. Nature Reviews Microbiology 5: 518–528. 12. Burattini MN, Chen M, Chow A, Coutinho FA, Goh KT, et al. (2008) Modelling the control strategies against dengue in Singapore. Epidemiol Infect 136: 309–319. . Burattini MN, Chen M, Chow A, Coutinho FA, Goh KT, et al. (2008 5. Eisen L, Beaty BJ, Morrison AC, Scott TW (2009) Proactive Vector Control Strategies and Improved Monitoring and Evaluation Practices for Dengue Prevention. J Med Entomol 46: 1245–1255. 5. Eisen L, Beaty BJ, Morrison AC, Scott TW (2009) Proactive Vector Control Strategies and Improved Monitoring and Evaluation Practices for Dengue Prevention. J Med Entomol 46: 1245–1255. 13. Reiter P (2010) Yellow fever and dengue: a threat to Europe? Euro Surveill 15(10): 19509. 14. Favier C, Schmit D, Muller-Graf CD, Cazelles B, Degallier N, et al. (2005) Influence of spatial heterogeneity on an emerging infectious disease: the case of dengue epidemics. Proc Biol Sci 272: 1171–1177. 6. y g 23. Luz PM, Vanni T, Medlock J, Paltiel AD, Galvani AP (2011) Dengue vector control strategies in an urban setting: an economic modelling assessment. The Lancet 377: 1673–1680. 22. Reiter P (2007) Oviposition, dispersal, and survival in Aedes aegypti: implications for the efficacy of control strategies. Vector Borne Zoonotic Dis 7: 261–273. 21. Sheppard PM, Macdonald WW, Tonn RJ, Grab B (1969) The dynamics of an adult population of Aedes aegypti in relation to dengue haemorrhagic fever in Bangkok. Journal of Animal Ecology 38: 661–702. 25. Kawada H, Higa Y, Nguyen Y, Tran S, Nguyen H, et al. (2009) Nationwide investigation of the pyrethroid susceptibility of mosquito larvae collected from used tires in Vietnam. PLoS Negl Trop Dis 3(3): e391. 24. Ponlawat A, Scott J, Harrington L (2005) Insecticide susceptibility of Aedes aegypti and Aedes albopictus across Thailand. J Med Entomol 42: 821–825. References Gubler DJ (1989) Aedes aegypti and Aedes aegypti-borne disease control in the 1990s: top down or bottom up. Charles Franklin Craig Lecture. Am J Trop Med Hyg 40: 571–578. 6. Gubler DJ (1989) Aedes aegypti and Aedes aegypti-borne disease control in the 1990s: top down or bottom up. Charles Franklin Craig Lecture. Am J Trop Med Hyg 40: 571–578. 15. Balmaseda A, Hammond SN, Tellez Y, Imhoff L, Rodriguez Y, et al. (2006) High seroprevalence of antibodies against dengue virus in a prospective study of schoolchildren in Managua, Nicaragua. Tropical Medicine and International Health 11: 935–942. 7. World Helath Organiztion (2009) Dengue Guideline For Diagnosis, Treatment, Prevention and Control. Available: http://whqlibdoc.who.int/publications/ 2009/9789241547871_eng.pdf. Accessed on 2011 Apr 20. 16. Ooi E, Hart T, Tan H, Chan S (2001) Dengue seroepidemiology in Singapore. The Lancet 357: 685–686. 8. Esu E, Lenhart A, Smith L, Horstick O (2010) Effectiveness of peridomestic space spraying with insecticide on dengue transmission; systematic review. Tropical Medicine and International Health 15: 619–631. 17. Luz PM, Codec¸o CT, Medlock J, Struchiner CJ, Valle D, et al. (2009) Impact of insecticide interventions on the abundance and resistance profile of Aedes aegypti. Epidemiol Infect 137: 1203–1215. 9. Paz-Soldan VA, Plasai V, Morrison AC, Rios-Lopez EJ, Guedez-Gonzales S, et al. (2011) Initial Assessment of the Acceptability of a Push-Pull Aedes aegypti October 2011 \| Volume 5 \| Issue 10 \| e1367 October 2011 \| Volume 5 \| Issue 10 \| e1367 6 www.plosntds.org Optimal Timing of Fogging to Minimize Dengue Cases Optimal Timing of Fogging to Minimize Dengue Cases 18. Focks DA, Haile DG, Daniels E, Mount GA (1993) Dynamic life table model for Aedes aegypti (Diptera: Culicidae): analysis of the literature and model development. J Med Entomol 30: 1003–1017. p J 19. Focks DA, Haile DG, Daniels E, Mount GA (1993) Dynamic life table model for Aedes aegypti (diptera: Culicidae): simulation results and validation. J Med Entomol 30: 1018–1028. 20. Focks DA, Daniels E, Haile DG, Keesling JE (1995) A simulation model of the epidemiology of urban dengue fever: literature analysis, model development, preliminary validation, and samples of simulation results. Am J Trop Med Hyg 53: 489–506. 21. Sheppard PM, Macdonald WW, Tonn RJ, Grab B (1969) The dynamics of an adult population of Aedes aegypti in relation to dengue haemorrhagic fever in Bangkok. Journal of Animal Ecology 38: 661–702. www.plosntds.org October 2011 \| Volume 5 \| Issue 10 \| e1367 7 7 www.plosntds.org www.plosntds.org
https://openalex.org/W2779981722	https://journal.unismuh.ac.id/index.php/exposure/article/download/764/pdf	English	null	THE IMPLEMENTATION OF “ARIAS” STRATEGY TO INCREASE STUDENTS’ SPEAKING SKILL AT SMA NEGERI 1 BUNGORO	Exposure: Jurnal Pendidikan Bahasa Inggris/Exposure	2,012	cc-by-sa	6,533	THE IMPLEMENTATION OF “ARIAS” STRATEGY TO INCREASE STUDENTS’ SPEAKING SKILL AT SMA NEGERI 1 BUNGORO Implementasi Strategy “ARIAS” Untuk Meningkatkan Kemampuan Berbicara Siswa SMA Negeri 1 Bungoro. Ismail Sangkala English Education Department, Faculty of Teacher Training and Education Muhammadiyah University of Makassar ismail@unismuh.ac.id ABSTRACT The Implementation of “ARIAS” Strategy to Increase Students’ Speaking Skill at SMA Negeri 1 Bungoro. The objective of the research is intend to know the increasing of the students’ accuracy and fluency in speaking through ARIAS strategy at third year students’ of SMA Negeri 1 Bungoro. The method of this research is classroom action research consist of two cycles. Both of cycles consist of four meetings. It means that there are eight meetings for two cycles. The subject in this research is X.3 Senior High School in 2011-2012 academic years. That consists of 17 women and 35 men. Instruments are speaking test and observation. Keywords: Speaking, Assurance, Relevance, Interest, Assessment, Accuracy, Fluency and Satisfaction. Penerapan Strategi "ARIAS" untuk Meningkatkan Keterampilan Siswa Berbicara di SMA Negeri 1 Bungoro. Tujuan dari penelitian ini adalah ingin mengetahui peningkatan akurasi siswa dan kefasihan dalam berbicara melalui strategi ARIAS pada siswa tahun ketiga dari SMA Negeri 1 Bungoro. Metode penelitian ini adalah penelitian tindakan kelas terdiri dari dua siklus. Kedua siklus terdiri dari empat pertemuan. Ini berarti bahwa ada delapan pertemuan selama dua siklus. Subjek dalam penelitian ini adalah X.3 SMA tahun 2011-2012 tahun akademik. Yang terdiri dari 17 perempuan dan 35 laki-laki. Ada 2 intrumen yang digunakan tes berbicara dan observasi. Kata Kunci: Berbicara, Jaminan, Relevansi, Bunga, Pengkajian, Akurasi, Kefasihan dan Kepuasan. Learning a foreign language is an integrated process that the learner should study the four basic skills: listening, speaking, reading, and writing. We use it to understand our world through listening and reading and to communicate our feeling, need, and desires through speaking and writing. By having more knowledge about language skill we have much better chance of understanding and being understood and getting what we want and need from these around us. Speaking plays an important role in learning especially in foreign language; in this case a teacher should choose appropriate method, technique and a good atmosphere as an effort to make teaching fresh and interest in order to motivate students to learn English. Rasyid in A. Najmawati (1992:1) stated that the focal point of English language teaching in Indonesia is in the classroom because this is English Education Department Vol. 1 No. 1 Mei 2012 Vol. 1 No. 1 Mei 2012 Vol. 1 No. 1 Mei 2012 English Education Department where contact among teacher, students, materials and method/approaches occurred. ABSTRACT However, it cannot be denied that the way of teachers in teaching the students effects the achievement or the result of the learning. The students will be easier to learn in the class if the teachers use appropriate method or strategy in the class. The method or strategy must cover some elements in learning process. In increasing speaking skill, the teachers sometimes find difficulties to encourage students to speak. This problem shows that the teachers are not merely demanded to teach and explain, but also pay attention to some things which will help much more in teaching. These things are the assurance, relevance of the material, the interest of the students in learning process, and also the way to measure their ability or assessment. All these components will support the teachers’ effort to encourage students and improve their speaking skill ability. Considering that, the researcher have taken an action research on the students’ increasing in speaking, so that the solution is by teaching speaking using ARIAS strategy. The ARIAS offers a complete method that motivated the students to speak and increase their ability in speaking. It grows the students’ confidence to speak and encourage them to be active in the classroom. ARIAS COMPONENT ARIAS learning model consists of assurance, relevance, interest, assessment, satisfaction which is arranged based on learning theories. The five components are one unity require learning process. The short descriptions of each component and some samples can be done to intrigue and increase learning as below. DEFINITION OF ARIAS According to Keller and Kopp (1987: 2-9), ARIAS learning model is interesting because it is developed based on learning and teaching theories. The evaluation or assessment is held not only in the end of the learning process but also during the learning process. Evaluation is held to measure how far the students’ learning achievement. Evaluation which is held during the learning process according to Saunders et.al. Will influence the result of students’ learning achievement. Based on that prominent reason, ARIAS is modified by adding evaluation component in the process of learning. This learning model involves five components, they are; Attention, Relevance, Confidence, Satisfaction, and Assessment. Modification is done by changing the “confidence” becomes “assurance” because “assurance” is a synonym of “self-confidence” (Morris, 1981: 80). In learning process, the teacher doesn’t only believe that the students will be able to learn easier but also they can be more confidence with their own Vol. 1 No. 1 Mei 2012 English Education Department capability. The changing of the word “attention” becomes “interest” involve the definition of attention. “Interest” word is raising the students’ attention during the learning process. To get the better acronym, the arranging of the word is modified becomes assurance, relevance, interest, assessment and satisfaction. The meaning of this modification is the first effort in learning process to build students’ self- confidence. Learning activities have relevancy toward students’ life, try to effect and conserve students’ attention. Then, evaluation is done to grow students’ proudness to give reinforcement. A. Assurance Assurance is related to self-confidence, be sure for being successor related to expectation to achieve something. (Keller, 1987:2-9). According to Bandura, a man who has self- confidence prone to be success with what she or he has. Attitude for being confidence, believe can be success to achieve something will influence them to behave for a success. This attitude encourages the students to behave to achieve success in learning activity (Petri, 1986: 218). In other word, education in the globalization era is more emphasize the development of students in all aspects. The teacher doesn’t only pay attention to speech ability, coordination, and social skill. The teacher encourages each individual to solve emotional or physical problem of them. C. Interest Interest is related to students’ attention and willingness to study. Woodruff (1966: 23) state that the learning process will not happen without interest and willingness. Keller also state that in learning process interest is not only needed to be assist but also be maintained during the process of learning. Interest is a useful tool in increasing students’ achievement. Some ways can be used to increase students’ willingness to study: the teacher use a story, analogy, picture series or something new in to teach, the teacher make some variations in learning activity as Lesser state variations from serious to humorist, from slow to be fast, and style in teaching. B. Relevance Relevance is related to students’ life, whether about their experiences in this present time or in the past which are related with career needs for this time being or for the future (Keller, 1987; 2-9). Students feel the learning activities that they follow have contributions, and advantages for their life. Students will be assisted to learn something if they know the relevance of the lessons with their life. In learning process, the teacher needs to pay attention English Education Department Vol. 1 No. 1 Mei 2012 Vol. 1 No. 1 Mei 2012 Vol. 1 No. 1 Mei 2012 English Education Department English Education Department toward the substantial of relevance. Some of ways can be used to increase the relevance in learning; explain the benefits of the lessons to the students, explain the goals will be achieved, use clear languages to explain and real samples based on environment experiences and also use strategy and appropriate tools in teaching. D. Assessment Assessment is related to the evaluations. Evaluation is one of main part in learning process whether for the teachers or the students. Among the macro skills of language, it has been widely recognized that speaking, particularly in a second or foreign language, is the most difficult language skill to assess? The various directions and foci in the testing of speaking abilities of learners frequently Lack solid grounding on theory and pedagogy and reliable test designs (Pennington, 1999; Murcia, Brinton, & Goodwin, 1996). This is due, for the most part, to the difficult matching of the testing goals and the appropriate instruments and tasks for assessment. Speaking as a major construct for testing is likewise divided into different criteria with highly diverse applications. The testing of pronunciation (both segmental and supra segmental), spoken grammar, spoken vocabulary, and even sociolinguistic applications of speech all fall into the construct of speaking but largely require discrete test designs and measures. This is a challenge for classroom teachers and researchers of learners' speaking abilities. Knowing what to test specifically and how to conduct the testing process require English Education Department Vol. 1 No. 1 Mei 2012 Vol. 1 No. 1 Mei 2012 Vol. 1 No. 1 Mei 2012 English Education Department applicable theories and valid procedures that map out the direction of the assessment strategy. As a result, drawing upon applied linguistic theories. Some ways can be used to evaluate students’ result in learning. Does the evaluative feed- back toward students’ achievement, give the objective evaluation and inform the result to the students. Give a chance for students to evaluate their self and their friend. B. Body language This element of pronunciation involves various parts of the body; the way you stand or sit when talking, the angle of your shoulders, your stance. Your head / face - where you look when you speak, how often you look at the people you are speaking to in the eye and how long you hold their gaze, whether you move your head up, down or side to side. Your hands / arms - your hand gestures and arm movements. E. Satisfaction Satisfaction is related to the gloried of the students with their achievement in learning. Success and proudness is reinforcement for the students for the next success (Gagne and Driscoll, 1988: 70). Reinforcement will give satisfaction to the students is prominent in learning process. Some ways can be used to give satisfaction to the students; give a chance to the students to apply their skill or knowledge in the real situation or simulation, ask the students to help their friends who find difficulties in learning activities. A. Pronunciation Pronunciation is the act of pronouncing a word or words (The new shorter Oxford English Dictionary: 1993). Pronunciation consists of a number of different elements. Each of these elements is important and contributes to a speaker's ability to speak clearly and fluently so that they can be understood by many different people in many different situations. C. Voice quality This relates to how your voice sounds. Your voice might be quiet, loud, and high or low pitched, husky, squeaky, etc. How you breathe also Vol. 1 No. 1 Mei 2012 English Education Department English Education Department English Education Department affects your voice quality. The speed of your speaking, whether very quick or very slow can have an effect on your voice quality. This last thing is related to the rhythm of you speech. D. Rhythm - pausing and stress, - linking and reduction Rhythm in speech involves many things. It includes where you pause in a sentence and which words you stress, or which parts of words (syllables) that you stress. 'Stress' relates to how loud you say a word, or how much emphasis you put on that word or syllable. Related to rhythm is linking. Fluent speakers ‘run’ their words together and this sometimes make it difficult for learners of English to understand native speakers. As a speaker you need to link words and to also reduce or weaken some words or parts of words. (For example when the phrase "night and day" is said by native speakers, they usually do not pronounce 'and' fully but make it sound like 'n'. This is an example of a reduced or weakened word. In the sentence, “Look out the window!” there is linking (look-out) and weakening or reduction (the). E. Intonation This is the use of different pitch and changes in pitch to convey meaning in a sentence. The same words can be said with different pitch and the listener understands something different. e.g. "She's finished" said with a rise in pitch at the end becomes a question. Said without this rise it is a statement. Intonation is used to express a great number of different meanings, including emotions and attitudes. F. Sounds The individual sounds of English may be different to the sounds in your first language. Or perhaps more importantly, they may be combined with other sounds in different ways or appear in different parts of a word. The vowels and consonants of English are important elements of pronunciation, each of these elements contributes to a person being a competent and clear speaker of English and no single element alone is the key to good pronunciation. However, achieving competence in all of these elements is important and should be each learner's goal. English Education Department Vol. 1 No. 1 Mei 2012 Vol. 1 No. 1 Mei 2012 English Education Department Vol. 1 No. 1 Mei 2012 English Education Department F. Accuracy Accuracy is the state of being correct or exact and without error especially as result of careful effect. (Oxford Advanced Learner’s Dictionary 1995: 9). Accuracy is the ability to produce correct sentences using correct grammar and vocabulary. Accuracy is relative. A child in early primary isn't capable of the same level of accuracy as an adult. Teachers who concentrate on accuracy help their students to produce grammatically correct written and spoken English. G. Fluency Fluency is the state of being able to speak the language smoothly and easily (Oxford Learns Pocket Dictionary, 1991) and students are to communicate easily to other friends. Fluency is the ability to read, speak, or write easily, smoothly, and expressively. In other words, the speaker can read, understand and respond in a language clearly and concisely while relating meaning and context. Fluency generally increases as learner’s progress from beginning to advanced readers and writers. Language teachers who concentrate on fluency help their students to express themselves in fluent English. They pay more attention to meaning and context and are less concerned with grammatical errors. There are four characteristics of fluency activity, they are: 1. The facts are usually whole piecesof discourse: conversation, stories, etc. 2. Performance is assessed and how well ideas are expressed or understood. 3. Texts are usually used as they would be in real life. 4. Tasks are often simualted real like situation. 4. Tasks are often simualted real like situation. 4. Tasks are often simualted real like situation. H. Vocabulary H. Vocabulary Vocabulary is the range of language of a particular author, group, discipline book and etc. (The New Shorter Oxford English Dictionary: 1993) 1. Definition of Vocabulary Vocabulary is the range of language of a particular author, group, discipline book and etc. (The New Shorter Oxford English Dictionary: 1993) 1. Definition of Vocabulary It is important to know what vocabulary is. There are many definitions of vocabulary. According to Oxford Learner Dictionary of Current English, Vol. 1 No. 1 Mei 2012 Vol. 1 No. 1 Mei 2012 English Education Department English Education Department vocabulary is ‘total number of words which (with rules for combining them) make up language (rang of) words to use by, a person in trade, profession etc” (Hornby, 1986). Webster (1980) point out that the vocabulary is a list of words and sometimes phrase, usually arranged in alphabetical may be categorized as having for separate but largely overlapping components indicate how vocabulary is processed and how it is used. Each of us has receptive and productive capacity and within capacity we processed and utilized both spoken and written language 2. Kinds of vocabulary 2. Kinds of vocabulary Harmer (1991: 157) divides vocabulary into types namely: a. Active vocabulary; refers to vocabulary which students have learned and which they are expected to be able to use. b. Passive vocabulary; refers to vocabulary which students will recognize when they meet them, but which they will probably no able to produce METHODOLOGY This research used classroom action research that contains of four stages, they are: Planning, Implementation of Action, Observation, and Reflection. This research conducted two cycles, they are first and second cycle and each cycle is the series of activity which has close relation. Where, the realization of the second cycle is continuing and repairing from the first cycle. 1. The Planning The activities that have done in this stage as follows: a. Studying and understanding the material that have been taught b. Making the lesson plan for the implementation of action c. Making the sheet of students’ assessment, to measure the students’ speaking ability with ARIAS Cycle II The stages that have done in the second cycle are almost same with the first cycle by doing several repairmen or adding several activities based on reality in the class. This research conducted on Januari until March, 2012 at SMA Negeri 1 Bungoro, Pangkep Region, and South Sulawesi. This research used two variables, they are: 3. Observation Observation is collecting data activity related with the learning English process which had solving problem and learning strategy which is improving stated by Adnan Latief (2009: 27). So in this stage the researcher prepared collection data, instrument data collector have used, data source have explained, and collection data and data collection technique have used. Analyze all of the data which had been collected from observation, to assess the teaching program’s achievement after given an action at the first cycle. The result can be a basic to formulate the next repairing lesson plan. Analyze all of the data which had been collected from observation, to assess the teaching program’s achievement after given an action at the first cycle. The result can be a basic to formulate the next repairing lesson plan. 2. Implementation of action 2. Implementation of action The activities that have done in this stage are: a. The teacher explained the lesson based on the curriculum at school Vol. 1 No. 1 Mei 2012 Vol. 1 No. 1 Mei 2012 English Education Department English Education Department b. The students have been given speaking conversation script based on their amount in a group of speaking c. The students memorize the text based on their part d. The students performed their conversation in front of the classroom, based on their group a. Independent variable The independent variable is implementation of ARIAS strategy. It is as the method used by the teacher when teaching the material. b. Dependent variable b. Dependent variable The dependent variable is the students’ speaking proficiency which covers fluency and accuracy. The research instrument of this research, there are two. They are; The research instrument of this research, there are two. They are; a. Observation List a. Observation List a. Observation List Observation sheet is used to watch out the situation of teaching and learning process which covers the method applied by a teacher in the class. Observation sheet is used to watch out the situation of teaching and learning process which covers the method applied by a teacher in the class Observation sheet is used to watch out the situation of teaching and Observation sheet is used to watch out the situation of teaching and learning process which covers the method applied by a teacher in the class. learning process which covers the method applied by a teacher in the class. learning process which covers the method applied by a teacher in the class. Vol. 1 No. 1 Mei 2012 English Education Department English Education Department b. Test The test will be used in the observation stage of every cycle to measure the students’ motivation in speaking. In this case, the researcher will ask the students to play a role in speaking based on their turn in conversation. Collecting data is done with the following procedures: Collecting data is done with the following procedures: a. Data source: the data source in this research is the students’ motivation in speaking before getting the material through ARIAS strategy. b. The students are given an oral test to the students. It is done after implementing the ARIAS strategy in the class or in the observation stage of classroom action research which will be done in every cycle. The following activities are: 1. The researcher explained material use the strategy of ARIAS. 2. The researcher is given script of conversation based on the number of students in a group. 3. The students performed their speaking activity or conversation in front of the class. There are two components that to be concern of the researcher in this research to increase students’ motivation in speaking. 1. Data analysis To analyze the data in the classroom action research is done by quantitative. The quantitative data used descriptive analysis. The descriptive analyses that used are mean score, table distribution of frequency, minimum value, maximum value, and percentage. 1. Data analysis 2. To calculate the mean score of the students’ test result. The researcher used the following formula: X = N X  X = N X  Where: Where: = the number of sample (Tiro, Muhammad Arif and Baharuddin Ilyas, 2002: 69) English Education Department Vol. 1 No. 1 Mei 2012 English Education Department Vol. 1 No. 1 Mei 2012 To describe the students’ motivation in speaking in every cycle, the researcher used table distribution of frequency and percentage with the following steps that is explained by Tiro, Muhammad Arif (2000: 116): 1. Deciding the range value, with the following formula: R= the highest score – The lowest score R= the highest score – The lowest score 2. Deciding the interval class is needed. The amount of the class depends on the necessity, but usually used five classes until twelve. 3. Deciding distance of the class p, with the following procedure: Where: p = Distance of the class n = Amount of the class 4. b. Test Choosing the lowest class as the first interval class and the table finished by using the values on the data. 5. Then, the data also made in the percentage form. 5. Then, the data also made in the percentage form. FINDINGS AND DISCUSSION This chapter consists of findings of the research and its discussion. The findings of the research present the result of the students’ activeness observation in teaching and learning process, the improvement of the students’ speaking proficiency, and the students’ attitude toward the application of communicative approach, and the discussion of the research covers further explanation of the findings. The findings of classroom action research deal with the answer of the problem statement which its aim is to improve students’ accuracy and fluency in speaking. The findings consist of students’ ability in speaking and observation result. The data of speaking accuracy consists of three items namely: vocabulary, pronunciation and grammar. While the data of speaking fluency consist of one item namely: smoothness. 1. The Students’ speaking accuracy The following table shows the students’ speaking accuracy of cycle 1 and cycle 2. 1. The Students’ speaking accuracy The following table shows the students’ speaking accuracy of cycle 1 and cycle 2. English Education Department Vol. 1 No. 1 Mei 2012 Vol. 1 No. 1 Mei 2012 English Education Department Table 1. The students’ speaking accuracy from cycle 1 to cycle 2 Cycle Variables Accuracy Vocabulary Pronunciation Grammar I 6.4 6.1 6.0 6.1 II 8.0 7.6 7.3 7.6 Table 1. The students’ speaking accuracy from cycle 1 to cycle 2 Both the diagrams above show the students’ mean score of accuracy in speaking which covers three items namely vocabulary, pronunciation, and grammar. The diagram shows the improvement of students’ mean score of accuracy after the implementation of the ARIAS startegy as an action in the first cycle and second cycle. This graphic presents the students’ mean score in cycle 1 and cycle 2 which focused on speaking accuracy. From the graphic it’s known that there is an improvement of the mean score in the cycle 1 to cycle 2. It can show the process of the mean score from 6.1 to 7.6. a. Vocabulary Table 2. Percentage of Vocabulary Assessment in Cycle I and II Table 2. Percentage of Vocabulary Assessment in Cycle I and II Table 2. Percentage of Vocabulary Assessment in Cycle I and II g y y Classification Score Cycle I Cycle II F % F % Excellent 9.6 – 10 0 0 0 0 Very good 8.6 – 9.5 7 23,33 9 30,00 Good 7.6 – 8.5 10 33,33 11 36,66 Fairly good 6.6 – 7.5 7 23,33 8 26,66 Fair 5.6 – 6.5 4 13,33 2 6,66 Poor 3.6 – 5.5 2 06,66 0 0 Very poor 0.0 – 3.5 0 0 0 0 30 100 30 100 Based on the table above, it can showed that in Cycle I there was no students got classification score very poor, there were 7 students (23,33 %) got poor, 4 students (13,33 %) got fair, 7 students (23,33 %) got fairly good, 10 students (33,33 %) got good and there were no students got classification scores very good and excellent. The results in Cycle I indicates that some of students still lack of vocabulary. In Cycle II there were no students got classification score very poor and poor, there were 2 students (6,66%) got fair, 8 students (26,66%) got fairly good, Vol. 1 No. 1 Mei 2012 English Education Department 11 students (36,66%) got good, 9 students (30,00%) got very good and there was no students got excellent. The result in Cycle II indicates that some of students experienced an improvement than Cycle I. b. Pronunciation Table 3. Percentage of Pronunciation Assessment in Cycle I Table 3. Percentage of Pronunciation Assessment in Cycle I Classification Scores Cycle I Cycle II F % F % Excellent 9.6 – 10 0 0 1 3,33 Very good 8.6 – 9.5 2 6,66 5 16,66 Good 7.6 – 8.5 8 26,66 15 53,33 Fairly good 6.6 – 7.5 11 13,33 6 20 Fair 5.6 – 6.5 6 20 2 6,66 Poor 3.6 – 5.5 3 10,00 1 3,33 Very poor 0.0 – 3.5 0 0 0 0 30 100 30 100 Based on the table above, it can show that in Cycle I there was no students got classification score very poor, there were 3 students (10,00 %) got poor, 6 students ( 20 %) got fair, 11 students (13,33 %) got fairly good, only 8 students (26,66 %) got good, 2 students (6,66%) got very good, and there were no students classification scores excellent. The results in Cycle I indicate that some of students still lack of pronunciation. In Cycle II there were no students got classification score very poor,there were 1 student (3,33%) still got poor score, there were 2 students (6,66%) got fair, 6 students (20%) got fairly good, 15 students (53,33%) got good, 5 students (16,66%) got very good and there was 1 student (3,33) got excellent. The results in Cycle II indicate that some of students experienced an improvement than Cycle I. c. Grammar Table 4. Percentage of Grammar Assessment in Cycle I Classification Score Cycle I Cycle II F % F % Excellent 9.6 – 10 0 0 0 0 Very good 8.6 – 9.5 2 6,66 9 30,00 Good 7.6 – 8.5 8 26,66 15 50 Fairly good 6.6 – 7.5 11 36,66 3 10,00 Fair 5.6 – 6.5 6 20 3 10,00 Poor 3.6 – 5.5 3 10,00 0 0 Very poor 0.0 – 3.5 0 0 0 0 30 100 30 100 Based on the table above, it can showed that in Cycle I there was no students got classification score very poor, there were 3 students (10,00 %) got English Education Department Vol. 1 No. 1 Mei 2012 Vol. 1 No. b. Pronunciation 1 Mei 2012 English Education Department poor, 6 students (20 %) got fair, 11 students (36,66 %) got fairly good, 8 students (26,66 %) got good, 2 students (6,66%) got very good and there were no students got classification scores excellent. The results in Cycle I indicate that some of students still lack of grammar. In Cycle II there were no students got classification score very poor and poor, there were 3 students (10,00%) got fair, 3 students (10,00%) got fairly good, 15 students (50%) got good, 9 students (30,00%) got very good and there was no students got excellent. The result in Cycle II indicates that some of students experienced an improvement than Cycle I. Table 5. The students’ speaking fluency of cycle 1 and cycle 2 Table 5. The students’ speaking fluency of cycle 1 and cycle 2 Cycle Fluency (Smoothness) Cycle I 6.06 Cycle II 7.46 The graphic above shows the students’ mean score of speaking fluency of the students of the third year students of SMP Muhammadiyah 5 Mariso. This graphic presents that the students’ mean score in cycle 1 and cycle 2 which focuses on speaking fluency. From the graphic it’s known that there is an improvement of the mean score in the cycle 1 to cycle 2. It can show the process of the score from 6.8to 8.0. 3. The students’ mean score of speaking fluency The following table shows the students’ mean score of speaking fluency of cycle1 and cycle 2. a. Smoothness Table 6. Percentage of Smoothness Assessment in Cycle I and II ble 6. Percentage of Smoothness Assessment in Cycle I and Classification Score Cycle I Cycle II F % F % Excellent 9.6 – 10 0 0 1 3,33 Very good 8.6 – 9.5 3 10 5 26,66 Good 7.6 – 8.5 3 10 7 23,33 Fairly good 6.6 – 7.5 6 20 4 13,33 Fair 5.6 – 6.5 8 26,66 6 20 Poor 3.6 – 5.5 9 30 7 23,33 Very poor 0.0 – 3.5 1 3,33 0 0,00 30 100 30 100 Based on the table above, it can showed that in Cycle I, there was 1 student (3, 33%) got classification score very poor, there were 9 students (30 %) got poor, 8 students (26, 66 %) got fair, 6 students (20 %) got fairly good, only 3 Vol. 1 No. 1 Mei 2012 Vol. 1 No. 1 Mei 2012 English Education Department students (12 %) got good, there were 3 students (10%) got very good, and there were no students got classification score excellent. The results in Cycle I indicate that some of students still lack of smoothness. In Cycle II there were no students got classification score very poor, there were still 7 students (23,33) got poor, there were 6 students (20%) got fair, 4 students (13,33%) got fairly good, 7 students (23,33%) got good, 5 students (26,66%) got very good and there was 1 student (3,33) got excellent. The result in Cycle II indicates that some of students experienced an improvement than Cycle I. 5. Observation Result The following table shows the students’ participation in learning speaking of cycle 1 and cycle 2. Table 7. The students’ observation in learning speaking Cycles Participation 1st Meeting % 2nd Meeting % 3rd Meeting % 4th Meeting % Cycle 1 50 55 63 67 Cycle 2 73 77 80 83 Based on the table above the researcher can explain that the students’ observation in learning process through talking chips every meeting in cycle 1 is still low with percentage of first meeting till fourth meeting are 50, 55, 63, and 67. The Presentations of the first meetings till fourth meeting of the cycle 2 are 73, 77, 80, and 83. It means that there is an improvement can be shown in students’ observation process from cycle 1 to cycle 2. students’ observation in learning process through talking chips every meeting in cycle 1 is still low with percentage of first meeting till fourth meeting are 50, 55, 63, and 67. The Presentations of the first meetings till fourth meeting of the cycle 2 are 73, 77, 80, and 83. It means that there is an improvement can be shown in students’ observation process from cycle 1 to cycle 2. The discussion aims to answer the question as follows: The discussion aims to answer the question as follows: 1. How is the increasing of students’ speaking fluency through ARIAS Strategy? 2. How is the increasing of students’ speaking accuracy through ARIAS Strategy? a. The students’ improvement of mean score in speaking accuracy through ARIAS strategy. a. The students’ improvement of mean score in speaking accuracy through ARIAS strategy. In applying the ARIAS strategy in learning process in the class, the researcher found that the mean score of test in cycle 2 of students’ proficiency in speaking accuracy was greater than test in cycle 1, in table 1 page 27 shows that in test of cycle 1 students’ mean score got 6, 1 and after repairing In applying the ARIAS strategy in learning process in the class, the researcher found that the mean score of test in cycle 2 of students’ proficiency in speaking accuracy was greater than test in cycle 1, in table 1 page 27 shows that in test of cycle 1 students’ mean score got 6, 1 and after repairing Vol. 1 No. 1 Mei 2012 English Education Department English Education Department the action in cycle 2 got 7.6. 5. Observation Result Therefore the researcher indicates that there was a significant improvement of speaking accuracy though ARIAS strategy. b. The students’ percentage in speaking accuracy b. The students’ percentage in speaking accuracy 2. Pronunciation Based on the results of data indicated that some of the students have an improvement in pronunciation in Cycle II than Cycle I, especially in Good classification score there are 8 students (26, 66) % but in the second cycle become 53,33 % got good, and poor score being less 3,33% from 10,00 %, even there are 5 students got very good score and 1 of them got excellent score. 1. Vocabulary Based on the data in previous findings the results indicated that some of the students have improvement in vocabulary than the first cycle. In the first cycle 33,33 % got good classification score, but in the second cycle become 36,66 % got good, and very fair being less 6,66 % from 13,33 %, even there were 9 students got very good score where the first cycle there were only 7 studnets d. The students’ percentage in speaking fluency d. The students percentage in speaking fluency Based on the data in previous findings indicated that some of the students have improvement of smoothness in Cycle II than Cycle I, especially in Good classification score there were 5 students (12%) in cycle I got good but in cycle II become 28.5 %, and very fair being less 12 % from 40.4 %, even that there are 10 students got very good score. Based on the data in previous findings indicated that some of the students have improvement of smoothness in Cycle II than Cycle I, especially in Good classification score there were 5 students (12%) in cycle I got good but in cycle II become 28.5 %, and very fair being less 12 % from 40.4 %, even that there are 10 students got very good score. e. The observation result of the students’ participation in learning speaking through natural approach. e. The observation result of the students’ participation in learning speaking through natural approach. Based on the data analysis as result of observation sheet of students’ participation in learning process in previous findings shows that the participation of students from the first meeting till fourth meetingswere50, 55, 63, and 67 with mean score of four meetings as cycle 1 was 58.75. Percentage of the first till the fourth meeting of the cycle 2 were 73, 77, 80, and 83 with the mean score 78. From the data analysis shows that the students’ participation in cycle 1 in process learning is still low. So that’s why the researcher did repairing in cycle 2 so that there was a significant improvement in cycle 2 of students’ participation. Based on the all result of data analysis above, researcher concludes that there are significant improvements of students’ speaking accuracy, fluency and participation of students in learning process. 3. Grammar Based on the data in previous findings indicated that some of the students have improvement in grammar than the first cycle, especially in Good classification score there were 8 students 26,66 % but in the second cycle become 50 % got good, and very fair being less 10% from 30 %, even there were 9 students got very good score. c. The students’ improvement of mean score in speaking fluency through ARIAS strategy c. The students’ improvement of mean score in speaking fluency through ARIAS strategy From the data analysis in the previous findings, the researcher found that the mean score of test in cycle 2 of students’ ability in speaking fluency was greater than test in cycle 1, in table 2 page 31 showed that in test of cycle 1 got 6.8, after repairing the action in cycle 2 got 8.0. Therefore the researcher indicates that there was significant improvement of speaking fluency through natural approach. English Education Department Vol. 1 No. 1 Mei 2012 Vol. 1 No. 1 Mei 2012 English Education Department English Education Department d. The students’ percentage in speaking fluency Brazil D, 1992. Classroom and spoken discourse. Birmingham: the University of Birmingham. Brazil D, 1992. Classroom and spoken discourse. Birmingham: the University of Birmingham. Brown, H. D. 1994. Principle of language learning and teaching. Third edition. New Gersey: Prantice Hall Regents. Byrne, Donn. 1987. Teaching Oral English. Longman Publishing Group. Byrne, Donn. 1987. Teaching Oral English. Longman Publishing Group. Coulthard, M. 1985. An introduction to discourse analysis. Second edition. Harlow: Longman. Coulthard, M. 1985. An introduction to discourse analysis. Second edition. Harlow: Longman. Dalle, Basri. 2010. Fundamentals of Research Methodology. Makassar: Universitas Muhammadiyah Makassar. Dalle, Basri. 2010. Fundamentals of Research Methodology. Makassar: Universitas Muhammadiyah Makassar. Ferre. P. John. 1983. Merill Guide to the Research Paper. States of America: Charles E. Merill Publishing Company. Ferre. P. John. 1983. Merill Guide to the Research Paper. States of America: Charles E. Merill Publishing Company. Vol. 1 No. 1 Mei 2012 English Education Department English Education Department Goodman, CV. 1973. Dictionary of Education. New York: Mc. Grow Hill, Inc. Harmer, J. 1987. The Practice of English language teaching. New York: Longman Inc. Manser Martin, H. 1983. Oxford Learner’s Pocket Dictionary. New Edition. Oxford University Press. Tiro, Muhammad Arif. 2000. Dasar-Dasar Statistika. Makassar: State University of Makassar Press. Tiro, Muhammad Arif and Baharuddin Ilyas. 2002. Statistika Terapan untuk Ilmu Ekonomi dan Ilmu Sosial. Makassar: Andira Publisher. Webster’. University Dictionary. Massachusettss, USA: A & C Merriam Company, publishers. Vol. 1 No. 1 Mei 2012 Vol. 1 No. 1 Mei 2012 English Education Department English Education Department
https://openalex.org/W4320715852	https://shura.shu.ac.uk/31432/7/ElFakir-KinshipGenderSocial%28VoR%29.pdf	English	null	Kinship, gender and social links impact on micro group lending defaults	International journal of finance & economics/International journal of finance and economics	2,023	cc-by	11,167	Published version EL FAKIR, Adil, FAIRCHILD, Richard, LAMRANI ALAOUI, Youssef, CHAN, Dora, TKIOUAT, Mohamed and AMER, Zaid (2023). Kinship, gender and social links impact on micro group lending defaults. International Journal of Finance and Economics. Kinship, gender and social links impact on micro group lending defaults EL FAKIR, Adil <http://orcid.org/0000-0002-0922-7274>, FAIRCHILD, Richard, LAMRANI ALAOUI, Youssef, CHAN, Dora, TKIOUAT, Mohamed and AMER, Zaid Available from Sheffield Hallam University Research Archive (SHURA) at: http://shura.shu.ac.uk/31432/ Kinship, gender and social links impact on micro group lending defaults EL FAKIR, Adil <http://orcid.org/0000-0002-0922-7274>, FAIRCHILD, Richard, LAMRANI ALAOUI, Youssef, CHAN, Dora, TKIOUAT, Mohamed and AMER, Zaid Kinship, gender and social links impact on micro group lending defaults EL FAKIR, Adil <http://orcid.org/0000-0002-0922-7274>, FAIRCHILD, Richard, LAMRANI ALAOUI, Youssef, CHAN, Dora, TKIOUAT, Mohamed and AMER, Zaid Available from Sheffield Hallam University Research Archive (SHURA) at: http://shura.shu.ac.uk/31432/ Available from Sheffield Hallam University Research Archive (SHURA) at: http://shura.shu.ac.uk/31432/ This document is the author deposited version. You are advised to consult the publisher's version if you wish to cite from it. R E S E A R C H A R T I C L E R E S E A R C H A R T I C L E Copyright and re-use policy See http://shura.shu.ac.uk/information.html See http://shura.shu.ac.uk/information.html Sheffield Hallam University Research Archive http://shura.shu.ac.uk Received: 28 May 2020 Revised: 20 November 2022 Accepted: 13 December 2022 Received: 28 May 2020 Revised: 20 November 2022 Accepted: 13 December 2022 DOI: 10.1002/ijfe.2786 Adil EL Fakir1 \| Richard Fairchild2 \| Youssef Lamrani Alaoui3 \| Dora Chan1 \| Mohamed Tkiouat4 \| Zaid Amer5 1College of Business Technology and Engineering, Sheffield Business School, Finance Accounting and Business Systems Department (FABS), Sheffield Hallam University, Sheffield, UK 2Management Accounting, Finance & Law, Centre for Business, Organisations and Society (CBOS), University of Bath, Bath, UK 1College of Business Technology and Engineering, Sheffield Business School, Finance Accounting and Business Systems Department (FABS), Sheffield Hallam University, Sheffield, UK Correspondence Correspondence Adil EL Fakir, College of Business Technology and Engineering, Sheffield Business School, Finance Accounting and Business Systems Department (FABS), Sheffield Business School, Sheffield Hallam University, Sheffield, UK. K E Y W O R D S default, gender, kinship, machine learning, microfinance, social links Funding information Superior Institutions of Science and Technology (SIST) - Associate College of Cardiff Metropolitan University, Funding Recipients: Adil EL Fakir and Zaid Amer Funding information Superior Institutions of Science and Technology (SIST) - Associate College of Cardiff Metropolitan University, Funding Recipients: Adil EL Fakir and Zaid Amer Kinship, gender and social links impact on micro group lending defaults Adil EL Fakir1 \| Richard Fairchild2 \| Youssef Lamrani Alaoui3 \| Dora Chan1 \| Mohamed Tkiouat4 \| Zaid Amer5 Int J Fin Econ. 2023;1–16. Abstract Joint liability aspires to improve micro-loan performance through the support of, and pressure from, the group borrowers. This paper examines how the group composition, in terms of the mixture of kinship (Family) ties and social (Friends and Neighbours) ties among the borrowers, affects the default rates. Using binary logistics regression and three machine learning models, responses from 507 group micro-loan borrowers from four major Moroccan cit- ies were analysed. The results show that the stronger the family and kinship ties are within a loan group, the higher is the default rate. On the contrary, the stronger the social ties are among the group, the lower is the default rate. Other key findings include that the diversion of fund usage from investment to consumption is not found to significantly cause default. Also, loan default can be a consequence of borrowers' strategic choice rather than financial distress. As compared to the female members, male borrowers are found to be causing higher default rates. Interestingly, the gender-related default rates are lower when more of the male borrowers are only socially related. Finally, group size is found to be positively associated with default. Our findings can help micro- finance institutions refine their lending policies and guidance on groups' com- position and size to reduce default rates. Finance Accounting and Business Systems Department (FABS), Sheffield Hallam University, Sheffield, UK 2Management Accounting, Finance & Law, Centre for Business, Organisations and Society (CBOS), University of Bath, Bath, UK 3Laboratory of Reseach in Applied Mathematics, IFE-Lab, Mohammadia School of Engineering, Mohammed V University in Rabat, Rabat, Morocco 4Laboratory of Research in Applied Mathematics, IFE-Lab, Mohammadia School of Engineering, Mohammed V University, Rabat, Morocco 5Superior Institutions of Science and Technology (SIST) - Associate College of Cardiff Metropolitan University, Casablanca, Morocco 2Management Accounting, Finance & Law, Centre for Business, Organisations and Society (CBOS), University of Bath, Bath, UK 3Laboratory of Reseach in Applied Mathematics, IFE-Lab, Mohammadia School of Engineering, Mohammed V University in Rabat, Rabat, Morocco 4Laboratory of Research in Applied Mathematics, IFE-Lab, Mohammadia School of Engineering, Mohammed V University, Rabat, Morocco 5Superior Institutions of Science and Technology (SIST) - Associate College of Cardiff Metropolitan University, Casablanca, Morocco 1.2 \| How group joint liability is linked to social and family and kinship ties? MF's operations bridge the social and economic spectrums (Bello-Bravo & Amoa-Mensa, 2019) and aim to serve the dual objective of financial sustainability and social outreach (Wijesiri et al., 2017, p. 63). While working towards their social and economic mission, MF provision is hindered by the risk of repayment default (Guha & Chowdhury, 2013; McIntosh & Wydick, 2005; Mia & Lee, 2017; Mohammed & Wobe, 2019; Murthy & Mariadas, 2017). For example, McIntosh and Wydick (2005) found a high credit risk facing the MF institutions (MFIs) in Bangladesh, illus- trated by 32% of the Grameen Bank's MF loans in the Tangail area being overdue by 2 years or more. Mohammed and Wobe (2019) study in Ethiopia revealed that 45% of the borrowers in the study area of Wondo Genet Woreda either did not or could not repay their loans following the credit schedules. The relationship between the members of a social net- work varies in tie strength. A network which is made up of members who have strong ties, such as family or kin- ship, is likely to develop a strong sense of solidarity and trust which can be used to leverage opportunistic behav- iour and build resources and resilience which are particu- larly critical at the times of uncertainty, adversity and/or hardship (Jackson & Young, 2016). Bourdieu (1973) sug- gested that families possess their own symbolic and/or material resources which can be used to generate benefits for their own members. Bonding capital manifests itself in family businesses. The bond among the owners and managers creates informal self-regulating, self- reinforcing control mechanisms which complement, and in some cases replace, the formal control systems which are emphasised by the Agency Theory to protect the interests of the principals (Mustakallio et al., 2002). Fam- ily businesses are often governed by a dual control sys- tem. These characteristics have empowered family businesses with a higher-than-average ability to survive, even in difficult times. Based on the above literature, the following two hypotheses are established: The sustainability of the MF sector relies on the loan repayment which, on one hand, replenishes the MFIs' credit capacity and, on the other hand, demonstrates bor- rowers' success in using the funds on productive eco- nomic activities. Funding information 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. g p p y © 2023 The Authors. International Journal of Finance & Economics published by John Wiley & Sons Ltd. wileyonlinelibrary.com/journal/ijfe wileyonlinelibrary.com/journal/ijfe 1 1.1 \| Why group lending? Obtaining start-up finance is challenging given that the sources are restrictive (Jackson & Young, 2016). Funds often come from personal savings and the support from family and friends Kotha and George (2012).This group(Mia & Lee, 2017) of entrepreneurs is often forced either to borrow via informal means, such as money lenders, at exceptionally high interest rates, or accept the exclusion of formal credit, thus losing out on investment opportunities (De Aghion, 1999). Micro-finance (MF) has been introduced as a mecha- nism which provides small sums of credit and other finan- cial services to the poor and vulnerable who do not have access to formal financial institutions (Mohammed & Wobe, 2019; Wijesiri et al., 2017). Micro loans are lent to the poor who lack the common lending criteria adopted by mainstream commercial banks, such as collateral, guar- antors and/or prior credit history. MF represents an inclu- sive approach for the financially excluded and can be an effective mechanism to alleviate poverty and promote socio-economic development and growth (Mohammed & Wobe, 2019; WorldBank, 2004). 1 \| INTRODUCTION 1.1 \| Why group lending? 2 EL FAKIR ET AL. (Abbink et al., 2006; Chowdhury et al., 2014). The pop- ularity of group lending can be explained by its reach to more people, thus widening the benefits to the wider community. Also, group lending can provide MFIs with a kind of social collateral as group members pro- vide guarantee to one another (Grameen System; Singh et al., 2017). When any member defaults, the rest of the group share the outstanding repayments, or all lose access to future loans (De Aghion, 1999). When a mem- ber defaults, they suffer a loss of reputation, social shame, deprived access to some or all of the social activities and resources, and ultimately the exclusion from the social group. Joint liability allows the MFIs to shift some of the costs and risk associated with group screening, loan monitoring and repayment enforce- ment to the borrowers (Besley & Coate, 1995; De Aghion, 1999; Dufhues et al., 2012; Mohammed & Wobe, 2019). Given the stated benefits of group lend- ing, why do MFIs face increasing rates of loan default? The question has driven this research study to explore whether and how the social and family ties among group members affect loan default. EL FAKIR ET AL. 3 H2. : The stronger the family ties between group members, the lower the probability of micro loan default H2. : The stronger the family ties between group members, the lower the probability of micro loan default fully aligned with the business needs (Mustakallio et al., 2002). The close ties between group members can have a negative effect on the rescheduling of loan repayment (Dufhues et al., 2012). Ahlin and Townsend (2007) study in Thailand revealed that the close bond and regular interaction between the group loan members can conceal important project information from the lender, impede social sanctions and promote collusion. All group mem- bers can work together to default on repayments and shield one another from the social pressure coming from the wider community (Chowdhury et al., 2014). While Mustakallio et al. (2002) refers to close ties as family links, the definition and measurement of tie strength have not been specified in the work of (Ahlin & Townsend, 2007; Dufhues et al., 2012). Since previous studies have found that close ties could exacerbate loan default, the following two hypotheses are established to further examine the nature and strength of ties on loan default: More recent research have switched their focus from quantitative to qualitative factors and examined the effect of different types of social capital and/or the balance between the social bonds and bridges on sustainable development (Fransen, 2015; Hunt et al., 2015; Jackson & Young, 2016; Serra, 2011). As Narayan and Pritchett (1999) argued, while strong social ties promote cohesion and inter-dependency among members, the lack of diversity in the group impedes innovation, skills, outlook, financial resources and opportunities. In a similar way, Jackson and Young (2016) observed that over-connected members tend to lack the variety of resources and opportunities to drive the transformation into high return produc- tion groups. Extending social interaction and relationships to dissimilar groups or individuals helps promote social tolerance and bridge social divides (Fransen, 2015). Intra-group interactions extend individuals' reach to a wider pool of information, intellectual capital, financial resources, power and/or opportunities which result in stronger bridging social capital (Fransen, 2015; Hunt et al., 2015; Nahapiet & Ghoshal, 1998; Paxton, 2002; Putnam, 2004; Putnam et al., 1994; Woolcock & Narayan, 2000) contributing to better social and eco- nomic outcomes (Hunt et al., 2015). Based on the above literature review, the following two hypotheses are drawn up for testing: H5. The stronger the family ties, the higher is the probability of micro loan default. H6. The stronger the social ties, the higher is the probability of micro loan default. In terms of the strength of social ties, we have hypothesised that neighbourhood ties are stronger than friendship ties as the former can carry out higher social sanction in the case of group loan default than the latter due to the proximity of locations (Besley & Coate, 1995). Besides the nature of the ties (Family and Kinship versus Social links) which forms the key variables of this study, a few other determinants of loan default are adopted as control variables. Explanations of the choice are illustrated below. H3. Micro loan groups with a higher diver- sity of social and family ties between mem- bers leads to a lower default rate than groups consist of only family members H3. Micro loan groups with a higher diver- sity of social and family ties between mem- bers leads to a lower default rate than groups consist of only family members H4. Micro loan groups with a higher diver- sity of social and family ties between mem- bers leads to a lower default rate than groups consist of only social members. 1.2 \| How group joint liability is linked to social and family and kinship ties? The CGAP 2009 states that a loan is in default when a borrower can or will not pay back the loan and when the MF institutions (MFIs) no longer expect the loan to be repaid (WorldBank, 2004). H1. : A micro loan group made up of family members (Kinship) has a lower probability of default than non-family (social ties) groups. Although there has been a movement towards indi- vidual lending over the past decade or so, group lend- ing has been widely adopted by MFIs worldwide H8. Financial hardship leads to group micro loan default. Loan purpose is claimed to be a determinant of loan default (Baesens et al., 2005). For example, Okorie (1986) found that borrowers who received a non-cash loan for investment purposes, such as seeds, fertilizer and equipment, demonstrated higher repayment rates than other borrowers who received cash loans. This was because some borrowers misused the cash, divert- ing it into personal consumption instead of investing the money in making their business more productive (Okorie, 1986). To examine the effect of age on loan default, this study will focus on respondents who are aged between 30 and 49. The reason behind the choice is that this age bracket covers the dominant segment, 46%, of the micro loan beneficiaries in Morocco (CentreMohammed, 2020), while the rest of the total percentage is almost equally distributed among three other age brackets. Nonetheless, some studies found that a higher loan default risk is associated with borrowers who used the funds for small business rather than non-small business purposes (Serrano-Cinca et al., 2015). This argument is supported by citepcader2011small who found more than 40% of the 90,134 small business samples observed failed after 3 years in business. In contrast to this high failure rate, Agarwal et al. (2007) found only 3.59% of the car loans (non-small business loans) defaulted out of a sam- ple of 6996 observations. Previous results on the impact of loan purpose on default were inconsistent. Other determinants of loan default, such as group size, gender, loan purpose and financial hardship, are adopted by this study. Group size is unique to group lend- ing rather than individual loans. Increased group size is believed to be more effective from the resource sharing perspective which benefits project performance and repayment ability (Ahlin, 2015). Along with this line, it is claimed that joint liability induces homogeneous match- ing (Ghatak, 1999, 2000). Nonetheless, there is a counter argument against large group size. For example, Ahlin (2015) found that homogeneous matching is lost when the group size is larger than two (n > 2). The study has also shown that if information deteriorates sufficiently with the growth of group size, an intermediate group size is better, for outreach and efficiency purposes, than both extremes (Ahlin, 2015). H9. Deviating funds from investment to per- sonal consumption leads to micro loan default. H9. Deviating funds from investment to per- sonal consumption leads to micro loan default. A group size of five is used by the Grameen Bank along with many of its replications, such as Green Bank (Giné & Karlan, 2014). Some lenders use slightly smaller groups, for example some Al Amana groups were 3–4 members (Crépon et al., 2015), while others use larger groups. As group lending can potentially benefit from lia- bility and resource sharing, the following hypothesis is established: It has been argued that female borrowers are better payers (Dinh & Kleimeier, 2007; Roslan & Karim, 2009; Salazar et al., 2008; Schreiner, 2004; Vigano, 1993) than their male counterparts. This is possibly due to their stronger work ethics and financial discipline (Bhatt & Tang, 2002; Pitt & Khandker, 1998). Compared to the male borrowers, females tend to be risk averse (Croson & Gneezy, 2009), and thus more likely to engage in less risky projects (Sharma & Zeller, 1997) and spend money on productive expenditure to enhance income and empower their family (Mohammed & Wobe, 2019). These characteristics in turn increase female borrowers' ability in loan repayment. The following hypothesis is estab- lished for testing: 1.3 \| Control factors affecting loan default rates We have chosen to include some of determinants identi- fied by previous studies in our study as control variables to test the relationship between the loan default rate and the main predictive variables (Family and Kinship and Social links). Mustakallio et al. (2002) argued that the close-tie emotions and relationships embedded in the gover- nance structure and mechanisms of family businesses can have both a supportive and destructive effect on the operation and strategic decision making, hence the economic performance. The owners and man- agers of family businesses are brought together by family ties, rather than by choice. Hence, their skill- set, vision, attitude, and commitment may not be Education level has been identified as a factor which influences loan repayment performance (Mohammed & Wobe, 2019). In our survey, micro loan borrowers are all having a lower level of education (sec- ondary school and below). This commonality has 4 EL FAKIR ET AL. enabled us to exclude education level as a determinant of default rate in this study. group composition before granting the loans (Nawai & Shariff, 2010). The following hypothesis is drawn up to test this aspect: The effect of age on loan default is mixed. For exam- ple, Mokhtar et al. (2012) found that borrowers in the 46–55 age group had a higher probability of having repayment problems. Mohammed and Wobe (2019) also found more defaulters in the 38–47 years group than in the younger age groups. These findings contradicted the common view which sees older borrowers as more responsible for loan repayment, whereas young and inex- perienced borrowers, due to their age and immaturity, would increase the default rate (Dorfleitner et al., 2017). H8. Financial hardship leads to group micro loan default. Since one of the main objectives of micro loans is to initiate or grow small businesses, moving away from the main investment objective seems more likely to be leading to non-productive activities and hence loan default. We therefore propose to test the following hypothesis: EL FAKIR ET AL. 5 H10. Male dominated micro loan groups are more likely to default than female dominated groups. H10. Male dominated micro loan groups are more likely to default than female dominated groups. The remainder of the paper is organised in the follow- ing way: Methodology of this paper is explained in Section 2. The analysis and findings are illustrated in Section 3 and finally a summary, recommendations, pol- icy implications and extensions are presented in the con- clusion section. 2 \| METHODOLOGY Facing increasing default rates and criticism of inefficiency (Wijesiri et al., 2017), over reliance on private funds and even mission drift (Mia & Lee, 2017), MFIs need to find ways of improving their policies and approach to accom- plish the dual social and financial inclusion mission. This study has pulled together the literature on social capital theory and loan repayment performance and default with the aim of identifying how the characteristics of the bor- rowing groups influence loan default. The key objective of this study is to help MFIs refine their lending policies and guidance to reduce the loan default rates and develop sus- tainable credit capacity. The study also aims to support the borrowers in partner selection and group relationship management. In order to accomplish the research objec- tive, this study aims at using the logistics regression method as many credits default studies have used it where the dependent variable is binary Vallini et al. (2008). How- ever, the logistics regression itself, as a traditional statisti- cal method, suffers from relying on strong assumptions, such as the type of error distribution, additivity of the parameters within the linear predictor, and proportional hazards Rajula et al. (2020). This is not the case when using machine learning (ML) methods. The later methods have the advantage of training statistical models from his- torical data citepmuller2016introduction. This feature makes machine-learning models focused on making pre- dictions as accurate as possible, while traditional statistical models are aimed at inferring relationships between vari- ables Rajula et al. (2020). ML is at the intersection of many other disciplines like statistics and computer science. While traditional statistical models focus on metrics such as R2, p-values and statistical significance, the ML tech- niques focus on out-of-sample forecasting and the bias- variance trade-off (Gogas & Papadimitriou, 2021).Unlike traditional statistical methods, the ML techniques have rel- evant importance due to their limited dependence on assumptions and their importance in processes automation (Li et al., 2020).The application of machine learning in our study is also supported by the fact that this is widely used in the literature of credit risk evaluation in microfinance (Bakshi, 2021; Beketnova, 2020; Bhatore et al., 2020; Condori-Alejo et al., 2021; Ruiz et al., 2017). Given the qualities that machine learning possesses, we contrast the result from the traditional logistic regression with that H7. Bigger group size decreases micro loan default rate. H7. Bigger group size decreases micro loan default rate. Prior studies, such as Greenbaum et al. (2019), Hoque (2010), Coyle (2000), Özdemir and Boran (2004), Mokhtar et al. (2012) have shown that loan default may be a result of borrowers' unwillingness and/or inability to repay. These results have reflected the importance of initial screening of borrowers' ability and commitment and the were h ln P Y ð Þ 1P Y ð Þ h i is the log odds of loan defaults.where Y is the dichotomous outcome which represents credit default (whether the loan was repaid or not) while Xi. i {1…n} are the predictor variables which are Strong family links (SFL), Weak Family Links(WFL), Neigh- bourhood (NEI) and Friendship (FRI). They represent the number of members in each category in the whole group. 6 ln P Y ð Þ 1P Y ð Þ ¼ β0 þβ1SFLþβ2WFLþβ3NEI þβ4FRI ð1Þ ln P Y ð Þ 1P Y ð Þ ¼ β0 þβ1SFLþβ2WFLþβ3NEI þβ4FRI Four cities were included in the survey. Casablanca is taking the largest part in terms of responses given its pop- ulation size and comparative economic power compared to the rest of the cities. With the exception of the year 2020 which has limited data (due to Corona Virus out- break and lock-downs), the data are fairly distributed along the period of the study. ð1Þ 2.1 \| Data collection Data for this Study were collected in Morocco and obtained from 507 clients who had an experience of being part of a joint liability micro-loan group. An Ini- tial target of 1000 survey was planned but such a target had proved to be difficult to reach given the reluctance of some of the respondents to engage fully or partici- pate in the survey. However, we have managed to col- lect a sample of 507 respondent, representing a response rate of 50.7%. The data collection period was initially set to be 3 years, yet the difficulty in collecting the data was an incentive to extend the study period to 6 years (2015–2020). The data collection started ini- tially at SIST-Cardiff Metropolitan University Branch in Morocco and continued in 2020 in collaboration with Sheffield Hallam University. Due to the geograph- ical dispersion of the survey, students from the four dif- ferent campuses in the country [Marrakesh (South), Casablanca (West), Rabat (West), and Tangier (North)] provided the necessary support in running the survey and collecting its data. The survey targeted respondents with low education level (primary to Secondary) and age range of 30–49. This is to exclude the age and edu- cation effect on default rate. The survey questions (see Appendix) are divided to three parts: I. Dependent variable questions, II. Control variable questions, III. Predictive Variable questions. The survey starts by asking the subjects about the main dependent variable question. That is, whether their group loan experienced a default. The second part of the survey focuses on the control variable questions: The purpose of the loan, Financial Hardship in paying the loan, Group Gender composition, and the size of the group. And finally, the third part of the survey focused on the predictive variables of interest. Strong family links (SFL) represents group members who are of the immedi- ate family of the interviewee. Due to the variety of defini- tion around members to include in this category, we restrict it to include (Spouse, Parents, Grandparents, Children, Grandchildren, Siblings and In-laws mother, father, brother, sister, daughter and son). 6 6 EL FAKIR ET AL. EL FAKIR ET AL. 6 TABLE 1 Survey Responses by year and location City District 2015 2016 2017 2018 2019 2020 Total Casablanca Lissassfa 13 5 5 8 5 7 43 Oulfa 7 4 4 5 9 6 35 Derb seltan 3 6 8 4 2 8 31 Hay mohammad 5 5 5 3 4 4 26 Derb Omar 7 5 7 2 9 5 35 Derb Ghallaf 5 3 2 4 2 3 19 Subtotal 40 28 31 26 31 33 189 Rabat Diour jamaa 5 9 5 10 2 3 34 Douar ihajja 5 6 10 4 8 3 36 Hay taqaddoum 4 7 5 9 4 3 32 Subtotal 14 22 20 23 14 9 102 Marrakesh Azzouzia 4 9 6 6 3 0 28 Bab Doukkala 3 8 7 8 8 0 34 Rahba Kedima 5 4 9 4 5 0 27 Bab Taghzout 4 5 2 9 6 0 26 Subtotal 16 26 24 27 22 0 115 Tangier Beni Makada 5 3 12 2 2 0 24 Charf Souani 8 8 4 4 5 0 29 Boukhalef 7 7 7 3 3 0 27 Charf Mghogha 7 8 2 2 2 0 21 Subtotal 27 26 25 11 12 0 101 Total 97 102 100 87 79 42 507 regression method is the choice adopted for this study. This method is widely used in credit default studies where the dependent variable is binary Vallini et al. (2008). This default is assessed in two stages. In stage 1, the probability of default is assessed through the pri- mary predictive variables of this study: Kinship variables (WFL, SFL) and Social links variables (NEI, FRI). This stage excludes the control variables. The probability of default, P, is given as: Weak family links (WFL) refer to members of the group who are part of the extended family. This category includes all family member other than the immediate family members described in the strong family category. Friends (FRI) are members in the group who do not fit in the neighbourhood category or family category of anyone member in the group. Neighbours (NEI) are members in the group who do not fit neither in the friends category nor in the family category of anyone member in the group1. We provide the number of responses by year and location (see Table 1). 3 \| RESULTS The second stage includes all the primary predic- tive variables in addition to the control variables: Loan Purpose (LoanPurpose) default due to financial hardship (FinHardship) and number of males in the group (MaleGr) and the number of group members (GroupSize). 2.2 \| Model specification The response variable, Default, is a binary variable (1 for default or 0 for no default). Therefore, the logistic 7 EL FAKIR ET AL. 7 FIGURE 1 Members type weighted presence in a group [Colour figure can be viewed at wileyonlinelibrary.com] FIGURE 1 Members type weighted presence in a group [Colour figure can be viewed at wileyonlinelibrary.com] URE 1 Members type weighted presence in a group [Colour figure can be viewed at wileyonlinelibrary.com EL FAKIR ET AL. 8 8 TABLE 2 Model goodness of fit Model summary Hosmer and Lemshow test Model predictability 2-Log likelihood Cox and Snell R square Neglekerke R square Chi- square σ Sig Observed default (training set) Predicted default % Observed no default (training set) Predicted no default % Overall percentage accuracy 412 0.304 0.405 8.379 8 0.397 188 80.3% 217 77.9% 79% TABLE 3 Logit model: Control variables included Variable β SE Wald Sig Odds EXP(β) WFL 0.299 0.205 2.118 0.146 1.348 SFL 0.423* 0.0.253 2.802 0.094 1526 FRI 0.437* 0.264 2.750 0.097 0.646 NEI 0.533* 0.192 7.690 0.006 0.587 Loan Purpose 0.273 0.292 0.872 0.350 0.761 FinHardship 0.084 0.251 0.113 0.737 0.919 MaleGr 0.445* 0.154 8.319 0.004 1.560 Constant 0.558 0.641 0.757 0.384 0.572 Significant at 10%. Significant at 1%. TABLE 2 Model goodness of fit TABLE 4 Hypothesis validation using χ2 test Variable χ 2 df Sig WFL 19.4* 4 0.001 SFL 96.9* 4 0.00 FRI 38.44* 2 0.00 NEI 117.4* 4 0.00 Loan Purpose 3.3 1 0.68 FinHardship 0.98 1 0.32 MaleGr 87.36* 3 0.00 GroupSize 11.182 2 0.04 Significant at 5%. ***Significant at 1%. TABLE 4 Hypothesis validation using χ2 test The sample also shows that groups are mainly com- posed of strong family links members and friends than from neighbours or weak family links groups (see Figure 1b). In terms of Control variables, male presence (58%) in groups dominates their female (42%) counter- parts (see Figure 1f). In terms of the objective of the loan (see Figure 1d), the sample shows that the majority (66%) had the intention to use the loan for investment pur- poses. While this is the main purpose of a micro-finance loan, 34% have shown a deviation from this purpose and that their intention was to buy consumer goods rather than starting a small business. In terms of financial dis- tress (see Figure 1e), 57% of the groups genuinely suf- fered from a financial distress while 43% did not. This probably could suggest that default can at times be volun- tary rather than a forced outcome of financial distress. ties on the risk of default. We will be using a training set of 80% and a testing set of 20%. ties on the risk of default. We will be using a training set of 80% and a testing set of 20%. 3.2.1 \| Robustness of the model In terms of the explanatory power of the model both Pseudo R2(Cox & Snell = 0.304 and Negelkerke = 0.405 in Table 2) show that independent variables explain an important percentage of the dependent variable (default). In terms of the number of members in a typical group (see Figure 1c), nearly half the sample shows that group are composed of four members or higher. This is followed by groups of three members at (31%) and then groups with two members (21%). 3.1 \| Descriptive results We describe the distribution of the studied sample in terms of the predictive variable and the control variables (see Figure 1). Loan purpose is a binary variable that takes two values: 1 if the loan is for investment purposes and 0 oth- erwise. The same applies to the ‘Financial hardship’ vari- able. It takes 1, if the group suffered financial hardship or 0 otherwise. The probability of default, P, in this stage, is given as: In terms of members type presence, the weighted average presence of each type in a group is presented. We can note, in this sample, a slight dominance of the social groups (52%) in group lending compared to the kinship groups presence (48%; see Figure 1a). In terms of sub-categorical formation, we can note the following: From a social link side point, groups are mainly formed of friends than of neighbours. With an overall presence of 38% in all groups, friends' members represent more than three times the presence of neigh- bours (see Figure 1b). ln P Y ð Þ 1P Y ð Þ ¼ β0 þβ1SFLþβ2WFLþβ3NEI þβ4FRI þ β5LoanPurposeþβ6FinHardship þ β7MaleGr þβ7GroupSize ð2Þ ln P Y ð Þ 1P Y ð Þ ¼ β0 þβ1SFLþβ2WFLþβ3NEI þβ4FRI þ β5LoanPurposeþβ6FinHardship þ β7MaleGr þβ7GroupSize ð2Þ From a kinship link, we can note the dominance of strong family links as opposed to weak family links. With an overall presence of 30% in all groups, Strong family links members represents more than two times the presence of weak family links (see Figure 1b). Given the qualities that machine learnings possess, as mentioned in the introduction, we will contrast the logis- tics regression results with those from the logistics regression. 8 4 \| DISCUSSION Based on the χ2 there was sufficient statistical signifi- cance to accept or reject all but 2 of the hypothesis (see Table 5). Based on the χ2 there was sufficient statistical signifi- cance to accept or reject all but 2 of the hypothesis (see Table 5). We discuss these hypotheses and the decisions around them in two steps: (1) primary Predictive vari- ables and (2) control variables. 4.1 \| Hypothesis testing results: Primary predictive variables H1–H6 are hypothesis around the primary predictive var- iables (Social links and Kinship Lonks) impact on the Group lending default rate. In terms of the first hypothesis, the results in Tables 3 and 1 show that the family links variables (SFL, WFL) are having higher odds of default in comparison with the social links variables (FRI, NEI). This result goes against the findings of (Bourdieu, 1973; Mustakallio et al., 2002) who are advocates of strong family ties as a positive fac- tor in business success. Our results, then, suggests that for a successful group liability loan, a higher weighting in terms of group members should be given to social mem- bers, such as friends and neighbours, rather than family members. In terms of goodness of fit, The Hosmer and Leme- show test (0.397 < 0.5) is not significant. This means that the model has a good fit. In terms of the predictive ability of the model, the classification table shows that the model classifies groups correctly in their original groups with a rate of 79%. Approximately 80% of defaulted groups and 78% of solvent groups are cor- rectly classified. In terms of H2, the results shows that the stronger the family links are, the higher are the odds of loan default. This is against the findings of Jackson and Young (2016) who suggests that stronger family ties provide stronger solidarity and trust among members which can used to leverage opportunistic behaviour and build resources and business resilience. On the other hand, our findings are aligned with those of Dufhues et al. (2012), and Ahlin and Townsend (2007) revealed that the close bond and regular interaction between the group loan members can conceal important project information, impede social sanctions, and promote collusion. Our results then sug- gest that if a group is to be composed of family members, then members who are of strong family ties (immediate family) should not be part of the group. Possible explana- tion of this, could be that each member of the family strongly relies on the performance of the other members. Therefore, even if default occurs, social penalty is less severe as it is self-contained within the household and members shield one another from the social pressure EL FAKIR ET AL. TABLE 5 Hypothesis testing results Hypothesis Decision H1: A group composed of family members (Kinship) have a lower probability of default than non-family (social ties) member groups. Reject H2: The stronger the family ties are, the lower is the probability of default. Reject H3: Diversity in term of social and kinship ties in a group lead to lower default rate than Family groups only. Accept H4: Diversity in terms of social and kinship ties in a group lead to lower default rate than social groups only. Reject H5: The stronger the family ties are, the higher is the probability of default. Accept H6: The stronger the social ties are, the higher is the probability of default. Reject H7: Higher group size decreases the default rate. Reject H8: Financial Hardship leads to group default. Not enough evidence to accept H9: Deviating funds to consumption rather than investments leads to default. Not enough evidence to accept H10: Male dominated groups are more likely to default than their female counterparts. Accept The p-values are not significance values for the null hypothesis themselves. Therefore, we run a Chi-square test (see Table 4) and use the significance of such test to validate the hypothesis: 3.2 \| Results of the logit model The aim of this logistic model is to investigate the effect of the group composition in terms of kinship and social H8, tests whether financial hardships lead to default. Our results are quite surprising as this factor impact was not found significant in leading to default. This suggests that group default can be voluntary rather than an inevi- table consequence of financial hardship. There is not enough evidence to suggest that financial hardship can lead to default in group lending. H8, tests whether financial hardships lead to default. Our results are quite surprising as this factor impact was not found significant in leading to default. This suggests that group default can be voluntary rather than an inevi- table consequence of financial hardship. There is not enough evidence to suggest that financial hardship can lead to default in group lending. H9, verifies whether deviating funds to consump- tion, rather than investment, leads to default. our results shows that there is not enough evidence to sug- gest that loan purpose affects default rate. Our results, therefore, do not support the views of Baesens et al. (2005) and Okorie (1986) who claim that deviating funds to consumption leads to default. Our results do not, as well, support the findings of Serrano-Cinca et al. (2015), Cader and Leatherman (2011), Agarwal et al. (2007) who claim that the odds of investments (small businesses) to default are higher than those of con- sumption (car loans). H9, verifies whether deviating funds to consump- tion, rather than investment, leads to default. our results shows that there is not enough evidence to sug- gest that loan purpose affects default rate. Our results, therefore, do not support the views of Baesens et al. (2005) and Okorie (1986) who claim that deviating funds to consumption leads to default. Our results do not, as well, support the findings of Serrano-Cinca et al. (2015), Cader and Leatherman (2011), Agarwal et al. (2007) who claim that the odds of investments (small businesses) to default are higher than those of con- sumption (car loans). While H5 has been discussed in conjunction with H2, H6 is largely aligned with H4. While H6 emphasises the importance of social ties over kinship ties, it adds an extra layer: that of the social strength. As previously stated, while laying out the foundation for the hypothesis in the introduction section, we assumed that neighbours have stronger ties than friends as they carry higher social pressure and social sanction (Besley & Coate, 1995). From Tables 3 and 1, we can see that neighbours have the highest contribution to ‘no default’ compared to friends. This means that the stronger the social ties are, the lower is the probability of default. Our finding contra- dicts those of Dufhues et al. (2012) and Ahlin and Town- send (2007), even though the strength of the ties, in their study, is not specified. That is, whether the ties are family ones (kinship) or social ones (friends and Neighbours). H6 is therefore rejected. In terms of H10, we verify whether males are having more odds to default than females. From Table 3, this result is confirmed as males are nearly three times more likely to default. This result is consistent with previous literature (Bhatt & Tang, 2002; Croson & Gneezy, 2009; Dinh & Kleimeier, 2007; Pitt & Khandker, 1998; Roslan & Karim, 2009; Salazar et al., 2008; Schreiner, 2004; Sharma & Zeller, 1997; Vigano, 1993). Many possible reasons for this outcome have been sug- gested by the former literature where females have higher risk aversion, higher hard work ethics and finan- cial discipline compared to males. We would also add that in developing countries, such as Morocco, females with low education level, are usually housewives, and spend more time engaging and socialising with their female neighbours. The high social engagement of females, compared to males, make them more socially concerned about the social sanction they can be subject to in case they default on their joint liabilities. 10 EL FAKIR ET AL. come from the wider community. While this result rejects H2 it does confirm our H5 that stronger family ties increase the odds of default. H7, tests whether or not an increase in the group size, should lead to lower default rate. Our findings suggest that an increase in the size of the group increases the default rate. This result is inconsistent with the findings of (Ahlin, 2015; Cassar et al., 2007; Ghatak, 1999; Ghatak, 2000) who claim that an increase in group size induces more social pressure to pay and foster homoge- neous matching. Our results are however consistent with the findings of Ahlin (2015) (homogeneous matching is lost with bigger size) and (Ahlin, 2015; In intermediate group are more efficient). Our findings therefore reject H7. A possible explanation for this result is that the higher is the group size, the more likelihood that the group suffers from free riders who rely on others to pay the group loan. We recommend lender to exercise more precautionary measures for larger groups. In terms of H3, the results in Tables 3 and 1, show that family members contribute positively to default while Neighbours and friends reduce it. This suggest that, by introducing members of the social category (friends and neighbours), default will be reduced than if the groups is composed of family members only. We there- fore accept H3. H4 is different from H3 as it tries to test whether diversity in the group leads to a lower default rate than if the groups are composed of social members only (friends and Neighbours). The results from Tables 3 and 1 show that family members contribute positively to default while Neighbours and friends reduce it. This suggests that including family members in a diversified group should exacerbate default rather than reduce it. While the covered literature recommends diversity in the group (Fransen, 2015; Fransen, 2015; Hunt et al., 2015; Nahapiet & Ghoshal, 1998; Paxton, 2002; Putnam et al., 1994; Putnam, 2004; citepwoolcock2000social Hunt et al., 2015), our recommendation is rather conditional. We recommend diversity in a group that contains family members so that the defaulting odds are mitigated by social pressures from friends and members. We do not recommend diversity in a group that contains friends and neighbours as introducing family members would simply exacerbate the odds of default. We therefore reject H4. 3.2.2 \| Regression results The Logit model results (see Table 3) show, based on the odds ratio, that strong family links in a group have the highest contribution to default followed by groups with weaker family links. On the other hand, social links reduces the default rate. Groups composed of neighbours are better than groups composed of friends in lowering default. ‘Loan purpose’ and ‘Financial Hardship’, on the other hand, have a negative β but was not significant. However, the p-values of the logit fit give the significance of the given variate in influencing the predicted result. 11 EL FAKIR ET AL. TABLE 6 Logistics regression compared with other machine learning models Model Accuracy score Sensitivity AUC Logistics regression 0.77 0.82 0.81 SVM 0.76 0.8 0.81 CART 0.74 0.78 0.72 KNN 0.77 0.8 0.78 FIGURE 2 Receiver operating characteristic curve for different models [Colour figure can be viewed at wileyonlinelibrary.com] TABLE 6 Logistics regression compared with other machine learning models KNN is based on features similarity. Its main idea is to assign a new object to the most frequent class among its K nearest neighbours. Model Accuracy score Sensitivity AUC Logistics regression 0.77 0.82 0.81 SVM 0.76 0.8 0.81 CART 0.74 0.78 0.72 KNN 0.77 0.8 0.78 Ten-folds cross validation is used to optimise the hyper parameters of the different machine learning models. The models are evaluated using four metrics (see Table 6): Accuracy score, Sensitivity, area under curve (AUC) and receiver operating characteristic curve (ROC; see Figure 2). The accuracy score is the number of the classes correctly predicted to the total number of samples. The sensitivity represents the percentage of actual defaulted groups that are predicted as defaulted group. The area under curve (AUC) is a metric that ranges from 0 to 1. The higher the value of the AUC is, the higher the performance of the model is. FIGURE 2 Receiver operating characteristic curve for different models [Colour figure can be viewed at wileyonlinelibrary.com] The obtained results show that the logistic regression is marginally superior to the other three models (much closer to the SVM model). The ROC curve (see Figure 2) plots the sensitivity (the percentage of well-classified defaulted groups) against 1- specificity (the proportion of false defaulted outcomes) at various probability thresholds. In our case, we noticed that all the ROC curves are close to the upper left corner, which indicates a high level of overall accu- racy. Based on the model prediction, the area under the ROC curves (AUC = 81% for the logistics regression and SVM) are also calculated; they show that the models have a good capacity to discriminate between defaulted and solvent groups. FIGURE 2 Receiver operating characteristic curve for different models [Colour figure can be viewed at wileyonlinelibrary.com] 4.4 \| Further investigation of the control variables Our study focuses on the use of the logistics regression to predict credit default in microfinance. However, the suit- ability of this method needs to be contrasted with machine learning methods given the qualities they pos- sess as discussed earlier in the introduction. Below is a description of three widely machine learning models that are going to be used for this purpose: The results around the control variable shows that two variables (financial hardship, loan purpose) are insignifi- cant to default while Gender and Group Size are signifi- cant. Due to their significance, we analyse Gender and Group Size further. • The support vector machine (SVM) is a predictive model that can be used to solve both linear and non- linear problems. The idea of SVM is to find the best separation hyper-plane to separate two or different classes using the training data. 4.2 \| Hypothesis testing results: Primary predictive variables H7–H10 are hypothesis around the control variables (Gender, Financial Hardship, Loan Purpose, Group Size) impact on the Group lending default rate. 4.4.1 \| Further investigation: Gender We show the default rate for females and males in differ- ent social and kinship categories (see Figure 3). Interest- ing results emerged typically for males in different social and kinship categories. We can see clearly that the high- est default rate of males happens in a group dominated by strong family links. The default is lessened as the strength of the family links weaken (WFL). Males have their lowest rates of default when they are part of a socially dominated group (Friends and Neighbours). • The classification and regression tree (CART) classifier is a popular predictive if-else algorithm that works for both continuous and categorical variables. One of the most import advantages of CART algorithm is that it allows simple interpretation and visualisation of data patterns. • The K-nearest neighbours (KNN) is one of the simplest and the popular machine learning algorithms. The 12 12 EL FAKIR ET AL. 12 FIGURE 3 Default by gender [Colour figure can be viewed at wileyonlinelibrary.com] FIGURE 3 Default by gender [Colour figure can be viewed at wileyonlinelibrary.com] FIGURE 3 Default by gender [Colour figure can be viewed at wileyonlinelibrary.com] FIGURE 4 Default by group size [Colour figure can be viewed at wileyonlinelibrary.com] FIGURE 4 Default by group size [Colour figure can be viewed at wileyonlinelibrary.com] The visual remark is not enough to draw an empirical conclusion. We therefore run an association test between males defaulting and their membership to either social or kinship groups. In this regard, the Chi-squared tests are significant at 1% level suggesting a strong association between males defaulting and kinship or social ties (see Figure 3c). We can then support the graphical visualisa- tion that as we go from a family dominated group (SFL and WFL) to a Social Group (FRI and NEI), male defaults decreases. to a female in a developing country. We can conclude from this, that males care more about the social sanction coming out from friends and neighbour, than they do in a family context. To reduce the default rate, it is then recommended that males are to be included in a social group rather than a family group. Females on the other hand have maintained relatively their high level of ‘no default’. This suggests that females can fit into any of the social and kinship groups while maintaining a very low default rate. 4.4.2 \| Further investigation: Group size We show that larger groups carry the highest default rate (see Figure 4a). This is consistent with our decision to reject H7 that higher group size decreases default. Adding the subcategories of default together (Kinship links = WFL + SFL, Social links = FRI + NEI), we can assess default of social and kinship cat- egories according to group size (see Figure 4b). We can see once again that default is higher in larger group size, and it is apparent more in social links groups than it is in Kinship groups. This shows that social links group may lose their default reducing ability if a group size is large (in our case N > 3). We can conclude from Figure 4 that large group size exacerbates default for both social and kinship groups with more impact on social groups. The results from this study can have important policy implications for micro-finance institutions. Namely, groups are to be either socially dominated groups (first best case) or diverse in terms of the composition (Social and Kinship). Groups which are kinship dominated are not recommended as they exacerbate the default rate. Another policy implication is the confirmation of female's better repayments rates compared to males. but we suggest a solution for male's high default rate. Males are recommended to be part of a socially dominated group (Friend and Neighbours) instead of a family domi- nated group. 13 EL FAKIR ET AL. different group sizes. On the other, males default rate is shown to decrease as the group size increases (see Figure 3b). it. However, what we found is (1) that females main- tained their superior repayment ability across different social and kinship groups and group sizes; and (2) that males default rate is increased if they are part of a kin- ship dominated group and is reduced if they are part of a Socially dominated group. To conform that there is an association between group size and males default we run a Chi-squared test. The latter test is significant at 1% level suggesting a strong association between males defaulting and group size (see Figure 3d). We can then support the graphical visualisation that as group size increases male defaults decreases. We can conclude that to decrease the males default rate, it is advisable that they are included in a large socially dominated groups (friends and Neighbours). Another control variable, Group size, was con- firmed to increase the default rate. What we found however is that group size has a bigger impact on the default rate when the group is socially dominated (Friends and Neighbours) and that a normal group size is better, in terms of lower default rate, than the two extremes. The other two variables were found insignif- icant: Financial Hardship and Loan Purpose. Namely, the fact that, groups genuinely incurred a financial hardship was not an explanatory factor of default. This means that group defaults could be voluntary rather than an inevitable consequence of financial hardship. Similarly, diverting funds to consumption rather than its original investment destination, was not found to be explaining defaults. This suggests that even if the funds were not invested as originally planned in small business activities, defaults may not necessarily occur. 4.4.1 \| Further investigation: Gender When it comes to group size, we can notice that females have shown a very low default rate across the This could be explained by a cultural factor in terms of males exercising a relative dominant power compared 14 EL FAKIR ET AL. 14 Beketnova, Y. M. (2020). Analysis of possibilities to automate detec- tion of unscrupulous microfinance organizations based on machine learning methods. Finance: Theory and Practice, 24(6), 38–50. ENDNOTE Crépon, B., Devoto, F., Duflo, E., & Parienté, W. (2015). Estimating the impact of microcredit on those who take it up: Evidence from a randomized experiment in Morocco. American Eco- nomic Journal: Applied Economics, 7(1), 123–150. 1 If a member is not a family member then the interviewee was ver- bally asked to specify if the member is a friend or neighbour. Croson, R., & Gneezy, U. (2009). Gender differences in preferences. Journal of Economic Literature, 47(2), 448–474. ACKNOWLEDGEMENTS The authors are indebted to the anonymous referees and the editor for their extensive and valuable comments. The authors would like to thank Sheffield Business School at Sheffield Hallam University, SIST- Associate College of Cardiff Metropolitan University, EMI School of Engineering, LERMA and IFE-lab, Uni- versity of Bath and IDB for their support in delivering this work. Special thanks go to Robert Marshall (who has now left the university), Denzil Watson, Firoz Bhaiyat, Rob Wilson, Mark Thompson, Damion Taylor, Jayne Revill, Lucian Tipi, Sean Kemp and everyone in the FABS team at Sheffield Hallam University for all their support and extensive efforts in helping to deliver this work. Bello-Bravo, J., & Amoa-Mensa, S. (2019). Scaffolding entrepreneur- ship: A local SME-NGO partnership to enable cocoa production in Ghana. Journal of Small Business & Entrepreneurship, 31(4), 297–321. Besley, T., & Coate, S. (1995). Group lending, repayment incentives and social collateral. Journal of Development Economics, 46(1), 1–18. Bhatore, S., Mohan, L., & Reddy, Y. R. (2020). Machine learning techniques for credit risk evaluation: A systematic literature review. Journal of Banking and Financial Technology, 4(1), 111–138. Bhatt, N., & Tang, S.-Y. (2002). Determinants of repayment in microcredit: Evidence from programs in the United States. International Journal of Urban and Regional Research, 26(2), 360–376. 5 \| CONCLUSIONS In this study, we tried to investigate the impact of group composition in terms of Social (friends and neighbours) and Kinship (strong family and weak fam- ily) ties on the default rate of Joint Liability Loans. We have found statistical evidence that Kinship ties con- tribute positively to default while social ties reduce it. more specifically, the stronger the kinship ties, the higher is the probability of default. Control variables (Loan Purpose, Gender, Financial Hardship, Group Size) were introduced in this study. In terms of Group Size, it is recommended that groups are of normal size (in our case N = 3). This case is shown to perform better than the extremes (N = 2 or N > 3). Increasing then size above the normal size, may nullify the benefit of a lower default rate characterising a socially dominated group and exacerbates the default of a kinship dominated group. Finally, Loan Purpose and Financial Difficulty are found not significant in identifying the default rate, therefore we recommend giving them the least scoring in loan and default evaluation. The introduction of the control variables did not change the impact of the main predictive variables of our study (Social and Kinship ties variables), but rather confirm some literature results and give rise to new ones. For example, females were confirmed, consistent with literature, to reduce default while males increase For further extension of this work, we propose extending the survey in different developing countries. The findings should confirm whether the current results are extendable and, therefore, whether policies can be applicable in different counties. ORCID Adil EL Fakir https://orcid.org/0000-0002-0922-7274 Adil EL Fakir https://orcid.org/0000-0002-0922-7274 Coyle, B. (2000). Framework for: Credit risk management. Global Professional Publishing. 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https://openalex.org/W2806968298	https://www.nature.com/articles/s41598-018-23565-2.pdf	English	null	CD4+ and CD8+ T Cells Exert Regulatory Properties During Experimental Acute Aristolochic Acid Nephropathy	Scientific reports	2,018	cc-by	10,684	CD4+ and CD8+ T Cells Exert Regulatory Properties During Experimental Acute Aristolochic Acid Nephropathy Apart from AAN, the role of the immune system has been largely studied in both ischemic and other toxic acute kidney injury (AKI) models. In response to acute tubular necrosis (ATN), inflammatory cytokines produced by PTEC, resident and infiltrating leukocytes contribute to the accumulation of inflammatory cells into the interstitium12. The roles of various immune cells, including dendritic cells (DC), natural killer T-cells (NKT), T and B lymphocytes, neutrophils and mφ have been described12, all of which ultimately lead to tubular atrophy and interstitial fibrosis. However, the respective role of each of these cell types remains controversial since the observed data depends on the type of model used. Moreover, the role of these different T-cell types has never been studied in experimental AAN. We therefore decided to investigate the potential role of CD4+ and CD8+ T-cells during the acute phase of toxic nephropathy, by studying the effects of selective depletion using monoclonal antibodies. 1Laboratory of Experimental Nephrology, Faculty of Medicine, Université libre de Bruxelles, Brussels, Belgium. 2Service of Nephrology, Dialysis and Renal Transplantation, Erasme Hospital, Université Libre de Bruxelles, Brussels, Belgium. 3Molecular Physiology Research Unit — URPhyM, NARILIS (Namur Research Institute for Life Sciences), University of Namur (UNamur), Namur, Belgium. Joëlle L. Nortier and Jean-Michel Hougardy contributed equally to this work. Correspondence and requests for materials should be addressed to T.B. (email: tbaudoux@ulb.ac.be) Human aristolochic acid nephropathy (AAN) was formerly known as “Chinese herb nephropathy”. This tubulointersti- tial nephritis was first reported in Belgian women after ingestion of herbal slimming remedies containing aristolochic acids (AA). These nitrophenanthrene derivatives were found responsible for the so-called Balkan endemic nephropa- thy and for hundreds of cases of chronic renal failure in China and Taiwan where traditional herbal medicines are still widely used1–3. Histologically, AAN displays an interstitial fibrosis with a typical corticomedullary gradient and tubular atrophy4,5. AAN was reproduced in several animal models including rabbits, mice and rats6–9. Prior time courses studies of our Wistar rat model demonstrated a biphasic evolution of tubulo-interstitial lesions10,11. During the acute phase, a necrosis of proximal tubular epithelial cells (PTEC) followed by a macrophage, CD4+ and CD8+ T-cell infiltration were observed. Thereafter, interstitial fibrosis and tubular atrophy were the hallmark of the chronic phase. In this model, inflammatory infiltrate (macrophages (mφ) followed by CD4+ and CD8+ T-cells) were proposed as the physiopatho- logical link between the acute and the chronic phases10. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 15 December 2017 Accepted: 14 March 2018 Published: xx xx xxxx CD4+ and CD8+ T Cells Exert Regulatory Properties During Experimental Acute Aristolochic Acid Nephropathy Received: 15 December 2017 Accepted: 14 March 2018 Published: xx xx xxxx Experimental aristolochic acid nephropathy is characterized by transient acute proximal tubule necrosis and inflammatory cell infiltrates followed by interstitial fibrosis and tubular atrophy. The respective role of T-cell subpopulations has never been studied in the acute phase of the mouse model, and was heretofore exclusively investigated by the use of several depletion protocols. As compared to mice injected with aristolochic acids alone, more severe acute kidney injury was observed after CD4+ or CD8+ T-cells depletion. TNF-alpha and MCP-1 mRNA renal expressions were also increased. In contrast, regulatory T-cells depletion did not modify the severity of the aristolochic acids induced acute kidney injury, suggesting an independent mechanism. Aristolochic acids nephropathy was also associated with an increased proportion of myeloid CD11bhighF4/80mid and a decreased proportion of their counterpart CD11blowF4/80high population. After CD4+ T-cell depletion the increase in the CD11bhighF4/80mid population was even higher whereas the decrease in the CD11blowF4/80high population was more marked after CD8+ T cells depletion. Our results suggest that CD4+ and CD8+ T-cells provide protection against AA-induced acute tubular necrosis. Interestingly, T-cell depletion was associated with an imbalance of the CD11bhighF4/80mid and CD11blowF4/80high populations. 1 SCIENTIFIC REPOrTS \| (2018) 8:5334 \| DOI:10.1038/s41598-018-23565-2 the CD11bhighF4/80mid and CD11blowF4/80high populations. Human aristolochic acid nephropathy (AAN) was formerly known as “Chinese herb nephropathy”. This tubulointersti- tial nephritis was first reported in Belgian women after ingestion of herbal slimming remedies containing aristolochic acids (AA). These nitrophenanthrene derivatives were found responsible for the so-called Balkan endemic nephropa- thy and for hundreds of cases of chronic renal failure in China and Taiwan where traditional herbal medicines are still widely used1–3. Histologically, AAN displays an interstitial fibrosis with a typical corticomedullary gradient and tubular atrophy4,5. AAN was reproduced in several animal models including rabbits, mice and rats6–9. Prior time courses studies of our Wistar rat model demonstrated a biphasic evolution of tubulo-interstitial lesions10,11. During the acute phase, a necrosis of proximal tubular epithelial cells (PTEC) followed by a macrophage, CD4+ and CD8+ T-cell infiltration were observed. Thereafter, interstitial fibrosis and tubular atrophy were the hallmark of the chronic phase. In this model, inflammatory infiltrate (macrophages (mφ) followed by CD4+ and CD8+ T-cells) were proposed as the physiopatho- logical link between the acute and the chronic phases10. Results However, there was no statistical difference between AA and AA + αCD4 group (Supplementary Figure 2) I ddi i l f i i d l h i f T ll d l i d i h h i g p ,f g p ( pp y g ) In an additional set of experiments, we aimed to evaluate the impact of T-cell depletion during the chronic phase of experimental AAN. AA was injected for 5, 15 or 22 days. However, a significant mortality rate was observed in the AA + αCD4 group as compared to the AA group (p < 0.0032) (Supplementary Figure 3a). No mice died in the αCD4 group. Again, a significant increase in pCr was observed in the AA + αCD4 group as com- pared to the AA group after 5 days of depletion (p < 0.014). Abnormal but non-significant pCr was still observed after 15 and 22 days of injections. The pCr determination showed normal levels in the αCD4 group after 22 days of injections (Supplementary Figure 3b). Given the high mortality rate induced by AA in the depleted group, we decided to focus on day 5 of our protocol for further experiments. Experimental AAN is associated with an increase in regulatory T-cells in spleen and kidneys but regulatory T-cells depletion does not increase AA induced acute kidney injury. Flow cytometry analyses of inflammatory kidney infiltrates after 5 days of AA injection revealed no increase in the absolute num- ber of lymphocytic kidney (CD3+, CD8+ or CD4+ T-cells) infiltrates as compared to controls (p > 0.5) (Fig. 2a). However, an increase in the percentage of regulatory T-cells (T-regs) was observed in the kidneys (p = 0.0026) and spleen (p = 0.0133) of the AA group as compared to CTRL group (Fig. 2b,c). T-regs have been shown to be protective in many models of AKI such as the ischemia reperfusion injury (IRI) model14,15 or the toxic model16–18. The exacerbation of AKI in the AA + αCD4 group could be linked to the loss of T-regs. Therefore, the role of T-regs was assessed in a separate experiment. Mice were injected with anti-CD25 depleting Ab (i.e PC61) and AA (AA + αCD25 group) or with AA and control Ab (AA group). A control group was injected with AA vehicle and anti-CD25 Ab (αCD25 group). Results CD4+ depletion aggravates acute kidney injury in experimental AAN. Mice were injected for 5 days with AA and were sacrificed 24 hours after the last injection. This time point is interesting as it corresponds to the AKI phase with maximal tubular necrosis and plasma creatinine (pCr) in our model13. In addition, mice received anti-CD4 depleting antibody (AA + αCD4 group) or control isotype (AA group). A control group received AA vehicle and anti-CD4 (αCD4 group). Mice groups were also compared to baseline mice receiving no injection (CTRL group). A significant decrease in the percentage of CD4 T-cells was observed in the spleen (about 95%) (Supplementary Figure 1a,b) and in the kidney (more than 90%) on day 5. T-cell depletion was associated with a significant increase in pCr (p < 0.0053)) (Fig. 1a) and blood urea nitrogen (BUN) (p < 0.0004) (Fig. 1b) in the AA + αCD4 group as compared to the AA group. Next, histological lesions were assessed in each group. No lesion was observed in the CTRL or αCD4 groups (Fig. 1c,d). However, a severe tubular necrosis was observed in the AA + αCD4 and AA groups (Fig. 1e,f). Tubular necrosis score was also significantly increased in the AA + αCD4 group as compared to the AA group (p < 0.0019) (Fig. 1g). Finally, CD4+ T-cell depletion was associated with an increased expression of intrarenal proinflammatory mRNA cytokines: a significant increase of MCP-1 (p = 0.0240) and MIP-1α (p = 0.0289) and, as well as a non significant increase of TNF-α, IL-6, CXCL10 and IL-1β were observed (Fig. 1h,i). Renal expression of KIM-1 and NGAL mRNA were also assessed in this model. A significant increase of KIM-1 and NGAL mRNA was observed after AA injection as compared to CTRL group. However, there was no statistical difference between AA and AA + αCD4 group (Supplementary Figure 2) In an additional set of experiments, we aimed to evaluate the impact of T-cell depletion during the chronic phase of experimental AAN. AA was injected for 5, 15 or 22 days. However, a significant mortality rate was observed in the AA + αCD4 group as compared to the AA group (p < 0.0032) (Supplementary Figure 3a). No mice died in the αCD4 group. Again, a significant increase in pCr was observed in the AA + αCD4 group as com- pared to the AA group after 5 days of depletion (p < 0.014). Results A sig- nificant decrease in the percentage of Tregs (about 75%) was observed in the spleen (Supplementary Figure 1c,d) and the kidney (more than 45%) on day 5. However, after 5 days of AA injection, Treg depletion did not result in any increase in pCr (Fig. 3a) or BUN (Fig. 3b). Tubular necrosis lesions (Fig. 3c–f) and tubular necrosis score (Fig. 3g) were almost identical to those observed in the AA group. CD8+ T-cells depletion aggravates acute kidney injury induced by AA. To complete our under- standing of the roles of the inflammatory infiltrate in our AAN model, we evaluated the role of CD8+ T-cells using depleting Ab. Mice were injected with anti-CD8+ YTS 169.4 and with AA (AA + αCD8 group), while another group was injected with AA vehicle and anti-CD8 (αCD8 group) or with AA and control Ab (AA group). A significant decrease in the percentage of CD8+ T-cells (about 90%) was observed in the spleen (Supplementary Figure 1c,d) and the kidney (more than 85%) on day 5. As with CD4+ T-cells depletion, worse AKI was observed as demonstrated by the significant increase in both pCr (p = 0.0415) (Fig. 4a) and BUN (p = 0.0273) (Fig. 4b). In addition, a non-significant increase in tubular necrosis and necrosis score was also observed (Fig. 4c–g). Finally, CD8+ T-cells depletion was associated with an increase in the expression of intrarenal pro-inflammatory cytokines, in particular a significant increase in TNF-α expression was observed (p < 0.044); in addition, MCP-1 mRNA expression tended to increase but without reaching the statistical significance (Fig. 4h). CD4+ and CD8+ T-cell depletions are associated with modifications in CD11bhighF4/80mid and CD11blowF4/80high myeloid populations. AA injections did not induce changes in the absolute number of kidney lymphocytes in the early phases of the model. However, previous studies have suggested an impor- tant role for macrophages in AKI and AAN pathophysiology19. Therefore, we studied this population in our model. Immunohistochemistry studies revealed that F4/80 positive cells were significantly diminished after AA injections as compared to CTRL group. In addition, no difference between AA + αCD4 or AA + αCD8 and AA + CTRL groups (Fig. 5a–f) was observed.i g g As F4/80 is non-specific for macrophages and because macrophages and dendritic cells constitute heteroge- neous populations with marked differences in their subpopulation, we decided to study the kidney myeloid pop- ulations more closely using flow cytometry analysis. CD4+ and CD8+ T Cells Exert Regulatory Properties During Experimental Acute Aristolochic Acid Nephropathy Apart from AAN, the role of the immune system has been largely studied in both ischemic and other toxic acute kidney injury (AKI) models. In response to acute tubular necrosis (ATN), inflammatory cytokines produced by PTEC, resident and infiltrating leukocytes contribute to the accumulation of inflammatory cells into the interstitium12. The roles of various immune cells, including dendritic cells (DC), natural killer T-cells (NKT), T and B lymphocytes, neutrophils and mφ have been described12, all of which ultimately lead to tubular atrophy and interstitial fibrosis. However, the respective role of each of these cell types remains controversial since the observed data depends on the type of model used. Moreover, the role of these different T-cell types has never been studied in experimental AAN. We therefore decided to investigate the potential role of CD4+ and CD8+ T-cells during the acute phase of toxic nephropathy, by studying the effects of selective depletion using monoclonal antibodies. 1Laboratory of Experimental Nephrology, Faculty of Medicine, Université libre de Bruxelles, Brussels, Belgium. 2Service of Nephrology, Dialysis and Renal Transplantation, Erasme Hospital, Université Libre de Bruxelles, Brussels, Belgium. 3Molecular Physiology Research Unit — URPhyM, NARILIS (Namur Research Institute for Life Sciences), University of Namur (UNamur), Namur, Belgium. Joëlle L. Nortier and Jean-Michel Hougardy contributed equally to this work. Correspondence and requests for materials should be addressed to T.B. (email: tbaudoux@ulb.ac.be) SCIENTIFIC REPOrTS \| (2018) 8:5334 \| DOI:10.1038/s41598-018-23565-2 1 www.nature.com/scientificreports/ Results Abnormal but non-significant pCr was still observed after 15 and 22 days of injections. The pCr determination showed normal levels in the αCD4 group after 22 days of injections (Supplementary Figure 3b). Given the high mortality rate induced by AA in the depleted group, we decided to focus on day 5 of our protocol for further experiments. CD4+ depletion aggravates acute kidney injury in experimental AAN. Mice were injected for 5 days with AA and were sacrificed 24 hours after the last injection. This time point is interesting as it corresponds to the AKI phase with maximal tubular necrosis and plasma creatinine (pCr) in our model13. In addition, mice received anti-CD4 depleting antibody (AA + αCD4 group) or control isotype (AA group). A control group received AA vehicle and anti-CD4 (αCD4 group). Mice groups were also compared to baseline mice receiving no injection (CTRL group). A significant decrease in the percentage of CD4 T-cells was observed in the spleen (about 95%) (Supplementary Figure 1a,b) and in the kidney (more than 90%) on day 5. T-cell depletion was associated with a significant increase in pCr (p < 0.0053)) (Fig. 1a) and blood urea nitrogen (BUN) (p < 0.0004) (Fig. 1b) in the AA + αCD4 group as compared to the AA group. Next, histological lesions were assessed in each group. No lesion was observed in the CTRL or αCD4 groups (Fig. 1c,d). However, a severe tubular necrosis was observed in the AA + αCD4 and AA groups (Fig. 1e,f). Tubular necrosis score was also significantly increased in the AA + αCD4 group as compared to the AA group (p < 0.0019) (Fig. 1g). Finally, CD4+ T-cell depletion was associated with an increased expression of intrarenal proinflammatory mRNA cytokines: a significant increase of MCP-1 (p = 0.0240) and MIP-1α (p = 0.0289) and, as well as a non significant increase of TNF-α, IL-6, CXCL10 and IL-1β were observed (Fig. 1h,i). Renal expression of KIM-1 and NGAL mRNA were also assessed in this model. A significant increase of KIM-1 and NGAL mRNA was observed after AA injection as compared to CTRL group. Results A severe tubular necrosis was observed in AA (e) and AA + αCD4 (f) groups (arrow). Original magnification x400, hematoxylin–eosin-stained kidney longitudinal sections. Histological analyses resulted in an increased necrosis score in AA + αCD4 acid as compared to AA group (g). Renal tissue qRT-PCR analysis of various cytokine mRNA levels on renal tissue (h,i) in the different groups. Statistical test used: Mann-Whitney. Results are expressed as the mean ± SEM, p < 0.05; p < 0.01; *p < 0.001. Number of mice per group: AA + αCD4 n = 13; AA n = 14; αCD4 n = 9; CTRL n = 6. Figure 1. Plasma creatinine (pCr) (a) and blood urea nitrogen (BUN) (b) levels from AA + αCD4 (black columns), AA (dark greys columns), αCD4 (light greys columns) and CTRL (white columns) groups on day 5 of the protocol. A significant increase in pCr and BUN was noted in the AA + αCD4 as compared to AA group. Representative photomicrographs of renal cortex longitudinal sections in each group after 5 days of AA injection (c–f). No lesion was observed in CTRL (c) and αCD4 (d) groups. A severe tubular necrosis was observed in AA (e) and AA + αCD4 (f) groups (arrow). Original magnification x400, hematoxylin–eosin-stained kidney longitudinal sections. Histological analyses resulted in an increased necrosis score in AA + αCD4 acid as compared to AA group (g). Renal tissue qRT-PCR analysis of various cytokine mRNA levels on renal tissue (h,i) in the different groups. Statistical test used: Mann-Whitney. Results are expressed as the mean ± SEM, p < 0.05; p < 0.01; p < 0.001. Number of mice per group: AA + αCD4 n = 13; AA n = 14; αCD4 n = 9; CTRL n = 6. particular, Ly6C expression profile was higher in the P1 population as compared to the P2 population in the steady state condition (Fig. 6c). Moreover, this was also the case after AA injections (AA or AA + αCD4 groups) (Fig. 6d,e), suggesting a different inflammatory profile. Finally, AA injections were associated with major particular, Ly6C expression profile was higher in the P1 population as compared to the P2 population in the steady state condition (Fig. 6c). Moreover, this was also the case after AA injections (AA or AA + αCD4 groups) (Fig. 6d,e), suggesting a different inflammatory profile. Results After gating on CD45+ renal cells and doublets elimination, myeloid populations were separated into Ly6G+SSC+ cells (i.e neutrophils). Then, after exclusion of neutrophils (NEU), CD11c positive cells were defined based on their high expression of this marker and, finally, remain- ing cells were separated into CD11bhighF4/80mid (P1) and CD11blowF4/80high population (P2) (Supplementary Figure 4). Ly6C analysis of these subpopulations revealed various expression of this marker (Fig. 6a,b). In SCIENTIFIC REPOrTS \| (2018) 8:5334 \| DOI:10.1038/s41598-018-23565-2 2 entificreports/ Figure 1. Plasma creatinine (pCr) (a) and blood urea nitrogen (BUN) (b) levels from AA + αCD4 (black columns), AA (dark greys columns), αCD4 (light greys columns) and CTRL (white columns) groups on day 5 of the protocol. A significant increase in pCr and BUN was noted in the AA + αCD4 as compared to AA group. Representative photomicrographs of renal cortex longitudinal sections in each group after 5 days of AA injection (c–f). No lesion was observed in CTRL (c) and αCD4 (d) groups. A severe tubular necrosis was observed in AA (e) and AA + αCD4 (f) groups (arrow). Original magnification x400, hematoxylin–eosin-stained kidney longitudinal sections. Histological analyses resulted in an increased necrosis score in AA + αCD4 acid as compared to AA group (g). Renal tissue qRT-PCR analysis of various cytokine mRNA levels on renal tissue (h,i) in the different groups. Statistical test used: Mann-Whitney. Results are expressed as the mean ± SEM, p < 0.05; p<0 01; p<0 001 Number of mice per group: AA+αCD4 n=13; AA n=14; αCD4 n=9; CTRL n=6 www.nature.com/scientificreports/ particular, Ly6C expression profile was higher in the P1 population as compared to the P2 population in the steady state condition (Fig. 6c). Moreover, this was also the case after AA injections (AA or AA + αCD4 groups) (Fig. 6d,e), suggesting a different inflammatory profile. Finally, AA injections were associated with major Figure 1. Plasma creatinine (pCr) (a) and blood urea nitrogen (BUN) (b) levels from AA + αCD4 (black columns), AA (dark greys columns), αCD4 (light greys columns) and CTRL (white columns) groups on day 5 of the protocol. A significant increase in pCr and BUN was noted in the AA + αCD4 as compared to AA group. Representative photomicrographs of renal cortex longitudinal sections in each group after 5 days of AA injection (c–f). No lesion was observed in CTRL (c) and αCD4 (d) groups. Discussion AAN is recognized as a worldwide public health problem: in addition to the 1993 Belgian outbreak, when hun- dreds of cases of the so-called Chinese herbs nephropathy were reported, the condition has been proven to be the cause of the Balkan endemic nephropathy and of thousands of cases of CKD and cancers in Asian countries where traditional Chinese medicine is widespread20. AAN was firstly described as paucicellular, with scarce lymphocytic infiltrate4. However, this paradigm was challenged after an inflammatory infiltrate had been described in human cases and animal models10,13,21. Moreover, a human pilot trial with steroids has been shown to slow down the progression towards renal failure, supporting the involvement of an immune-mediated process22.il p g pp g p In our AAN model, PTEC necrosis was followed by mφ infiltrate and by CD4+ and CD8+ T-cells influx10. It is important to note that the respective role of these cells has never yet been investigated before the present study. Although several other studies have demonstrated a “pathogenic” role for CD4+ or CD8+ T-cells with a less severe AKI in the absence of these cells23–26, we, on the other hand, surprisingly demonstrated that CD4+ or CD8+ T-cells depletion aggravated AKI after 5 days of AA injection with an increase in pCr, BUN and tubular necrotic lesions. Absence of significant difference for KIM-1 and NGAL levels between AA and AA + αCD4 group could be linked to the over-sensitivity of these markers regarding the importance of tubular necrosis observed. Moreover, when AA injections were prolonged during 22 days, the mortality rate was significantly higher in the CD4+ T-cells depleted group as compared to AA alone, suggesting a nephroprotective effect of CD4+ T-cells in long-term AA intoxication. In addition, AKI aggravation observed after CD4+ T-cells depletion has been found to be unrelated to Tregs subpopulation as their depletion did not increase AKI lesions in our study. Lower per- centage of Treg depletion obtained in the kidney may be considered as critical in explaining the absence of any effect of their depletion. However, this magnitude of depletion is likely relevant as more than 75% of Treg were depleted in the spleen. Moreover, other authors have earlier observed a negative effect of Treg depletion in their AKI model with a similar or lower magnitude of depletion in the kidney14,15,27,28. Results Finally, AA injections were associated with major SCIENTIFIC REPOrTS \| (2018) 8:5334 \| DOI:10.1038/s41598-018-23565-2 3 www.nature.com/scientificreports/ Figure 2. Absolute number of specific kidney lymphocyte subpopulations per gram of kidney tissue (a). No statistical change in the absolute number of CD3+, CD4+ or CD8+ lymphocytes was observed after AA injections (greys columns) as compared to CTRL group (white columns). However an increase in the percentage of splenic (b) and kidney (c) CD3+CD4+CD25highFOXP3+ T-reg cells per CD3+CD4+ T cells was observed after AA injection (greys columns) as compared to CTRL group (white columns). Statistical test used: Mann-Whitney. Results are expressed as the mean ± SEM, p < 0.05; *p < 0.01. Number of mice per group: AA n = 14; CTRL n = 6. Figure 2. Absolute number of specific kidney lymphocyte subpopulations per gram of kidney tissue (a). No statistical change in the absolute number of CD3+, CD4+ or CD8+ lymphocytes was observed after AA injections (greys columns) as compared to CTRL group (white columns). However an increase in the percentage of splenic (b) and kidney (c) CD3+CD4+CD25highFOXP3+ T-reg cells per CD3+CD4+ T cells was observed after AA injection (greys columns) as compared to CTRL group (white columns). Statistical test used: Mann-Whitney. Results are expressed as the mean ± SEM, p < 0.05; *p < 0.01. Number of mice per group: AA n = 14; CTRL n = 6. modifications in the leukocytic infiltrate with an increase in the absolute number of P1 population (p < 0.05) as compared to CTRL and a decrease in the absolute number of P2 population (p < 0.0001). However, no significant modification in the absolute number of NEU or CD11c were observed (Fig. 7a,b). In addition, as compared to the AA group, the increase in CD11c+ (p < 0.01) and P1 (p < 0.05) populations was higher in the AA + αCD4 group (Fig. 7a,b). After AA injection and CD8+ T-cell depletion, there was no increase in the P1 population as compared to the AA group, however a significant decrease of P2 population was noted (p < 0.0265) (Fig. 7c). Discussion Other authors have reported a protective role of CD4+ T-cells in a toxic injury model and in an unilateral ureteral obstruction (UUO) model29,30. Moreover, transfer of kidney-infiltrating lymphocytes, isolated 24 hours after renal IRI, into a T-cell-deficient SCIENTIFIC REPOrTS \| (2018) 8:5334 \| DOI:10.1038/s41598-018-23565-2 4 www.nature.com/scientificreports/ Figure 3. Plasma creatinine (pCr) (a) and blood urea nitrogen (BUN) levels (b) from AA + αCD25 (black columns), AA (dark greys columns), αCD25 (light greys columns) and CTRL (white columns) groups on day 5 of the protocol. There was no increase in pCr or BUN in the AA + αCD25 group as compared to AA group. Representative photomicrographs of renal cortex longitudinal sections in each group after 5 days of AA injection (c–f). No lesion was observed in CTRL and αCD25 groups (c,d). As with AA + αCD4, an important necrosis was observed in AA (e) and AA + αCD25 (f) groups (arrow). However necrotic lesions were not enhanced by T-regs depletion as compared to AA group. Original magnification x400, hematoxylin–eosin- stained kidney longitudinal sections. Histological analysis of necrosis injury score in the different groups (g). Statistical test used: Mann-Whitney. Results are expressed as the mean ± SEM, ns = no statistical difference observed. Number of mice per group: AA + αCD25 n = 7; AA n = 9; αCD25 n = 8; CTRL n = 6. Figure 3. Plasma creatinine (pCr) (a) and blood urea nitrogen (BUN) levels (b) from AA + αCD25 (black columns), AA (dark greys columns), αCD25 (light greys columns) and CTRL (white columns) groups on h Figure 3. Plasma creatinine (pCr) (a) and blood urea nitrogen (BUN) levels (b) from AA + αCD25 (black columns), AA (dark greys columns), αCD25 (light greys columns) and CTRL (white columns) groups on day 5 of the protocol. There was no increase in pCr or BUN in the AA + αCD25 group as compared to AA group. Representative photomicrographs of renal cortex longitudinal sections in each group after 5 days of AA injection (c–f). No lesion was observed in CTRL and αCD25 groups (c,d). As with AA + αCD4, an important necrosis was observed in AA (e) and AA + αCD25 (f) groups (arrow). However necrotic lesions were not enhanced by T-regs depletion as compared to AA group. Original magnification x400, hematoxylin–eosin- stained kidney longitudinal sections. Histological analysis of necrosis injury score in the different groups (g). Statistical test used: Mann-Whitney. Discussion Results are expressed as the mean ± SEM, ns = no statistical difference observed. Number of mice per group: AA + αCD25 n = 7; AA n = 9; αCD25 n = 8; CTRL n = 6. mouse conferred a relative protection against subsequent ischemia reperfusion injury, suggesting a protective effect of the lymphocytes induced by renal ischemia31. As these cells are present in the renal interstitium in the steady state, a role in the early stage of inflammation is likely, even before the leukocyte infiltrate influx32.i y y gl y yil Secondly, we described an increased P1 population infiltrate (i.e CD11bhighF4/80mid) following AA injection associated with a substantial decreased P2 population (i.e CD11blowF4/80high). This observation is consistent with that of other authors in alternative AKI models33–35. Moreover, the increase in the P1 population was even more important in the CD4+ depleted mice injected with AA, while a decrease in the P2 population was observed after CD8+ T-cells depletion. Interestingly, Harris and colleagues suggested, in their adriamycin nephropathy model, that increase in glomerular lesions and pCr, observed after CD4 depletion, could be linked to an increase in the macrophage infiltrate (MAC-3+ cells)29. Macrophages play an essential role in AKI. After activation/injury, they can differentiate into various macrophage subtypes under the influence of their local microenvironment, affecting both their surface proteins expression and cytokines production. Basically, two well-defined phenotypes are com- monly described: classically activated macrophages (M1 macrophages) and alternatively activated macrophages (M2 macrophages) with pro or anti-inflammatory properties respectively36. Imbalance between M1 and M2 mac- rophages in favour of M1 phenotype has been shown to increase AKI lesions37,38 whereas M2 phenotype is protec- tive35,38,39. Several studies focusing on myeloid population analyses have used cytometry gating strategies similar to those used in our experimental procedures, showing that P1 had M1 macrophage characteristics and P2 had M2 macrophage characteristics33,35. Our results also suggest that P1 cells have a higher inflammatory phenotype as reflected by their expression of the Ly6C marker as compared to P2 cells. The restricted characterization of both P1 and P2 populations by their basic phenotype is an additional limitation. SCIENTIFIC REPOrTS \| (2018) 8:5334 \| DOI:10.1038/s41598-018-23565-2 Discussion Indeed, we do not confirm that these populations are fully functional M1 and M2 macrophages as others have described34,35,37–39.i Finally, it was demonstrated recently that macrophages from CD4+ KO mice display an M1 profile, suggesting that CD4+ T-cells play a dominant role over other lymphocyte populations in providing the cytokine environ- ment for regulating macrophages towards an M2 profile under normal wild-type conditions40. The role of the CD11c+ population has not been investigated in this model. However, these cells did not express high level of SCIENTIFIC REPOrTS \| (2018) 8:5334 \| DOI:10.1038/s41598-018-23565-2 5 www.nature.com/scientificreports/ Figure 4. Plasma creatinine (pCr) (a) and blood urea nitrogen (BUN) (b) levels from AA + αCD8 (black columns), AA (dark greys columns), αCD8 (light greys columns) and CTRL (white columns) groups on day 5 (a). A significant increase in pCr and BUN was noted in the AA + αCD8 as compared to AA group. Representative photomicrographs of renal cortex longitudinal sections in each group after 5 days of AA injection (c–f). No lesion was observed in CTRL and αCD8 groups (data not shown). AA induced a massive necrosis in AA (e) and AA + αCD8 (f) groups (arrow). Original magnification x400, hematoxylin–eosin- stained kidney longitudinal sections. Histological analysis of necrosis injury score in the different groups (g). qRT-PCR analysis of various cytokine mRNA levels (h) in each group. Statistical test used: Mann-Whitney. Results are expressed as the mean ± SEM, NS = no statistical difference observed; p < 0.05. Number of mice per group: AA + αCD8 n = 13; AA n = 14; αCD8 n = 8; CTRL n = 6. www.nature.com/scientificreports/ Figure 4. Plasma creatinine (pCr) (a) and blood urea nitrogen (BUN) (b) levels from AA + αCD8 (black columns), AA (dark greys columns), αCD8 (light greys columns) and CTRL (white columns) groups on day 5 (a). A significant increase in pCr and BUN was noted in the AA + αCD8 as compared to AA group. Representative photomicrographs of renal cortex longitudinal sections in each group after 5 days of AA injection (c–f). No lesion was observed in CTRL and αCD8 groups (data not shown). AA induced a massive necrosis in AA (e) and AA + αCD8 (f) groups (arrow). Original magnification x400, hematoxylin–eosin- stained kidney longitudinal sections. Histological analysis of necrosis injury score in the different groups (g). qRT-PCR analysis of various cytokine mRNA levels (h) in each group. Discussion Statistical test used: Mann-Whitney. Results are expressed as the mean ± SEM, NS = no statistical difference observed; p < 0.05. Number of mice per group: AA + αCD8 n = 13; AA n = 14; αCD8 n = 8; CTRL n = 6. Figure 4. Plasma creatinine (pCr) (a) and blood urea nitrogen (BUN) (b) levels from AA + αCD8 (black columns), AA (dark greys columns), αCD8 (light greys columns) and CTRL (white columns) groups on day 5 (a). A significant increase in pCr and BUN was noted in the AA + αCD8 as compared to AA group. Representative photomicrographs of renal cortex longitudinal sections in each group after 5 days of AA injection (c–f). No lesion was observed in CTRL and αCD8 groups (data not shown). AA induced a massive necrosis in AA (e) and AA + αCD8 (f) groups (arrow). Original magnification x400, hematoxylin–eosin- stained kidney longitudinal sections. Histological analysis of necrosis injury score in the different groups (g). qRT-PCR analysis of various cytokine mRNA levels (h) in each group. Statistical test used: Mann-Whitney. Results are expressed as the mean ± SEM, NS = no statistical difference observed; p < 0.05. Number of mice per group: AA + αCD8 n = 13; AA n = 14; αCD8 n = 8; CTRL n = 6. Ly6C,and another study had suggested a protective role of this population in toxic AKI41. Moreover, we demon- strated that CD4+ or CD8+ T-cell depletion in AAN was associated with renal increase in the expression of pro-inflammatory cytokines (TNF-α and MCP-1). Recent studies have suggested that adaptive immune cells SCIENTIFIC REPOrTS \| (2018) 8:5334 \| DOI:10.1038/s41598-018-23565-2 6 www.nature.com/scientificreports/ Figure 5. Representative photomicrographs of renal cortex longitudinal sections showing immunolocalization of F4/80 positive cells in each group (a–d). Numerous F4/80 positive cells (arrow) were localized in the interstitium in CTRL group (a). A drastic reduction of the number of F4/80 positive cells was observed after 5 days of AA injection in AA (b), AA + αCD4 (c) and AA + αCD8 (d) groups. Original magnification x400, Luxol fast blue-stained kidney longitudinal sections and magnified details showing exact position of F4/80 positive cells. The number of F4/80 positive cells per high power field (hpf) (x400) was compared in different groups (e,f). As compared to CTRL group, a significant reduction of F4/80 positive cells was observed in AA group in both experiments. Discussion Taken together, our preliminary results suggest that the absence of CD4+ or CD8+ T-cells in the acute phase of our AAN model could lead to a modified inflammatory microenvironment responsible for an imbalance between M1 and M2 macrophage phenotypes and, ultimately, to more severe AKI. However, complementary investigations are necessary to confirm this hypothesis. Other physiopathological hypotheses should also be tested, such as the role of CD11c+ population or the role of Tr1 and gamma-delta lymphocytes, as these cells also have regulatory properties. were able to actively dampen initial innate responses42. Authors using a mouse model of TLR stimulation with poly(I:C) or LPS demonstrated that the absence of residential CD4+CD25−Foxp3− or CD8+ T-cells resulted in a TNF-α storm produced by CD11b+ or CD11c+ cells that was responsible for an increased mortality42. Others have confirmed that in vivo T-cell–macrophage interaction through CD40 ligation with CD40L was able to down-regulate excessive TNF production by macrophages through a mechanism implicating IRAK143. Taken together, our preliminary results suggest that the absence of CD4+ or CD8+ T-cells in the acute phase of our AAN model could lead to a modified inflammatory microenvironment responsible for an imbalance between M1 and M2 macrophage phenotypes and, ultimately, to more severe AKI. However, complementary investigations are necessary to confirm this hypothesis. Other physiopathological hypotheses should also be tested, such as the role of CD11c+ population or the role of Tr1 and gamma-delta lymphocytes, as these cells also have regulatory properties. p p In conclusion, we demonstrated that CD4+ or CD8+ T-cells lymphocyte depletion was associated with AKI worsening during acute experimental AAN. This observation has been related to a modified interstitial influx in the CD11bhighF4/80mid and CD11blowF4/80high subpopulations. Discussion However, no statistical difference was observed in the number of F4/80 positive cells between AA group and AA + αCD4 (e) or AA + αCD8 groups (f). Statistical test used: Kruskall-Wallis test followed by Mann-Whitney U-test and Bonferroni post-hoc test. Results are expressed as the mean ± SEM, *p < 0.001; p < 0.01; p < 0.05. Number of mice per group: AA + αCD4 n = 13; AA n = 14; αCD4 n = 9; AA + αCD8 n = 13; αCD8 n = 8; CTRL n = 6. Figure 5. Representative photomicrographs of renal cortex longitudinal sections showing immunolocalization of F4/80 positive cells in each group (a–d). Numerous F4/80 positive cells (arrow) were localized in the interstitium in CTRL group (a). A drastic reduction of the number of F4/80 positive cells was observed after 5 days of AA injection in AA (b), AA + αCD4 (c) and AA + αCD8 (d) groups. Original magnification x400, Luxol fast blue-stained kidney longitudinal sections and magnified details showing exact position of F4/80 positive cells. The number of F4/80 positive cells per high power field (hpf) (x400) was compared in different groups (e,f). As compared to CTRL group, a significant reduction of F4/80 positive cells was observed in AA group in both experiments. However, no statistical difference was observed in the number of F4/80 positive cells between AA group and AA + αCD4 (e) or AA + αCD8 groups (f). Statistical test used: Kruskall-Wallis test followed by Mann-Whitney U-test and Bonferroni post-hoc test. Results are expressed as the mean ± SEM, p < 0.001; p < 0.01; p < 0.05. Number of mice per group: AA + αCD4 n = 13; AA n = 14; αCD4 n = 9; AA + αCD8 n = 13; αCD8 n = 8; CTRL n = 6. were able to actively dampen initial innate responses42. Authors using a mouse model of TLR stimulation with poly(I:C) or LPS demonstrated that the absence of residential CD4+CD25−Foxp3− or CD8+ T-cells resulted in a TNF-α storm produced by CD11b+ or CD11c+ cells that was responsible for an increased mortality42. Others have confirmed that in vivo T-cell–macrophage interaction through CD40 ligation with CD40L was able to down-regulate excessive TNF production by macrophages through a mechanism implicating IRAK143. Methods Experimental protocols. All protocols were approved by the Ethical Committee for Animal Care (Faculty of Medicine, Université Libre de Bruxelles). In addition, all experiments were performed in accordance with SCIENTIFIC REPOrTS \| (2018) 8:5334 \| DOI:10.1038/s41598-018-23565-2 7 www.nature.com/scientificreports/ Figure 6. Representative dot plots showing the localization of neutrophils (NEU) (blue dots), CD11c + (orange dots), P1 (red dots) and P2 (green dots) populations on a CD11b-F4/80 diagram in a mice injected with AA (a). Representative histograms showing the Ly6C expression in the neutrophils (blue line), CD11c + (orange line), P2 (green line) and P1 (red line) populations in the AA group (b). P1 and P2 population differ from their respective Ly6C expression. Geometric mean of Ly6C MFI expression for neutrophils (black columns), CD11C + (white columns), P2 (light grey columns) and P1 populations (dark grey columns) in CTRL (c), AA (d) and AA + αCD4 (e) groups. A significant difference in the expression of Ly6c MFI was observed between P1 and P2 populations in different conditions. In addition, a significant increase in the geometric mean of Ly6C MFI expression in the P1 population was observed after AA injection as compared to controls (data not shown). Statistical test used: Mann-Whitney. Results are expressed as the mean ± SEM, *p < 0.0001 and p < 0.01. Number of mice per group: AA + αCD4 n = 13; AA n = 14; CTRL n = 6. Figure 6. Representative dot plots showing the localization of neutrophils (NEU) (blue dots), CD11c + (orange dots), P1 (red dots) and P2 (green dots) populations on a CD11b-F4/80 diagram in a mice injected with AA (a). Representative histograms showing the Ly6C expression in the neutrophils (blue line), CD11c + (orange line), P2 (green line) and P1 (red line) populations in the AA group (b). P1 and P2 population differ from their respective Ly6C expression. Geometric mean of Ly6C MFI expression for neutrophils (black columns), CD11C + (white columns), P2 (light grey columns) and P1 populations (dark grey columns) in CTRL (c), AA (d) and AA + αCD4 (e) groups. A significant difference in the expression of Ly6c MFI was observed between P1 and P2 populations in different conditions. In addition, a significant increase in the geometric mean of Ly6C MFI expression in the P1 population was observed after AA injection as compared to controls (data not shown). Statistical test used: Mann-Whitney. Methods After one week of acclimatization, 10-week-old C57BL/6 male mice (Elevage Janvier, Le Genest Saint-Isle, France) were randomly assigned to one of the 7 groups (Supplementary Figure 5). Depending on their groups, mice were injected with AA + anti-CD4 Ab (GK1.5; Bioxcell, West Lebanon, NH USA) (AA + αCD4 group); with AA + anti-CD8 Ab (YTS 169.4; Bioxcell) (AA + αCD8 group); with AA + con- trol isotype (LTF-2; Bioxcell) (AA group); with AA + anti-CD25 Ab (PC61; Bioxcell) (AA + αCD25 group); with AA vehicle (i.e polyethylene-glycol, PEG) (Fluka Chemie, Buchs, Switzerland) + anti-CD4 Ab (αCD4 group); with PEG + anti-CD8 Ab (αCD8 group); or PEG + anti-25 Ab (αCD25 group). In addition, a control group did not receive any injection and was used as baseline day 0 (CTRL). AA (5mgr/kg body weight) or an equiva- lent volume of PEG was administered by means of daily IP injections during five days and GK1.5 or YTS169.4 (0.250 mg) or control isotype (0.250 mg) were administered by means of a single IP injections 72 hours before the first injection of AA. PC61 (0.300 mg) was injected twice, i.e. 72 and 24 hours before the first AA injection. AA (Acros Organics Co., Geel, Belgium; 40% AAI, 60% AAII,) was given in 150 µl of PEG and sterile water. GK1.5, YTS 169.4 and LTF-2 were given in 150 µl of sterile dPBS. For the chronic phase experiment, mice were injected with AA for 5, 15 or 22 days as described above. Ab injections were repeated weekly until sacrifice. CD4+ T-cell depletion (>95%) was effective during all protocol (supplementary Figure 1a,b). Twenty four hours after the last AA injection, mice were anesthetised with ketamine-HCl (Merial, Brussels, Belgium) and 2% xylazine (Bayer, Brussels, Belgium) after which a blood specimen was obtained by cardiac puncture. Then, kidneys were flushed with 30 ml of NaCl 0,9% and kidneys were harvested for analysis. Left kidneys were used for flow cytometric anal- ysis whereas right kidneys were used for realtime polymerase chain reaction (rtPCR) (immediately snap frozen in liquid nitrogen and stored at −80 °C) and for histological analysis. Renal histopathology. 4% buffered formaldehyde fixed and paraffin embedded (FFPE)) sections (4 μm) were attached to poly-L-lysine pre-treated slides (Sigma-Aldrich, Bornem, Belgium). After air-drying, the par- affin from tissue sections was removed (xylene solution). Methods Results are expressed as the mean ± SEM, **p < 0.0001 and p < 0.01. Number of mice per group: AA + αCD4 n = 13; AA n = 14; CTRL n = 6. relevant guidelines and regulations. After one week of acclimatization, 10-week-old C57BL/6 male mice (Elevage Janvier, Le Genest Saint-Isle, France) were randomly assigned to one of the 7 groups (Supplementary Figure 5). Depending on their groups, mice were injected with AA + anti-CD4 Ab (GK1.5; Bioxcell, West Lebanon, NH USA) (AA + αCD4 group); with AA + anti-CD8 Ab (YTS 169.4; Bioxcell) (AA + αCD8 group); with AA + con- trol isotype (LTF-2; Bioxcell) (AA group); with AA + anti-CD25 Ab (PC61; Bioxcell) (AA + αCD25 group); with AA vehicle (i.e polyethylene-glycol, PEG) (Fluka Chemie, Buchs, Switzerland) + anti-CD4 Ab (αCD4 group); with PEG + anti-CD8 Ab (αCD8 group); or PEG + anti-25 Ab (αCD25 group). In addition, a control group did not receive any injection and was used as baseline day 0 (CTRL). AA (5mgr/kg body weight) or an equiva- lent volume of PEG was administered by means of daily IP injections during five days and GK1.5 or YTS169.4 (0.250 mg) or control isotype (0.250 mg) were administered by means of a single IP injections 72 hours before the first injection of AA. PC61 (0.300 mg) was injected twice, i.e. 72 and 24 hours before the first AA injection. AA (Acros Organics Co., Geel, Belgium; 40% AAI, 60% AAII,) was given in 150 µl of PEG and sterile water. GK1.5, YTS 169.4 and LTF-2 were given in 150 µl of sterile dPBS. For the chronic phase experiment, mice were injected with AA for 5, 15 or 22 days as described above. Ab injections were repeated weekly until sacrifice. CD4+ T-cell depletion (>95%) was effective during all protocol (supplementary Figure 1a,b). Twenty four hours after the last AA injection, mice were anesthetised with ketamine-HCl (Merial, Brussels, Belgium) and 2% xylazine (Bayer, Brussels, Belgium) after which a blood specimen was obtained by cardiac puncture. Then, kidneys were flushed with 30 ml of NaCl 0,9% and kidneys were harvested for analysis. Left kidneys were used for flow cytometric anal- ysis whereas right kidneys were used for realtime polymerase chain reaction (rtPCR) (immediately snap frozen in liquid nitrogen and stored at −80 °C) and for histological analysis. relevant guidelines and regulations. Methods Tubular damages were assessed after hematoxylin/ eosin coloration by scoring tubular necrosis in 10 non-overlapping fields (400× magnification) in the cortex and corticomedullary junction. Injury was scored by a pathologist (TB) blinded for the groups on a 5-point SCIENTIFIC REPOrTS \| (2018) 8:5334 \| DOI:10.1038/s41598-018-23565-2 8 www.nature.com/scientificreports/ Figure 7. As illustrated on this representative CD11b-F4/80 dots plot, ATN lesions observed after AA injections were associated with a sharp decrease in the P2 population and a significant increase in the P1 population (a). Absolute number of neutrophils (NEU), CD11c+, P1 and P2 population per gram of kidney in AA + αCD4 (black columns), AA (dark greys columns), αCD4 (light greys columns) and CTRL (white columns) groups on day 5 (b). As compared to CTRL group, a non significant increase in the absolute number of CD11c+, a significant increase in the absolute number of P1 populations and a significant reduction in the number of P2 population were observed after AA injection. Moreover, in the AA + αCD4 the increase in CD11c+ and P1 populations was even greater than those found in AA group. After CD8 depletion, a significant decrease in P2 population was also observed as compared to AA group (c). Statistical test used: ANOVA followed by t-test and Bonferroni correction for multiple comparisons or Mann-Whitney U-test for simple comparisons. Results are expressed as the mean ± SEM, **p < 0.0001, p < 0.01, p < 0.05; NS = no statistical difference observed. Number of mice per group: AA + αCD4 n = 13; AA n = 14; αCD4 n = 9; AA + αCD8 n = 8; αCD8 n = 8; CTRL n = 6. Figure 7. As illustrated on this representative CD11b-F4/80 dots plot, ATN lesions observed after AA injections were associated with a sharp decrease in the P2 population and a significant increase in the P1 population (a). Absolute number of neutrophils (NEU), CD11c+, P1 and P2 population per gram of kidney in AA + αCD4 (black columns), AA (dark greys columns), αCD4 (light greys columns) and CTRL (white columns) groups on day 5 (b). As compared to CTRL group, a non significant increase in the absolute number of CD11c+, a significant increase in the absolute number of P1 populations and a significant reduction in the number of P2 population were observed after AA injection. Methods Moreover, in the AA + αCD4 the increase in CD11c+ and P1 populations was even greater than those found in AA group. After CD8 depletion, a significant decrease in P2 population was also observed as compared to AA group (c). Statistical test used: ANOVA followed by t-test and Bonferroni correction for multiple comparisons or Mann-Whitney U-test for simple comparisons. Results are expressed as the mean ± SEM, *p < 0.0001, p < 0.01, *p < 0.05; NS = no statistical difference observed. Number of mice per group: AA + αCD4 n = 13; AA n = 14; αCD4 n = 9; AA + αCD8 n = 8; αCD8 n = 8; CTRL n = 6. semi-quantitative score: 0 = no damage, 1 less than 10% of the cortex and corticomedullary junction injured, 2 = 11–25%, 3 = 26–50%, 4 = 51–75%, 5 more than 75% of the cortex and corticomedullary junction injured. Immunohistochemistry. PBS was used for all washing steps. The FFPE sections (4 μm) were deparaffined, rehydrated and immersed in a retrieval sodium citrate buffer (pH 6.0) solution to unmask antigen. Endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide in a methanol solution (30 min). After blocking non-specific site, tissue sections were incubated overnight with primary antibodies (rat anti-mouse Ab F4/80, clone RM0029–11H3, Abcam, UK). Then, slides were exposed for 30 min to the secondary Ab, kidney sections were finally incubated with strepta-HRP complex for 30 min, and bound peroxidase activity was detected with the DAB kit (Dako, Heverlee, Belgium). Counterstaining with Luxaol Fast blue completed the processing. The specificity of Ab used was established by the producer. Normal serum (5% solution) instead of the primary Ab (used in order to exclude non-specific staining of kit reagents) showed no staining. Quantification of immunostainings. Quantifications were performed by two investigators (TB and IJ) who were blinded to the experimental groups. F4/80 positive cells were counted on ten photographs of non-overlapping 400× field, after which mean for the two investigators was calculated for each photograph. Biochemical evaluation of renal function. Plasma creatinine (Pcr) excretion levels were determined as previously described using an HPLC technique. Plasma BUN were determined using Quantichrom Urea Assay Kit (Bioassay systems, Gentaur, Brussels, Belgium). Isolation of lymphocytes from mouse kidneys. Kidney leukocytes were isolated using a technique described previously, with few modifications32. Methods In short, organs were disrupted mechanically in 5 ml RPMI 1640 SCIENTIFIC REPOrTS \| (2018) 8:5334 \| DOI:10.1038/s41598-018-23565-2 9 www.nature.com/scientificreports/ Protein Supplier Forward, 5′ -3′ Reverse, 5′ -3′ # IL-1b ABS na na Mm00434228_m1 IL-2 EG CAGGATGCTCACCTTCAAATT CAAGTTCATCTTCTAGGCACTGAA na CXCL10 ABS na na Mm00445235_m1 MCP-1 EG GCTCAGCCAGATGCAGTTAAC CCTACTCATTGGGATCATCTTG na IFNγ EG GGATGCATTCATGAGTATTGC GCTTCCTGAGGCTGGATTC na TNFα EG CAGACCCTCACACTCAGATCA CACTTGGTGGTTTGCTACGA na HPRT EG GGACCTCTCGAAGTGTTGGAT CCAACAACAAACTTGTCTGGAA na IL-6 ABS na na Mm00446190_m1 CXCL1 ABS na na Mm04207460_m1 TLR-2 EG CCTACATTGGCCATGGTGAC CCTCTATTGTATTGATTCTGCTGGA na TLR-4 EG TGTTGCTTGTATATGTGAACATCAG TCAACATTCACCAAGAACTGC na INOS EG CAGCTGGGCTGTACAAACCTT CATTGGAAGTGAAGCGTTTCG na MIP1-α EG AAGTCTTCTCAGCGCCATATG GTGGAATCTTCCGGCTGTAG na Table 1. ABS: applied biosystem; EG: Eurogentec; NA: non available. Table 1. ABS: applied biosystem; EG: Eurogentec; NA: non available. medium (PAA, Pasching, Austria)+ 10% FBS using a syringe plunger in a petri dish. To remove debris, samples were passed through a 40 micrometre filter that was then rinsed with 15 ml of RPMI 1640 medium. Next, cells were suspended in 5 ml of 36% Percoll (Sigma, Belgium Overijse), gently overlaid onto 72% Percoll and cen- trifuged at 1000 g for 25 min at room temperature. Cells were isolated from the Percoll interface and washed in medium at 300 g for 5 min at 4 °C, after which they were resuspended in 2 ml of RPMI and finally divided in two equal parts for immunostaining. Antibodies. The fluorochrome-conjugated mAb to mouse antigens used for flow cytometry analysis were from eBioscience (VWR, Haasrode, Belgium): anti-CD16/CD32, anti-Ly-6c (PE conjugated; clone HK1.4), anti F4/80 (APC conjugated; clone BM8), anti-Foxp3 (APC conjugated; clone FJK-16s); from Beckton Dickinson (Erembodegem, Belgium): anti-CD3 (PerCp conjugated; clone 145-2C11), anti-CD4 (FITC conjugated; clone RMA4-4), anti-CD8 (Pe-Cy7 conjugated; clone 53-6.7), anti-CD45 (APC-Cy7 conjugated; clone 30-F11), anti-CD25 (PE conjugated; clone 7D4), anti-CD11b (FITC conjugated; clone M1/70); or from Biolegend (Imtec, Antwerpen Belgium): anti-CD11c (Pe/Cy7 conjugated; clone N418), anti-Ly-6G (Brillant violet 421 conjugated; clone 1A8). GK1.5 mAb reportedly do not block binding of RM4-4 antibodies. Flow cytometry analysis. Lymphocytes were preincubated with anti-CD16/CD32 for 30 min to minimize nonspecific Ab binding. Cells were then incubated with various combinations of mAb for a minimum of 1 hour at 4 °C. One half was stained with anti-CD45, CD3, CD4, CD8 and CD25. After fixation and permeabilization with Fixpem solution (eBioscience) for 1 hour at 4 °C, cells were finally incubated with anti-Foxp3 Ab. The second half was stained with anti-CD45, CD11b, CD11c, Ly-6G, Ly6C and F4/80. References References 1. Vanherweghem, J. L. et al. Rapidly progressive interstitial renal fibrosis in young women: association with slimming regimen including Chinese herbs. Lancet 341, 387–391 (1993).i g ( ) 2. Vanhaelen, M., Vanhaelen-Fastre, R., But, P. & Vanherweghem, J. L. Identification of aristolochic acid in Chinese herbs. Lancet 343 174 (1994). 174 (1994). 3. Debelle, F. D., Vanherweghem, J. L. & Nortier, J. L. Aristolochic acid nephropathy: a worldwide problem. Kidney Int. 74, 158–169 3. Debelle, F. D., Vanherweghem, J. L. & Nortier, J. L. Aristolochic acid nephropathy: a worldwide problem. Kidney Int. 74, 158–169 (2008). ( ) 4. Depierreux, M., Van Damme, B., Vanden Houte, K. & Vanherweghem, J. L. 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Methods Eight-colours immunofluorescence staining was analyzed using a FACSCanto II instrument (BD Biosciences). The leukocytes were gated using CD45 and side-scatter (SSC) followed by exclusion of debris, dead cells and doublets. Gating strategies for myeloid pop- ulation are summarized in supplementary Figure 4. T-regs were defined as CD45+CD3+CD4+CD25highFOXP3+ cells. Cell count. The absolute number of kidney lymphocytes was determined using the Trucount (Beckton Dickinson) tubes following the instructions from the manufacturer. The absolute number of cells was expressed as number of cells per gram of kidneys (cell/organ) excepting T-regs which are a percentage of CD4+ T-cells. Messenger RNA quantification via Real-Time Reverse-transcriptase polymerase chain reac- tion. Frozen kidney samples were homogenized in a lysis solution with a MagNalyser (Roche Diagnostics, Brussels, Belgium). Total RNA was then extracted with the High Pure RNA Tissue Kit (Roche Diagnostics) according to the manufacturer’s protocol, which included DNase treatment. The mRNA quantification was performed using a 2-step real-time reverse-transcriptase polymerase chain reaction (LightCycler, Roche Diagnostics). The primers and probes were designed using Primer3 software (Whitehead Institute for Biomedical Research, Cambridge, MA) or were purchased as ready to use (Life technologies, Ghent Belgium) (Table 1). We used HPRT as the housekeeping gene. Data are expressed using the comparative threshold cycle (dCt) method, and mRNA ratios are given by 2−dCT as compared to CTRL group. Statistical analysis. Wilk-Shapiro test and Q-Q plots are used to verify if there is any deviation from the normality for the continuous variables. For group comparisons, Mann-Whitney tests, Kruskall-Wallis test followed by Mann-Whitney U-test or One Way ANOVA followed by t-test were used when appropriate. The Bonferroni correction has been used for multiple comparisons. For survival study, Log-rank (Mantel-Cox) test was used. All analyses were performed using Prism 5.0c software for Macintosh (GraphPad Software, San Diego California USA) or SPSS.v24 (SPSS Inc Chicago, Illinois, USA). SCIENTIFIC REPOrTS \| (2018) 8:5334 \| DOI:10.1038/s41598-018-23565-2 10 www.nature.com/scientificreports/ Acknowledgements g T. Baudoux is a fellow of the Fonds Erasme pour la Recherche Médicale, Brussels Belgium. The present study was in part supported by the Fonds Erasme. The authors are very grateful to the Association de Défense des Insuffisants Rénaux (ADIR, Belgium) for its financial support. Author Contributions All the authors have made substantial contributions to the intellectual content of the manuscript. T.B., J.M.H., J.L.N. participated in the design of the study and conceived the experiments. T.B., E.D.P., M.H.A. and C.H. performed the experiments. T.B. acquired the data. T.B. analyzed and interpreted the data (with J.M.H. for flow cytometric data and with I.J. for immunohistological data). T.B. wrote the manuscript. J.M.H. and J.L.N. revised the manuscript. References 72, 290–299 (2007).i 39. Cao, Q. et al. IL-10/TGF-{beta}-Modified Macrophages Induce Regulatory T Cells and Protect against Adriamycin Nephrosis. J. Am. Soc. Nephrol. (2010). p 0. Chan, T., Pek, E. A., Huth, K. & Ashkar, A. A. CD4(+) T-cells are important in regulating macrophage polarization in C57BL/6 wild-type mice. Cell. Immunol. 266, 180–186 (2011). y 41. Tadagavadi, R. K. & Reeves, W. B. Renal dendritic cells ameliorate nephrotoxic acute kidney injury. J. Am. Soc. Neph (2010). 41. Tadagavadi, R. K. & Reeves, W. B. Renal dendritic cells ameliorate nephrotoxic acute kidney injury. J. Am. Soc. Nephrol. 21, 53–63 (2010). ( ) 2. Kim, K. D. et al. Adaptive immune cells temper initial innate responses. Nat. Med. 13, 1248–1252 (2007). p p p 43. Inoue, M. et al. T cells down-regulate macrophage TNF production by IRAK1-mediated IL-10 expression and control innate hyperinflammation. Proc. Natl. Acad. Sci. USA 111, 5295–5300 (2014). SCIENTIFIC REPOrTS \| (2018) 8:5334 \| DOI:10.1038/s41598-018-23565-2 11 www.nature.com/scientificreports/ Acknowledgements SCIENTIFIC REPOrTS \| (2018) 8:5334 \| DOI:10.1038/s41598-018-23565-2 Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-23565-2 Competing Interests: The authors declare no competing interests. Competing Interests: The authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2018 SCIENTIFIC REPOrTS \| (2018) 8:5334 \| DOI:10.1038/s41598-018-23565-2 12
https://openalex.org/W4253896772	https://peerj.com/articles/9637v0.3/submission	English	null	Peer Review #3 of "Overexpression of KIAA1199 is an independent prognostic marker in laryngeal squamous cell carcinoma (v0.2)"	null	2,020	cc-by	11,277	Manuscript to be reviewed Overexpression of KIAA1199 is an independent prognostic marker in laryngeal squamous cell carcinoma Meixiang Huang 1 , Feifei Liao 2 , Yexun Song 1 , Gang Zuo 3 , Guolin Tan 1 , Ling Chu Corresp., 2 , Tiansheng Wang Corresp. 1 1 Department of Otolaryngology Head and Neck Surgery, The Third Xiangya Hospital, Central South University, Changsha, Hunan, China 2 Department of pathology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, Chian 3 Ministry of Education (Central South University), Key Laboratory of Metallogenic Prediction of Nonferrous Metals and Geological Environment Monitoring, Changsha, Hunan, China Overexpression of KIAA1199 is an independent prognostic marker in laryngeal squamous cell carcinoma Meixiang Huang 1 , Feifei Liao 2 , Yexun Song 1 , Gang Zuo 3 , Guolin Tan 1 , Ling Chu Corresp., 2 , Tiansheng Wang Corresp. 1 1 Department of Otolaryngology Head and Neck Surgery, The Third Xiangya Hospital, Central South University, Changsha, Hunan, China 2 Department of pathology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, Chian 3 Ministry of Education (Central South University), Key Laboratory of Metallogenic Prediction of Nonferrous Metals and Geological Environment Monitorin Changsha, Hunan, China Corresponding Authors: Ling Chu, Tiansheng Wang Email address: 1799417560@qq.com, tswheaven@hotmail.com Corresponding Authors: Ling Chu, Tiansheng Wang Email address: 1799417560@qq.com, tswheaven@hotmail.com Co espo d g u o s g C u, a s e g a g Email address: 1799417560@qq.com, tswheaven@hotmail.com Background: KIAA1199 is a recently identiﬁed novel gene that is up regulated in various human cancers with poor survival, but its role and the underlying mechanisms in laryngeal squamous cell carcinoma remain unknown. Here, we collected tissues from 105 cases of laryngeal squamous cell carcinoma (LSCC) to investigate the relationships between KIAA1199 protein expression and clinical factors. Methods: Western blotting and quantitative real-time PCR (qPCR) were utilized to detect the protein and mRNA expression of KIAA1199 in LSCC tissue. Immunohistochemistry (IHC) staining was used to detect the expression of KIAA1199. Patient clinical information, for instance sex, age, pathological diﬀerentiation, clinical region, T stage, N stage, clinical stage, operation type, neck lymph dissection, smoking status, and drinking status, were recorded. Kaplan-Meier survival analysis and Cox analysis were applied to ﬁnd out the relationship between KIAA1199 and LSCC. Results: Western blotting results showed KIAA1199 protein was signiﬁcantly higher in tumor tissues vs adjacent non cancerous tissues (0.9385±0.1363 vs1.838±0.3209, P=0.04). It revealed that KIAA1199 mRNA expression was considerably higher in tumor tissues (P<0.001) than in adjacent non cancerous tissues by qPCR. IHC results showed , the up-regulated KIAA1199 expression, which was related with some severe clinicopathological parameters: pathologic diﬀerentiation (P, 0.002), T stage (P<0.001), N stage (P<0.001), clinical stage (P<0.001), survival time (P, 0.008) and survival status (P<0.001). Kaplan- Meier survival analysis revealed that patients with high KIAA1199 protein expression had poor overall survival(OS) (P<0.05). Cox analysis suggested that the KIAA1199 protein expression was an independent prognostic marker for LSCC patients (P<0.001). Conclusion: Our ﬁndings revealed that KIAA1199 protein expression may be used to predict LSCC patient outcome. PeerJ reviewing PDF \| (2020:01:44662:2:0:NEW 29 Jun 2020) Manuscript to be reviewed 1 Overexpression of KIAA119 2 prognostic marker in laryn 3 carcinoma 4 5 Meixiang Huang1, Feifei Liao2, Yexun Song3, Gang Z 6 Wang7 7 8 9 1 Department of Otolaryngology Head and Neck Surg 10 South University, Changsha, Hunan, P.R. China 11 2 Department of pathology, The Third Xiangya Hosp 12 Hunan, P.R. China 13 3 Department of Otolaryngology Head and Neck Surg 14 South University, Changsha, Hunan, P.R. Overexpression of KIAA1199 is an independent prognostic marker in laryngeal squamous cell carcinoma Meixiang Huang 1 , Feifei Liao 2 , Yexun Song 1 , Gang Zuo 3 , Guolin Tan 1 , Ling Chu Corresp., 2 , Tiansheng Wang Corresp. 1 1 Department of Otolaryngology Head and Neck Surgery, The Third Xiangya Hospital, Central South University, Changsha, Hunan, China 2 Department of pathology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, Chian 3 Ministry of Education (Central South University), Key Laboratory of Metallogenic Prediction of Nonferrous Metals and Geological Environment Monitorin Changsha, Hunan, China China 15 4 Ministry of Education (Central South University), K 16 of Nonferrous Metals and Geological Environment Mo 17 5 Department of Otolaryngology Head and Neck Surg 18 South University, Changsha, Hunan, P.R. China 19 6 Department of pathology, The Third Xiangya Hosp 20 Hunan, P.R. China 21 7 Department of Otolaryngology Head and Neck Surg 22 South University, Changsha, Hunan, P.R. China 23 24 25 Corresponding Author: 26 Tiansheng Wang1, Ling Chu2 27 1Department of Otolaryngology Head and Neck Surg 28 South University, Changsha, Hunan, P.R. China 29 Email address: tswheaven@hotmail.com 30 2 Department of pathology, The Third Xiangya Hosp 31 Hunan, P.R. China 32 Email address: 1799417560@qq.com 33 34 1 Overexpression of KIAA1199 is an independen 2 prognostic marker in laryngeal squamous cel 3 carcinoma 4 5 Meixiang Huang1, Feifei Liao2, Yexun Song3, Gang Zuo4, Guolin Tan5, Ling Chu6, Tianshen 6 Wang7 7 8 9 1 Department of Otolaryngology Head and Neck Surgery, The Third Xiangya Hospital, Centra 10 South University, Changsha, Hunan, P.R. China 11 2 Department of pathology, The Third Xiangya Hospital, Central South University, Changsha 12 Hunan, P.R. China 13 3 Department of Otolaryngology Head and Neck Surgery, The Third Xiangya Hospital, Centra 14 South University, Changsha, Hunan, P.R. China 15 4 Ministry of Education (Central South University), Key Laboratory of Metallogenic Predictio 16 of Nonferrous Metals and Geological Environment Monitoring, Changsha, Hunan, P.R. China 17 5 Department of Otolaryngology Head and Neck Surgery, The Third Xiangya Hospital, Centra 18 South University, Changsha, Hunan, P.R. China 19 6 Department of pathology, The Third Xiangya Hospital, Central South University, Changsha 20 Hunan, P.R. China 21 7 Department of Otolaryngology Head and Neck Surgery, The Third Xiangya Hospital, Centra 22 South University, Changsha, Hunan, P.R. China 23 24 25 Corresponding Author: 26 Tiansheng Wang1, Ling Chu2 27 1Department of Otolaryngology Head and Neck Surgery, The Third Xiangya Hospital, Centra 28 South University, Changsha, Hunan, P.R. China 29 Email address: tswheaven@hotmail.com 30 2 Department of pathology, The Third Xiangya Hospital, Central South University, Changsha 31 Hunan, P.R. China 32 Email address: 1799417560@qq.com 33 34 7 8 9 1 Department of Otolaryngology Head and Neck Surgery, The Third Xiangya Hospital, Central 10 South University, Changsha, Hunan, P.R. China 11 2 Department of pathology, The Third Xiangya Hospital, Central South University, Changsha, 12 Hunan, P.R. Manuscript to be reviewed Manuscript to be reviewed Overexpression of KIAA1199 is an independent prognostic marker in laryngeal squamous cell carcinoma Meixiang Huang 1 , Feifei Liao 2 , Yexun Song 1 , Gang Zuo 3 , Guolin Tan 1 , Ling Chu Corresp., 2 , Tiansheng Wang Corresp. 1 1 Department of Otolaryngology Head and Neck Surgery, The Third Xiangya Hospital, Central South University, Changsha, Hunan, China 2 Department of pathology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, Chian 3 Ministry of Education (Central South University), Key Laboratory of Metallogenic Prediction of Nonferrous Metals and Geological Environment Monitorin Changsha, Hunan, China China 13 3 Department of Otolaryngology Head and Neck Surgery, The Third Xiangya Hospital, Central 14 South University, Changsha, Hunan, P.R. China 15 4 Ministry of Education (Central South University), Key Laboratory of Metallogenic Prediction 16 of Nonferrous Metals and Geological Environment Monitoring, Changsha, Hunan, P.R. China 9 1 Department of Otolaryngology Head and Neck Surgery, The Third Xiangya Hospital, Central 10 South University, Changsha, Hunan, P.R. China 11 2 Department of pathology, The Third Xiangya Hospital, Central South University, Changsha, 12 Hunan, P.R. China 13 3 Department of Otolaryngology Head and Neck Surgery, The Third Xiangya Hospital, Central 14 South University, Changsha, Hunan, P.R. China 15 4 Ministry of Education (Central South University), Key Laboratory of Metallogenic Prediction 16 of Nonferrous Metals and Geological Environment Monitoring, Changsha, Hunan, P.R. China 17 5 Department of Otolaryngology Head and Neck Surgery, The Third Xiangya Hospital, Central 18 South University, Changsha, Hunan, P.R. China 15 4 Ministry of Education (Central South University), Key Laboratory of Metallogenic Prediction 16 of Nonferrous Metals and Geological Environment Monitoring, Changsha, Hunan, P.R. China 17 5 Department of Otolaryngology Head and Neck Surgery, The Third Xiangya Hospital, Central 18 South University, Changsha, Hunan, P.R. China 19 6 Department of pathology, The Third Xiangya Hospital, Central South University, Changsha, 20 Hunan, P.R. China 19 6 Department of pathology, The Third Xiangya Hospital, Central South University, Changsha, 20 Hunan, P.R. China 20 Hunan, P.R. China 21 7 Department of Otolaryngology Head and Neck Surgery, The Third Xiangya Hospital, Central 22 South University, Changsha, Hunan, P.R. China 23 24 25 Corresponding Author: 26 Tiansheng Wang1, Ling Chu2 27 1Department of Otolaryngology Head and Neck Surgery, The Third Xiangya Hospital, Central 28 South University, Changsha, Hunan, P.R. China 29 Email address: tswheaven@hotmail.com 30 2 Department of pathology, The Third Xiangya Hospital, Central South University, Changsha, 31 Hunan, P.R. China 32 Email address: 1799417560@qq.com 33 34 21 7 Department of Otolaryngology Head and Neck Surgery, The Third Xiangya Hospital, Central 22 South University, Changsha, Hunan, P.R. China 23 PeerJ reviewing PDF \| (2020:01:44662:2:0:NEW 29 Jun 2020) Manuscript to be reviewed Over-expression of 79 KIAA1199 contributes to resistance to cell immortalization and cancerization in normal human 80 cells and is associated with cell death(Eriko Michishita, et al., 2006). Several researches have 81 illuminated that KIAA1199 is over-expressed in different cancers, including oral squamous cell 82 carcinoma(Pitak Chanthammachat, et al., 2013), breast cancer(Nikki A. Evensen, et al., 2013), 83 gastric cancer(Shinji Matsuzaki, et al., 2009), colorectal tumors((K Birkenkamp-Demtroder, et al., 84 2011; Lawrence C. LaPointe, et al., 2012), prostate cancer(Eriko Michishita, et al., 2006), ovarian 85 cancer(Fan Shena, et al., 2019) and hepatocellular carcinoma(Zhengchen Jiang, et al., 2018). The 86 workings proved, that KIAA1199, which regulates the proliferation, migration, and invasion of 87 tumors. So far, in LSCC remain an enigma, we have no report about KIAA1199 expression in 88 LSCC, and the clinical value and biological role of KIAA1199. 89 We implemented immunohistochemical detection of KIAA1199 protein expression to 90 investigate the clinical significance of KIAA1199 and to detect whether it plays a key role in the 91 progression of LSCC in 105 paired formalin-fixed and paraffin-embedded cancer and adjacent 92 noncancer tissues obtained from patients with LSCC. In the end, we illuminated the 93 clinicopathologic characteristics of LSCC patients was related to KIAA1199, which were 94 statistically evaluated. Manuscript to be reviewed 68 Laryngeal squamous cell carcinoma is the most common laryngeal cancer , and it is the second 69 highest incidence among head and neck cancer(Ling Gao, et al., 2018). In 2016, Laryngeal 70 malignancies accounted for approximately 13,400 cancer cases in U.S.A, of which an estimated 71 3600 patients succumbed to the disease(Rebecca L. Siegel, et al., 2019). The number of laryngeal 72 cancer has radically changed in the last 20 years in USA(S. Michael Rothenberg, et al., 2012). In 73 the available treatments, there have been improvements, but the patients still need identified novel 74 gene that is up-regulated in human cancer suffer from poor prognosis. So, in the pathogenesis of 75 LSCC, identification of the key molecules is urgently needed to improve the treatment of LSCC. 76 The KIAA1199 gene, which was first discovered in association with non-syndromic hearing 77 loss(Satoko Abe, et al., 2003). Nowadays, we have known that the KIAA1199 gene is expressed 78 in a wide range of normal human tissues(Yongsheng Zhang, et al., 2014). Over-expression of 79 KIAA1199 contributes to resistance to cell immortalization and cancerization in normal human 80 cells and is associated with cell death(Eriko Michishita, et al., 2006). Several researches have 81 illuminated that KIAA1199 is over-expressed in different cancers, including oral squamous cell 82 carcinoma(Pitak Chanthammachat, et al., 2013), breast cancer(Nikki A. Evensen, et al., 2013), 83 gastric cancer(Shinji Matsuzaki, et al., 2009), colorectal tumors((K Birkenkamp-Demtroder, et al., 84 2011; Lawrence C. LaPointe, et al., 2012), prostate cancer(Eriko Michishita, et al., 2006), ovarian 85 cancer(Fan Shena, et al., 2019) and hepatocellular carcinoma(Zhengchen Jiang, et al., 2018). The 86 workings proved, that KIAA1199, which regulates the proliferation, migration, and invasion of 87 tumors. So far, in LSCC remain an enigma, we have no report about KIAA1199 expression in 88 LSCC, and the clinical value and biological role of KIAA1199. 68 Laryngeal squamous cell carcinoma is the most common laryngeal cancer , and it is the second 69 highest incidence among head and neck cancer(Ling Gao, et al., 2018). In 2016, Laryngeal 70 malignancies accounted for approximately 13,400 cancer cases in U.S.A, of which an estimated 71 3600 patients succumbed to the disease(Rebecca L. Siegel, et al., 2019). The number of laryngeal 72 cancer has radically changed in the last 20 years in USA(S. Michael Rothenberg, et al., 2012). 35 Abstract 35 Abstract 36 Background: KIAA1199 is a recently identified novel gene that is upregulated in various human 37 cancers with poor survival, but its role and the underlying mechanisms in laryngeal squamous cell 38 carcinoma remain unknown. Here, we collected tissues from 105 cases of laryngeal squamous cell 39 carcinoma (LSCC) to investigate the relationships between KIAA1199 protein expression and 40 clinical factors. 41 42 Methods: Western blotting and real time quantitative PCR (RT- PCR) were used for detect the 43 protein and mRNA expression of KIAA1199 in LSCC tissue. Immunohistochemistry (IHC) 44 staining was used to detect the expression of KIAA1199. Patient clinical information, for instance 45 sex, age, pathological differentiation, clinical region, T stage, N stage, clinical stage, operation 46 type, neck lymph dissection, smoking status, and drinking status, were recorded. Kaplan-Meier 47 survival analysis and Cox analysis were applied to identify the relationship between KIAA1199 48 and LSCC. 49 50 Results: Western blotting results showed KIAA1199 protein was significantly higher in tumor 51 tissues vs adjacent non cancerous tissues (0.9385 ± 0.1363 vs 1.838 ± 0.3209, P=0.04). The 52 KIAA1199 mRNA expression was considerably higher in tumor tissues (P<0.001) than in adjacent 53 non cancerous tissues by RT-PCR. IHC results showed up-regulated KIAA1199 expression was 54 related with some severe clinicopathological parameters: pathologic differentiation (P, 0.002), T 55 stage (P<0.001), N stage (P<0.001), clinical stage (P<0.001), survival time (P, 0.008) and survival 56 status (P<0.001). Kaplan-Meier survival analysis showed that patients with high KIAA1199 57 protein expression had poor overall survival(OS) (P<0.05). Cox analysis suggested that the 58 KIAA1199 protein expression constituted an independent prognostic marker for LSCC patients 59 (P<0.001). 60 61 Conclusion: Our findings revealed that KIAA1199 protein expression may be used to predict 62 LSCC patient outcome. 63 64 Keywords: KIAA1199, CEMIP, Laryngeal squamous carcinoma, Prognostic marker 65 66 61 Conclusion: Our findings revealed that KIAA1199 protein expression may be used to predict 62 LSCC patient outcome. 63 PeerJ reviewing PDF \| (2020:01:44662:2:0:NEW 29 Jun 2020) Manuscript to be reviewed In 73 the available treatments, there have been improvements, but the patients still need identified novel 74 gene that is up-regulated in human cancer suffer from poor prognosis. So, in the pathogenesis of 75 LSCC, identification of the key molecules is urgently needed to improve the treatment of LSCC. 68 Laryngeal squamous cell carcinoma is the most common laryngeal cancer , and it is the second 69 highest incidence among head and neck cancer(Ling Gao, et al., 2018). In 2016, Laryngeal 70 malignancies accounted for approximately 13,400 cancer cases in U.S.A, of which an estimated 71 3600 patients succumbed to the disease(Rebecca L. Siegel, et al., 2019). The number of laryngeal 72 cancer has radically changed in the last 20 years in USA(S. Michael Rothenberg, et al., 2012). In 73 the available treatments, there have been improvements, but the patients still need identified novel 74 gene that is up-regulated in human cancer suffer from poor prognosis. So, in the pathogenesis of 75 LSCC, identification of the key molecules is urgently needed to improve the treatment of LSCC. 76 The KIAA1199 gene, which was first discovered in association with non-syndromic hearing 77 loss(Satoko Abe, et al., 2003). Nowadays, we have known that the KIAA1199 gene is expressed 78 in a wide range of normal human tissues(Yongsheng Zhang, et al., 2014). Over-expression of 79 KIAA1199 contributes to resistance to cell immortalization and cancerization in normal human 80 cells and is associated with cell death(Eriko Michishita, et al., 2006). Several researches have 81 illuminated that KIAA1199 is over-expressed in different cancers, including oral squamous cell 82 carcinoma(Pitak Chanthammachat, et al., 2013), breast cancer(Nikki A. Evensen, et al., 2013), 83 gastric cancer(Shinji Matsuzaki, et al., 2009), colorectal tumors((K Birkenkamp-Demtroder, et al., 84 2011; Lawrence C. LaPointe, et al., 2012), prostate cancer(Eriko Michishita, et al., 2006), ovarian 85 cancer(Fan Shena, et al., 2019) and hepatocellular carcinoma(Zhengchen Jiang, et al., 2018). The 86 workings proved, that KIAA1199, which regulates the proliferation, migration, and invasion of 87 tumors. So far, in LSCC remain an enigma, we have no report about KIAA1199 expression in 88 LSCC, and the clinical value and biological role of KIAA1199. 76 The KIAA1199 gene, which was first discovered in association with non-syndromic hearing 77 loss(Satoko Abe, et al., 2003). Nowadays, we have known that the KIAA1199 gene is expressed 78 in a wide range of normal human tissues(Yongsheng Zhang, et al., 2014). Manuscript to be reviewed 102 performed curative resection for LSCC were registered from 2009 to 2014. Patients with 103 recurrence of laryngeal cancer or multiple cancers were excluded. No anticancer therapy was given 104 before surgery. Postoperative pathological examination of patients diagnosed with laryngeal 105 squamous cell cancer. Patient clinical data such as sex, age, pathological differentiation, clinical 106 region, T stage, N stage, clinical stage, operation type, neck lymph dissection, smoking status, and 107 drinking status were collected. To investigate the prognostic value of KIAA1199 in postoperative 108 patients, we examined the overall survival rate (OS) of the LSCC patients. The average arrange 109 follow up cycle was 54 months (5 months extent to 10 years). Prior to the start of the study, we 110 obtained the written informed consent of all patients and the approval of the hospital's Human 111 Research Ethics Committee in accordance with the Helsinki Declaration Guidelines(NO. 2018- 112 S084). All tissue samples were treated and anonymous in accordance with ethical and legal 113 standards. The tumor stage was determined according to the TNM(tumor, lymph node, metastasis) 114 grading of the International Union Against Cancer (UICC, 2002). 115 116 RNA extraction and real-time RT-PCR 117 According to the manufacturer’s protocol, the total RNA was isolated from LSCC and matched 118 adjacent tissues by using TRIzol Reagent (Invitrogen). Nanodroplet spectrophotometer (Thermo 119 Scientific, Waltham, MA, USA) was used to measure the concentration and purity of total RNA. 120 According to the manufacturer's instructions, the total RNA was converted to cDNA using a 121 quantitative PCR (qPCR) reverse transcription kit (TOYOBO Life Science, Shanghai, P.R. China), 122 fresh tissues were used to synthesize cDNA. Real-time RT-PCR was applied three times using a 123 KOD SYBR qPCR Mix Fluorescent Quantitative PCR kit (TOYOBO Life Science, Shanghai, P.R. 124 China). PCR and data collection were conducted by using an EP Real-time PCR System 125 (Eppendorf Inc., Hauppauge, NY, USA). For standardization, we used GAPDH as an endogenous 126 control. The primers used in our study were purchased from Sangon Biotech (Shanghai, P.R. 127 China), and the following primer sequences were used: KIAA1199, F primer 5′- 128 CCAGTAACCTGCGAATGAAGA-3′ and R primer 5′-TGGTCCCAGTGGATGGTGTAG-3′. 129 GAPDH, F primer 5′-TTGGTATCGTGGAAGGACTCA-3′ and R primer 5′- 130 TGTCATCATATTTGGCAGGTT-3′. The reaction conditions were 95°C for 5 min, followed by 131 40 cycles at 95°C for 15 sec and 58°C for 30 sec. The relative expression level was determined by 132 the 2-ΔΔCt method. 97 Patient enrolment and arrange follow up 98 The research was implemented in the Department of ENT, the Third Xiangya Hospital, Central 99 South University. We collected 10 pairs of fresh specimens and their matched adjacent non 100 cancerous specimens, which were from patients diagnosed with human laryngeal squamous cell 101 carcinoma by pathological examination in February 2018. A total of 105 patients who had been PeerJ reviewing PDF \| (2020:01:44662:2:0:NEW 29 Jun 2020) 141 Immunohistochemistry 142 One hundred and five formalin-fixed, paraffin-embedded LSCC tissues were used for the 143 immunohistochemistry (IHC) studies. Briefly, the tissue was sliced continuously into 144 approximately 4 μm section, paraffin was removed from the sections using a graded alcohol series 145 of 100% and 95% in xylene, rehydrated in 75%, and finally washed with PBS. Subsequently, the 146 antigen was prepared with sodium citrate buffer PBS and incubated in 3% H2O2 deionized water 147 for 15 minutes to inactivate endogenous peroxidase. The sections were washed 3 times with PBS, 148 incubated with calf serum to block non-specific antigen for 10 min, incubated with polyclonal 149 rabbit anti-KIAA1199 antibody (1:70) at room temperature for 1 hour, washed with PBS three 150 times, and then incubated with secondary antibody at room temperature for 30 minutes. Sections 151 were washed with PBS 3 times, stained with diaminobenzidine (DAB) for 4 minutes, washed 3 152 more times with PBS, restained with haematoxylin for 30 seconds, washed with flowing water, 153 dried and sealed. Dried sections were observed with an optical microscope. The positive control 154 was gastric cancer tissue confirmed by pathological examination, and the adjacent normal tissues 155 from patients with LSCC were used as the negative control. 156 The positive expression of KIAA1199 was patchy with aggregates of brown granules in the 157 cytoplasm. Semiquantitative analysis was used to determine the percentage of positive cells under 158 the microscope and score the staining intensity. Two senior pathologists of the Department of 159 Pathology were assigned to read the slides in a double-blinded manner, and 3-5 different fields 160 were randomly selected from each IHC staining section for observation. The staining results were 161 semiquantitatively analysed in terms of staining intensity and percentage of cells with positive 162 expression. Evaluation of dyeing intensity: range, 0-3; colourless (negative) = 0, weak (pale 163 yellow) = 1, medium (brown-yellow) = 2, strong (tan) = 3. Percentage of stained cells: range, 0-3; 164 percent positive cells < 5% = 0, 0.5%-10% = 1, 10%-50% = 2, ≥50% = 3. The score of the two 165 was multiplied to show the positive grade: 0 is negative (-), ≤3 is low expression, and >3 is higher 166 expression. 156 The positive expression of KIAA1199 was patchy with aggregates of brown granules in the 157 cytoplasm. Manuscript to be reviewed 137 article(Wei Li, et al., 2012).Primary antibodies were used as follows: polyclonal rabbit anti- 138 KIAA1199 antibody (diluted 1:1,000), anti-GAPDH antibody (diluted 1:5000), and horseradish 139 peroxidase-conjugated secondary antibody (1:10,000). 137 article(Wei Li, et al., 2012).Primary antibodies were used as follows: polyclonal rabbit anti- 138 KIAA1199 antibody (diluted 1:1,000), anti-GAPDH antibody (diluted 1:5000), and horseradish 139 peroxidase-conjugated secondary antibody (1:10,000). 137 article(Wei Li, et al., 2012).Primary antibodies were used as follows: pol 138 KIAA1199 antibody (diluted 1:1,000), anti-GAPDH antibody (diluted 1:500 139 peroxidase-conjugated secondary antibody (1:10,000). 140 141 Immunohistochemistry 142 One hundred and five formalin-fixed, paraffin-embedded LSCC tissues 143 immunohistochemistry (IHC) studies. Briefly, the tissue was sliced 144 approximately 4 μm section, paraffin was removed from the sections using a g 145 of 100% and 95% in xylene, rehydrated in 75%, and finally washed with PBS 146 antigen was prepared with sodium citrate buffer PBS and incubated in 3% H2 147 for 15 minutes to inactivate endogenous peroxidase. The sections were washe 148 incubated with calf serum to block non-specific antigen for 10 min, incuba 149 rabbit anti-KIAA1199 antibody (1:70) at room temperature for 1 hour, was 150 times, and then incubated with secondary antibody at room temperature for 3 151 were washed with PBS 3 times, stained with diaminobenzidine (DAB) for 4 152 more times with PBS, restained with haematoxylin for 30 seconds, washed 153 dried and sealed. Dried sections were observed with an optical microscope. T 154 was gastric cancer tissue confirmed by pathological examination, and the adj 155 from patients with LSCC were used as the negative control. 156 The positive expression of KIAA1199 was patchy with aggregates of bro 157 cytoplasm. Semiquantitative analysis was used to determine the percentage of 158 the microscope and score the staining intensity. Two senior pathologists of 159 Pathology were assigned to read the slides in a double-blinded manner, and 160 were randomly selected from each IHC staining section for observation. The s 161 semiquantitatively analysed in terms of staining intensity and percentage of 137 article(Wei Li, et al., 2012).Primary antibodies were used as follows: polyclonal rabbit anti- 138 KIAA1199 antibody (diluted 1:1,000), anti-GAPDH antibody (diluted 1:5000), and horseradish 139 peroxidase-conjugated secondary antibody (1:10,000). 134 Western Blotting Analysis 135 Proteins were extracted from LSCC fresh tissue samples and adjacent non cancerous fresh 136 tissue samples. The Western blotting analysis was carried out according to our previous PeerJ reviewing PDF \| (2020:01:44662:2:0:NEW 29 Jun 2020) 141 Immunohistochemistry Semiquantitative analysis was used to determine the percentage of positive cells under 158 the microscope and score the staining intensity. Two senior pathologists of the Department of 159 Pathology were assigned to read the slides in a double-blinded manner, and 3-5 different fields 160 were randomly selected from each IHC staining section for observation. The staining results were 161 semiquantitatively analysed in terms of staining intensity and percentage of cells with positive 162 expression. Evaluation of dyeing intensity: range, 0-3; colourless (negative) = 0, weak (pale 163 yellow) = 1, medium (brown-yellow) = 2, strong (tan) = 3. Percentage of stained cells: range, 0-3; 164 percent positive cells < 5% = 0, 0.5%-10% = 1, 10%-50% = 2, ≥50% = 3. The score of the two 165 was multiplied to show the positive grade: 0 is negative (-), ≤3 is low expression, and >3 is higher 166 expression. 167 168 169 Statistical analysis 170 Our results were interpreted with GraphPad Prism version 7.0 (GraphPad Software, Inc., La 171 Jolla, CA, USA) and SPSS 23.0 software package (SPSS, 112 Y.-H. HAO ET AL. Chicago, IL, 172 USA). Chi-square test was used to analyze the associate with KIAA1199 protein expression and 173 clinicopathological characteristics in LSCC patients. Cox regression analysis estimated the risk of Manuscript to be reviewed 174 death associated with KIAA1199 protein expre 175 the total survival curve. Other data were ana 176 conducted to determine the differences in two o 177 ± SD with P < 0.05 (P<0.05, P<0.01, *P< 178 179 180 Results 181 1. Clinical data 182 To understand the clinical features of patients 183 pathological differentiation, clinical region, T st 184 lymph dissection, smoking status, and drinking 185 and these data are summarized in Table 1 for the 186 and 2 (1.9%) were women, ranging in age from 187 patients (66.6%), N1-N3 stage was detected in 188 time ranged from 6 to 108 months. 189 190 191 2. Increased Expression of KIAA1199 in Hum 192 To uncover the role of KIAA1199 expressio 193 and mRNA expression in 10 pairs of fresh hum 194 noncancerous specimens using Western blottin 195 1C). As shown in, KIAA1199 protein levels w 196 0.3209 vs 0.9385 ± 0.1363, P=0.04) (Fig.1B) 197 revealed that KIAA1199 mRNA expression w 198 tissues (P<0.001) than in cancer tissues (Fig 199 KIAA1199 in 105 LSCC tissues and t 200 immunohistochemistry. There was weak or 201 noncancerous tissue but high expression in th 202 staining and negative staining rates in LSCC t 203 (50/105), respectively. Semiquantitative analy 204 increased in LSCC tissues. Representative photo 205 3 A-F. RT-PCR results for KIAA1199 mRNA 206 IHC results, showing that KIAA1199 is increase 207 208 209 3 KIAA1199 i i i t d ith 174 death associated with KIAA1199 protein expression. Kaplan-meier method was used to analyze 175 the total survival curve. Other data were analysed using Student’s t-test, and ANOVA was 176 conducted to determine the differences in two or more groups. All data are presented as the mean 177 ± SD with P < 0.05 (P<0.05, P<0.01, P<0.005, **P<0.001). 174 death associated with KIAA1199 protein expression. Kaplan-meier method was used to analyze 175 the total survival curve. Other data were analysed using Student’s t-test, and ANOVA was 176 conducted to determine the differences in two or more groups. All data are presented as the mean 177 ± SD with P < 0.05 (P<0.05, P<0.01, P<0.005, **P<0.001). 174 death associated with KIAA1199 protein expression. Kaplan-meier method was used to analyze 175 the total survival curve. Other data were analysed using Student’s t-test, and ANOVA was 176 conducted to determine the differences in two or more groups. Manuscript to be reviewed Clinical data 182 To understand the clinical features of patients, the detailed data of the patients, such as sex, age, 183 pathological differentiation, clinical region, T stage, N stage, clinical stages, operation type, neck 184 lymph dissection, smoking status, and drinking status, were collected from their medical records, 185 and these data are summarized in Table 1 for the 105 patients in this study; 103 (98.1%) were men, 186 and 2 (1.9%) were women, ranging in age from 37 to 82 years. T1-T2 stage was detected in 70 187 patients (66.6%), N1-N3 stage was detected in 20 patients (19%), and the overall survival (OS) 188 time ranged from 6 to 108 months. 169 Statistical analysis y 170 Our results were interpreted with GraphPad Prism version 7.0 (GraphPad Software, Inc., La 171 Jolla, CA, USA) and SPSS 23.0 software package (SPSS, 112 Y.-H. HAO ET AL. Chicago, IL, 172 USA). Chi-square test was used to analyze the associate with KIAA1199 protein expression and 173 clinicopathological characteristics in LSCC patients. Cox regression analysis estimated the risk of PeerJ reviewing PDF \| (2020:01:44662:2:0:NEW 29 Jun 2020) Manuscript to be reviewed All data are presented as the mean 177 ± SD with P < 0.05 (P<0.05, P<0.01, P<0.005, **P<0.001). 178 179 180 Results 181 1. Clinical data 182 To understand the clinical features of patients, the detailed data of the patients, such as sex, age, 183 pathological differentiation, clinical region, T stage, N stage, clinical stages, operation type, neck 184 lymph dissection, smoking status, and drinking status, were collected from their medical records, 185 and these data are summarized in Table 1 for the 105 patients in this study; 103 (98.1%) were men, 186 and 2 (1.9%) were women, ranging in age from 37 to 82 years. T1-T2 stage was detected in 70 187 patients (66.6%), N1-N3 stage was detected in 20 patients (19%), and the overall survival (OS) 188 time ranged from 6 to 108 months. 189 190 191 2. Increased Expression of KIAA1199 in Human LSCC tissues 192 To uncover the role of KIAA1199 expression in LSCC, we first detected KIAA1199 protein 193 and mRNA expression in 10 pairs of fresh human LSCC specimens and their matched adjacent 194 noncancerous specimens using Western blotting (Fig. 1A, 1B), IHC (Fig. 2) and RT-PCR (Fig. 195 1C). As shown in, KIAA1199 protein levels were significantly higher in LSCC tissues (1.838 ± 196 0.3209 vs 0.9385 ± 0.1363, P=0.04) (Fig.1B) than in adjacent noncancerous tissues. RT-PCR 197 revealed that KIAA1199 mRNA expression was considerably lower in adjacent noncancerous 198 tissues (P<0.001) than in cancer tissues (Fig. 1C). Then, we compared the expression of 199 KIAA1199 in 105 LSCC tissues and their adjacent noncancerous tissues through 200 immunohistochemistry. There was weak or negative expression of KIAA1199 in adjacent 201 noncancerous tissue but high expression in the cytoplasm of LSCC tissue cells. The positive 202 staining and negative staining rates in LSCC tissues were 52.4% (55/105, Table 1) and 47.6% 203 (50/105), respectively. Semiquantitative analysis showed that KIAA1199 was significantly 204 increased in LSCC tissues. Representative photographs of the immunostaining are shown in figure 205 3 A-F. RT-PCR results for KIAA1199 mRNA levels were agree with the Western blotting and 206 IHC results, showing that KIAA1199 is increased in LSCC tissues. 207 208 209 3. KIAA1199 expression is associated with pathologic differentiation, T, N, clinical stage, 210 survival status and survival times of LSCC 181 1. 174 death associated with KIAA1199 protein expression. Kaplan-meier method was used to analyze 175 the total survival curve. Other data were analysed using Student’s t-test, and ANOVA was 176 conducted to determine the differences in two or more groups. All data are presented as the mean 177 ± SD with P < 0.05 (P<0.05, P<0.01, P<0.005, *P<0.001). 219 4. Survival assessment: A high level of KIAA1199 is predictive of poor prognosis in LSCC 220 patients The univariate 228 analysis results (Table 3) showed that age (HR =1.032, 95% CI: 1.001 - 1.063; P, 0.04), pathologic 229 differentiation (HR =0.643, 95% CI: 0.524 - 0.789; P<0.001), T stage (HR =1.402, 95% CI: 1.139 230 - 1.724; P<0.001), N stage (HR =1.679, 95% CI: 1.148 - 2.4577; P, 0.008), clinical stage (HR 231 =1.445, 95% CI: 1.180 - 1.769; P<0.001), operation type (HR =0.380, 95% CI: 0.222 - 0.650; 232 P<0.001) and KIAA1199 expression (HR =12.165, 95% CI: 5.434 - 27.233; P<0.001) were 233 significantly associated with the overall survival of LSCC patients. Multivariate survival analysis 234 (table 4) showed that KIAA1199 expression was statistically significant predictor of OS(HR 235 =27.937, 95% CI: 10.600–73.632; P<0.0001) and that age (HR =1.039, 95% CI: 1.003 - 1.077; P, 236 0.0354), clinical stage (HR =0.704, 95% CI: 0.581–0.960; P, 0.023), operation type (HR =0.285, 237 95% CI: 0.093-0.870; P, 0.027), T stage (HR =0.68, 95% CI: 0.529-0.874; P, 0.003) and smoking 238 status (HR =0.19, 95% CI: 0.057–0.630; P, 0.007) were independent predictive factors for OS. 191 2. Increased Expression of KIAA1199 in Human LSCC tissues 191 2. Increased Expression of KIAA1199 in Human LSCC tissues 192 To uncover the role of KIAA1199 expression in LSCC, we first detected KIAA1199 protein 193 and mRNA expression in 10 pairs of fresh human LSCC specimens and their matched adjacent 194 noncancerous specimens using Western blotting (Fig. 1A, 1B), IHC (Fig. 2) and RT-PCR (Fig. 195 1C). As shown in, KIAA1199 protein levels were significantly higher in LSCC tissues (1.838 ± 196 0.3209 vs 0.9385 ± 0.1363, P=0.04) (Fig.1B) than in adjacent noncancerous tissues. RT-PCR 197 revealed that KIAA1199 mRNA expression was considerably lower in adjacent noncancerous 198 tissues (P<0.001) than in cancer tissues (Fig. 1C). Then, we compared the expression of 199 KIAA1199 in 105 LSCC tissues and their adjacent noncancerous tissues through 200 immunohistochemistry. There was weak or negative expression of KIAA1199 in adjacent 201 noncancerous tissue but high expression in the cytoplasm of LSCC tissue cells. The positive 202 staining and negative staining rates in LSCC tissues were 52.4% (55/105, Table 1) and 47.6% 203 (50/105), respectively. Semiquantitative analysis showed that KIAA1199 was significantly 204 increased in LSCC tissues. Representative photographs of the immunostaining are shown in figure 205 3 A-F. RT-PCR results for KIAA1199 mRNA levels were agree with the Western blotting and 206 IHC results, showing that KIAA1199 is increased in LSCC tissues. 207 208 209 3. KIAA1199 expression is associated with pathologic differentiation, T, N, clinical stage, 210 survival status and survival times of LSCC 209 3. KIAA1199 expression is associated with pathologic differentiation, T, N, clinical stage, 210 survival status and survival times of LSCC PeerJ reviewing PDF \| (2020:01:44662:2:0:NEW 29 Jun 2020) Manuscript to be reviewed 211 In order to further reveal the character of KIAA1199 in LSCC, we evaluated the relationship 212 between its expression and the clinicopathological characteristics of LSCC. As shown in Table 2, 213 upregulation of KIAA1199 expression was associated with some clinicopathological parameters: 214 pathologic differentiation (P, 0.002), T stage (P<0.001), N stage (P<0.001), clinical stage 215 (P<0.001), survival time (P, 0.008) and survival status (P<0.001). However, KIAA1199 expression 216 was not correlated with age, sex, clinical region, smoking status, or drinking status. 217 219 4. Survival assessment: A high level of KIAA1199 is predictive of poor prognosis in LSCC 220 patients p 221 The survival curve was plotted by kaplan-meier method, and the survival time was tested by 222 log-rank test. The results showed that LSCC patients with high KIAA1199 expression had a lower 223 prognosis, and low KIAA1199 expression in LSCC patients (P<0.001, for OS) was related with 224 considerably longer OS compared with high KIAA1199 expression. The median OS for high 225 KIAA1199 expression was 60±4.113 months and that for low KIAA1199 expression was 226 96±7.928 months (Fig. 3 G). Then analyzed independent prognostic factors for survival in patients 227 with LSCC by using univariate and multivariate Cox proportional hazards analysis. The univariate 228 analysis results (Table 3) showed that age (HR =1.032, 95% CI: 1.001 - 1.063; P, 0.04), pathologic 229 differentiation (HR =0.643, 95% CI: 0.524 - 0.789; P<0.001), T stage (HR =1.402, 95% CI: 1.139 230 - 1.724; P<0.001), N stage (HR =1.679, 95% CI: 1.148 - 2.4577; P, 0.008), clinical stage (HR 231 =1.445, 95% CI: 1.180 - 1.769; P<0.001), operation type (HR =0.380, 95% CI: 0.222 - 0.650; 232 P<0.001) and KIAA1199 expression (HR =12.165, 95% CI: 5.434 - 27.233; P<0.001) were 233 significantly associated with the overall survival of LSCC patients. Multivariate survival analysis 234 (table 4) showed that KIAA1199 expression was statistically significant predictor of OS(HR 235 =27.937, 95% CI: 10.600–73.632; P<0.0001) and that age (HR =1.039, 95% CI: 1.003 - 1.077; P, 236 0.0354), clinical stage (HR =0.704, 95% CI: 0.581–0.960; P, 0.023), operation type (HR =0.285, 237 95% CI: 0.093-0.870; P, 0.027), T stage (HR =0.68, 95% CI: 0.529-0.874; P, 0.003) and smoking 238 status (HR =0.19, 95% CI: 0.057–0.630; P, 0.007) were independent predictive factors for OS. p 221 The survival curve was plotted by kaplan-meier method, and the survival time was tested by 222 log-rank test. The results showed that LSCC patients with high KIAA1199 expression had a lower 223 prognosis, and low KIAA1199 expression in LSCC patients (P<0.001, for OS) was related with 224 considerably longer OS compared with high KIAA1199 expression. The median OS for high 225 KIAA1199 expression was 60±4.113 months and that for low KIAA1199 expression was 226 96±7.928 months (Fig. 3 G). Then analyzed independent prognostic factors for survival in patients 227 with LSCC by using univariate and multivariate Cox proportional hazards analysis. Manuscript to be reviewed 249 and invasion and could be a new therapeutic target in breast cancer(Mohammad-Saeid Jami, et al., 250 2014). A similar analysis reported, KIAA1199 overexpression can predict poor survival in patients 251 with colon cancer(Jian Xu, et al., 2015). By several mechanisms, KIAA1199 protein can accelerate 252 cancer progression. Simultaneously, other research have shown that the KIAA1199 protein 253 expression level is elevated upon p53 activation(Shinji Matsuzaki, et al., 2009). KIAA1199 is also 254 related to angiogenesis in rheumatoid arthritis(Xinyu Yang, et al., 2015). However, the mechanism 255 of KIAA1199 tumor-promoting effects in LSCC is little known. 256 In our study, we first verified KIAA1199 protein and mRNA expression in 10 pairs of fresh 257 surgically resected LSCC samples by Western blotting, IHC and real-time RT-PCR. Our results 258 have drawn a conclusion that KIAA1199 was highly expressed in LSCC cancerous in contrast to 259 adjacent non cancerous tissue. In view of our data, we also can censor the hidden expression of 260 KIAA1199 by immunohistochemistry in 105 paraffin-embedded sections (2009-2014) to further 261 explore the relationship between KIAA1199 and clinicopathological characteristics. By our data 262 analysis, showed that KIAA1199 expression was not kenspeckle related with clinical parameters 263 which as age, sex, clinical region, smoking, or drinking. Interestingly, for some severe 264 clinicopathological parameters: pathologic differentiation (P, 0.002), T stage (P<0.001), N stage 265 (P<0.001), clinical stage (P<0.001), survival time (P, 0.008) and survival status (P<0.001), the 266 significant correlations were observed. Through our experiments, we obtained many data, which 267 provides a new evidence that KIAA1199 is highly expressed in primary LSCC tissues and its 268 immunoreactivity is higher in cancerous than adjacent noncancerous tissues, revealing that 269 KIAA1199 might help distinguish benign from malignant larynx tumors. Moreover, our results 270 and analysis illuminated that the expression of KIAA1199 was elevated in LSCC tissues with 271 aggressive clinicopathological characteristics, suggesting its potential as a marker of cancer 272 invasionality. 273 The abnormal expression of KIAA1199 has also been found in other cancer studies, such as oral 274 squamous cell carcinoma(Pitak Chanthammachat, et al., 2013), breast cancer(Nikki A. Evensen, 275 et al., 2013), gastric cancer(Shinji Matsuzaki, et al., 2009), colorectal tumors(Amit Tiwari, et al., 276 2013; K Birkenkamp-Demtroder, et al., 2011; Lawrence C. LaPointe, et al., 2012), prostate 277 cancer(Eriko Michishita, et al., 2006), ovarian cancer(Fan Shena, et al., 2019) and hepatocellular 278 carcinoma(Zhengchen Jiang, et al., 2018). 241 Discussion 242 In order to identify novel gene that is up-regulated in human cancer with poor prognosis, a 243 deeply cognition to the molecular biology profiles of LCSS is a vital work. To outcomes remain 244 elusive, the molecular pathways involved in LSCC incidence, progression and clinical yet. 245 Espeially the endoplasmic reticulum, KIAA1199, which is a glycosylated protein which located 246 in the cytoplasm and membrane(Amit Tiwari, et al., 2013; Eriko Michishita, et al., 2006; Nikki A. 247 Evensen, et al., 2013). The relationship between cancer and KIAA1199 have been studied in many 248 research directions. KIAA1199 is a recently identified novel gene that can regulate cell growth PeerJ reviewing PDF \| (2020:01:44662:2:0:NEW 29 Jun 2020) Manuscript to be reviewed 285 expression of KIAA1199 was also observably associated with tumor invasion, metastasis and 286 TNM staging. Increased mortality risks associated with overexpression of KIAA1199 in primary 287 hepatocellular cancer patient. Previous researches have demonstrated that up-regulation of 288 KIAA1199 motivates carcinogenesis, motility and apoptosis. Metastasis, invasion, and cell 289 movement of a variety of cell types are associated with KIAA1199 expression(Yongsheng Zhang, 290 et al., 2014). By the Wnt/β-catenin signalling pathway, EMT is one of the important processes 291 mediated, which plays a key role in cancer invasion and metastasis(Yanyuan Wu, et al., 2012). 292 Interestingly, the KIAA1199 signalling pathway also induces the development and progression of 293 tumor. Other researches showed that the cell proliferation and mobility of colorectal cancer cells 294 were inhibited by knocking down the expression of CEMIP in vitro, and the EMT process of 295 colorectal cancer cells is suppressed by shRNA-CEMIP via inactivation of the Wnt/β-catenin/Snail 296 pathway(Guodong Liang, et al., 2018). Collectively, our results demonstrated that the 297 overexpression of KIAA1199 mRNA may affect tumor spread, lymph node metastasis, tumor 298 differentiation and prognosis(Shinji Matsuzaki, et al., 2009). In a report, it was defined KIAA1199 299 as an carcinogenic protein induced by HPV infection and compositive NF-kB activity that 300 transmits pro-survival and aggressive signals via EGFR signalling(Kateryna Shostak, et al., 2014). 301 Research has suggested that KIAA1199 may promote the development of ovarian cancer by 302 regulating PI3K/AKT signalling(Fan Shena, et al., 2019). One study insisted, AMPK/GSK3β/β- 303 catenin cascade triggered KIAA1199 over-expression may promote migration and invasion in 304 anoikis-resistant prostate cancer cells by increasing PDK4-associated metabolic reprogramming, 305 which may provide a novel therapeutic target for the prostate cancer(Peng Zhang, et al., 2018). 306 Therefore, in the light of the upper research about the KIAA1199-related signalling pathway, we 307 can draw a conclusion that KIAA1199 can influence the occurrence and development of laryngeal 308 cancer, which may also be related to the Wnt/β-catenin, EGFR, PI3K/AKT, and AMPK/GSK3 309 signalling pathways and other pathways. Thence, we will carry out a molecular mechanism 310 research of KIAA1199 in LSCC cells and animal models in our future study. 311 Some limitations exist in our research. First, the sample size of this study was a little small. As 285 expression of KIAA1199 was also observably associated with tumor invasion, metastasis and 286 TNM staging. Manuscript to be reviewed It was reported(Xuehua Jiao, et al., 2019) that 279 KIAA1199 was abnormaly increased in the papillary thyroid tumor compared with normal 280 specimens tissues and that upregulation of KIAA1199 was positively correlated with more 281 advanced clinical variables. There was analysis showed that the cell invasion and migration were 282 related with KIAA1199. KIAA1199 silencing inhibited the invasive ability of papillary thyroid 283 cancer cells by affecting epithelial-mesenchymal transition (EMT) in vitro and in vivo. 284 Additionally, the same as our study, In clone cancer study(Jian Xu, et al., 2015) proved the PeerJ reviewing PDF \| (2020:01:44662:2:0:NEW 29 Jun 2020) Manuscript to be reviewed Increased mortality risks associated with overexpression of KIAA1199 in primary 287 hepatocellular cancer patient. Previous researches have demonstrated that up-regulation of 288 KIAA1199 motivates carcinogenesis, motility and apoptosis. Metastasis, invasion, and cell 289 movement of a variety of cell types are associated with KIAA1199 expression(Yongsheng Zhang, 290 et al., 2014). By the Wnt/β-catenin signalling pathway, EMT is one of the important processes 291 mediated, which plays a key role in cancer invasion and metastasis(Yanyuan Wu, et al., 2012). 292 Interestingly, the KIAA1199 signalling pathway also induces the development and progression of 293 tumor. Other researches showed that the cell proliferation and mobility of colorectal cancer cells 294 were inhibited by knocking down the expression of CEMIP in vitro, and the EMT process of 295 colorectal cancer cells is suppressed by shRNA-CEMIP via inactivation of the Wnt/β-catenin/Snail 296 pathway(Guodong Liang, et al., 2018). Collectively, our results demonstrated that the 297 overexpression of KIAA1199 mRNA may affect tumor spread, lymph node metastasis, tumor 298 differentiation and prognosis(Shinji Matsuzaki, et al., 2009). In a report, it was defined KIAA1199 299 as an carcinogenic protein induced by HPV infection and compositive NF-kB activity that 300 transmits pro-survival and aggressive signals via EGFR signalling(Kateryna Shostak, et al., 2014). 301 Research has suggested that KIAA1199 may promote the development of ovarian cancer by 302 regulating PI3K/AKT signalling(Fan Shena, et al., 2019). One study insisted, AMPK/GSK3β/β- 303 catenin cascade triggered KIAA1199 over-expression may promote migration and invasion in 304 anoikis-resistant prostate cancer cells by increasing PDK4-associated metabolic reprogramming, 305 which may provide a novel therapeutic target for the prostate cancer(Peng Zhang, et al., 2018). 306 Therefore, in the light of the upper research about the KIAA1199-related signalling pathway, we 307 can draw a conclusion that KIAA1199 can influence the occurrence and development of laryngeal 308 cancer, which may also be related to the Wnt/β-catenin, EGFR, PI3K/AKT, and AMPK/GSK3 309 signalling pathways and other pathways. Thence, we will carry out a molecular mechanism 310 research of KIAA1199 in LSCC cells and animal models in our future study. 311 Some limitations exist in our research. First, the sample size of this study was a little small. As 312 a retrospective study design that the selection bias might not be ignored. Second, our study did not Manuscript to be reviewed 319 In conclusion, our results revealed significant associations of KIAA1199 protein expression 320 with various clinicopathologic characteristics and the prognosis of LSCC patients. Moreover, 321 survival analysis illuminated KIAA1199 was an independent prognostic factor for overall survival 322 in LSCC. All of these findings indicate that the KIAA1199 protein might be used as a pathological 323 marker to identify individuals with poor outcomes and to provide a reference for clinical therapy 324 in the future. Further studies are required to investigate its rationality as a marker and the potential 325 pathways involved in KIAA1199-mediated cell invasion and metastasis. 326 319 In conclusion, our results revealed significant associations of KIAA1199 protein expression 320 with various clinicopathologic characteristics and the prognosis of LSCC patients. Moreover, 321 survival analysis illuminated KIAA1199 was an independent prognostic factor for overall survival 322 in LSCC. All of these findings indicate that the KIAA1199 protein might be used as a pathological 323 marker to identify individuals with poor outcomes and to provide a reference for clinical therapy 324 in the future. Further studies are required to investigate its rationality as a marker and the potential 325 pathways involved in KIAA1199-mediated cell invasion and metastasis. 326 327 Acknowledgements 328 This study was supported by the Hunan Provincial Science Foundation of China (No 329 2016JJ4104) and the National natural science foundation of China (NO.81702706). 328 This study was supported by the Hunan Provincial Science Foundation of China (No 329 2016JJ4104) and the National natural science foundation of China (NO.81702706). 330 We thank Dr. Sheng Xiao for assistance with the IHC experiments. 331 332 333 References 334 Eriko Michishita GGs, J. Carl Barrett, Izumi Horikawa. 2006. Upregulation of the KIAA11 335 gene is associated with cellular mortality. Cancer Lett 239:71-77. D 336 10.1016/j.canlet.2005.07.028. 337 Fan Shena Z-hZ, Yao Liuc, Shuo Chena, Xiu-jie Shenga, Yang Zhao. 2019. CEMIP promot 338 ovarian cancer development and progression via the PI3K/AKT signaling pathwa 339 Biomed Pharmacother 114:108787. DOI 10.1016/j.biopha.2019.108787. 340 Gu Changjiang NQ, Ni Kan, Zhang Shu, Qian Haixin. 2018. Expression and cliIlical significan 341 of KIAA1199 in primary hepatocellular carcinoma. Natl Med J China 98:1609-1613. D 342 10. 3760/cma. j. issn. 0376-2491. 2018. 20. 017. 343 Guodong Liang XF, Yubo Yang, Yan Song. 2018. Silencing of CEMIP suppresses Wnt 344 catenin/Snail signaling transduction and inhibits EMT program of colorectal cancer ce 345 Acta Histochemica 120:56–63. DOI 10.1016/j.acthis.2017.11.002. 346 Jian Xu YL, Xudong Wang, Jianfei Huang, Huijun Zhu, Zhiqian Hu, Defeng Wang. 201 347 Association between KIAA1199 overexpression and tumor invasion, TNM stage, and po 348 prognosis in colorectal cancer. Int J Clin Exp Pathol 8:2909-2918. 330 We thank Dr. Sheng Xiao for assistance with the IHC experiments. 332 333 References 334 Eriko Michishita GGs, J. Carl Barrett, Izumi Horikawa. 2006. Upregulation of the KIAA1199 335 gene is associated with cellular mortality. Cancer Lett 239:71-77. DOI 336 10.1016/j.canlet.2005.07.028. 337 Fan Shena Z-hZ, Yao Liuc, Shuo Chena, Xiu-jie Shenga, Yang Zhao. 2019. CEMIP promotes 338 ovarian cancer development and progression via the PI3K/AKT signaling pathway. 339 Biomed Pharmacother 114:108787. DOI 10.1016/j.biopha.2019.108787. 340 Gu Changjiang NQ, Ni Kan, Zhang Shu, Qian Haixin. 2018. Expression and cliIlical significance 341 of KIAA1199 in primary hepatocellular carcinoma. Natl Med J China 98:1609-1613. DOI 342 10. 3760/cma. j. issn. 0376-2491. 2018. 20. 017. 343 Guodong Liang XF, Yubo Yang, Yan Song. 2018. Silencing of CEMIP suppresses Wnt/β- 344 catenin/Snail signaling transduction and inhibits EMT program of colorectal cancer cells. 345 Acta Histochemica 120:56–63. DOI 10.1016/j.acthis.2017.11.002. 334 Eriko Michishita GGs, J. Carl Barrett, Izumi Horikawa. 2006. Upregulation of the KIAA1199 335 gene is associated with cellular mortality. Cancer Lett 239:71-77. 318 Conclusions PeerJ reviewing PDF \| (2020:01:44662:2:0:NEW 29 Jun 2020) 327 Acknowledgements DOI 336 10.1016/j.canlet.2005.07.028. 337 Fan Shena Z-hZ, Yao Liuc, Shuo Chena, Xiu-jie Shenga, Yang Zhao. 2019. CEMIP promotes 338 ovarian cancer development and progression via the PI3K/AKT signaling pathway. 339 Biomed Pharmacother 114:108787. DOI 10.1016/j.biopha.2019.108787. 340 Gu Changjiang NQ, Ni Kan, Zhang Shu, Qian Haixin. 2018. Expression and cliIlical significance 341 of KIAA1199 in primary hepatocellular carcinoma. Natl Med J China 98:1609-1613. DOI 342 10. 3760/cma. j. issn. 0376-2491. 2018. 20. 017. 343 Guodong Liang XF, Yubo Yang, Yan Song. 2018. Silencing of CEMIP suppresses Wnt/β- 344 catenin/Snail signaling transduction and inhibits EMT program of colorectal cancer cells. 345 Acta Histochemica 120:56–63. DOI 10.1016/j.acthis.2017.11.002. 346 Jian Xu YL, Xudong Wang, Jianfei Huang, Huijun Zhu, Zhiqian Hu, Defeng Wang. 2015. 347 Association between KIAA1199 overexpression and tumor invasion, TNM stage, and poor 348 prognosis in colorectal cancer. Int J Clin Exp Pathol 8:2909-2918. PeerJ reviewing PDF \| (2020:01:44662:2:0:NEW 29 Jun 2020) Manuscript to be reviewe Manuscript to be reviewed Discovery and validation of m 360 and cancer with application to b 361 10.1371/journal.pone.0029059. 362 Mohammad-Saeid Jami JH, Miao Liu, Mich 363 Dong,Liying Geng, Jing Wang, Fang Yu, 364 Rakesh K Singhand Shi-Jian Ding. 2014 365 involvement of KIAA1199 in breast cancer gr 366 14:194. DOI 10.1186/1471-2407-14-194. 367 Nikki A. Evensen CK, Hoang-Lan Nguyen, K 368 Kadam, You-jun Hu, Ashleigh Pulkoski-G 369 Cao. 2013. Unraveling the role of KIAA119 370 cancer cell migration. J Natl Cancer Inst 105 371 Peng Zhang YS, Yadong Sun, Xuechao Li, Lif 372 AMPK/GSK3beta/beta-catenin cascade-trig 373 migration and invasion in anoikis-resistant p 374 reprogramming. FASEB J:fj201701078R. D 375 Pitak Chanthammachat WP, Kowit Pruegsa 376 Srisomsap, Daranee Chokchaicham 377 Boonyaphiphat, Singkhamanan K, Pa 378 proteomic analysis of oral squamous cell ca 379 Thailand. Arch Oral Biol 58:1677-1685. DOI 349 K Birkenkamp-Demtroder AM, F Mansilla, K Thorsen, CL Andersen, B Øster, S Hahn, TF 350 Ørntoft. 2011. Repression of KIAA1199 attenuates Wnt-signalling and decreases the 351 proliferation of colon cancer cells. Br J Cancer 105:552-561. DOI 10.1038/bjc.2011.268. 352 Kateryna Shostak XZ, Pascale Hubert, Serkan Ismail Go¨ktuna, Zheshen Jiang, Iva 353 Klevernic, Julien Hildebrand, Patrick Roncarati, Benoit Hennuy, Aure´lie Ladang, 354 Joan Somja, Andre´ Gothot, Pierre Close, Philippe Delvenne, Alain Chariot. 2014. 355 NF-kappaB-induced KIAA1199 promotes survival through EGFR signalling. Nat Commun 356 5:5232. DOI 10.1038/ncomms6232. 357 Lawrence C. LaPointe SKP, Robert Dunne, Glenn S. Brown, Letitia Pimlott,Snigdha Gaur, 358 Aidan McEvoy, Melissa Thomas, David Wattchow, Peter L. Molloy, Graeme P. 359 Young. 2012. Discovery and validation of molecular biomarkers for colorectal adenomas 360 and cancer with application to blood testing. PLoS One 7:e29059. 361 10.1371/journal.pone.0029059. 362 Mohammad-Saeid Jami JH, Miao Liu, Michelle L Varney, Hesham Hassan, Jixin 363 Dong,Liying Geng, Jing Wang, Fang Yu, Xin Huang, Hong Peng, Kai Fu1, Yan Li, 364 Rakesh K Singhand Shi-Jian Ding. 2014. Functional proteomic analysis reveals the 365 involvement of KIAA1199 in breast cancer growth, motility and invasiveness. BMC Cancer 366 14:194. DOI 10.1186/1471-2407-14-194. 367 Nikki A. Evensen CK, Hoang-Lan Nguyen, Kevin Zarrabi, Antoine Dufour, Pournima 368 Kadam, You-jun Hu, Ashleigh Pulkoski-Gross, Wadie F. Bahou, Stanley Zucker, Jian 369 Cao. 2013. Unraveling the role of KIAA1199, a novel endoplasmic reticulum protein, in 370 cancer cell migration. J Natl Cancer Inst 105:1402-1416. DOI 10.1093/jnci/djt224. 371 Peng Zhang YS, Yadong Sun, Xuechao Li, Lifeng Chen, Likun Yang, Yifei Xing. 2018. Manuscript to be reviewed 349 K Birkenkamp-Demtroder AM, F Mansilla, K Thorsen, CL Andersen, B Øster, S Hahn, TF 350 Ørntoft. 2011. Repression of KIAA1199 attenuates Wnt-signalling and decreases the 351 proliferation of colon cancer cells. Br J Cancer 105:552-561. DOI 10.1038/bjc.2011.268. 352 Kateryna Shostak XZ, Pascale Hubert, Serkan Ismail Go¨ktuna, Zheshen Jiang, Iva 353 Klevernic, Julien Hildebrand, Patrick Roncarati, Benoit Hennuy, Aure´lie Ladang, 354 Joan Somja, Andre´ Gothot, Pierre Close, Philippe Delvenne, Alain Chariot. 2014. 355 NF-kappaB-induced KIAA1199 promotes survival through EGFR signalling. Nat Commun 356 5:5232. DOI 10.1038/ncomms6232. 357 Lawrence C. LaPointe SKP, Robert Dunne, Glenn S. Brown, Letitia Pimlott,Snigdha Gaur, 358 Aidan McEvoy, Melissa Thomas, David Wattchow, Peter L. Molloy, Graeme P. 359 Young. 2012. Discovery and validation of molecular biomarkers for colorectal adenomas 360 and cancer with application to blood testing. PLoS One 7:e29059. 361 10.1371/journal.pone.0029059. 362 Mohammad-Saeid Jami JH, Miao Liu, Michelle L Varney, Hesham Hassan, Jixin 363 Dong,Liying Geng, Jing Wang, Fang Yu, Xin Huang, Hong Peng, Kai Fu1, Yan Li, 364 Rakesh K Singhand Shi-Jian Ding. 2014. Functional proteomic analysis reveals the 365 involvement of KIAA1199 in breast cancer growth, motility and invasiveness. BMC Cancer 366 14:194. DOI 10.1186/1471-2407-14-194. 367 Nikki A. Evensen CK, Hoang-Lan Nguyen, Kevin Zarrabi, Antoine Dufour, Pournima 368 Kadam, You-jun Hu, Ashleigh Pulkoski-Gross, Wadie F. Bahou, Stanley Zucker, Jian 369 Cao. 2013. Unraveling the role of KIAA1199, a novel endoplasmic reticulum protein, in 370 cancer cell migration. J Natl Cancer Inst 105:1402-1416. DOI 10.1093/jnci/djt224. 371 Peng Zhang YS, Yadong Sun, Xuechao Li, Lifeng Chen, Likun Yang, Yifei Xing. 2018. 372 AMPK/GSK3beta/beta-catenin cascade-triggered overexpression of CEMIP promotes 373 migration and invasion in anoikis-resistant prostate cancer cells by enhancing metabolic 374 reprogramming. FASEB J:fj201701078R. DOI 10.1096/fj.201701078R. 375 Pitak Chanthammachat WP, Kowit Pruegsanusak, Sittiruk Roytrakul, Chantragan 376 Srisomsap, Daranee Chokchaichamnankit, Jisnuson Svasti, Pleumjit 377 Boonyaphiphat, Singkhamanan K, Paramee Thongsuksai. 2013. Comparative 378 proteomic analysis of oral squamous cell carcinoma and adjacent non-tumour tissue from 379 Thailand. Arch Oral Biol 58:1677-1685. DOI 10.1016/j.archoralbio.2013.08.002. 349 K Birkenkamp-Demtroder AM, F Mansilla, K Th 350 Ørntoft. 2011. Repression of KIAA1199 a 351 proliferation of colon cancer cells. Br J Canc 352 Kateryna Shostak XZ, Pascale Hubert, Serka 353 Klevernic, Julien Hildebrand, Patrick Ro 354 Joan Somja, Andre´ Gothot, Pierre Close 355 NF-kappaB-induced KIAA1199 promotes su 356 5:5232. DOI 10.1038/ncomms6232. 357 Lawrence C. LaPointe SKP, Robert Dunne, Glen 358 Aidan McEvoy, Melissa Thomas, David 359 Young. 2012. Manuscript to be reviewed 380 Rebecca L. Siegel KDM, Ahmedin Jemal, DVM. 2019. Cancer statistics, 2019. CA Canc 381 Clin 69:7-34. DOI 10.3322/caac.21551. 382 S. Michael Rothenberg LWE. 2012. The molecular pathogenesis of head and neck squam 383 cell carcinoma. J Clin Invest 122:1951-1957. DOI 10.1172/jci59889. 384 Satoko Abe S-iU, Yusuke Nakamura. 2003. Mutations in the gene encoding KIAA1199 pro 385 an inner-ear protein expressed in Deiters' cells and the fibrocytes, as the caus 386 nonsyndromic hearing loss. J Hum Genet 48:564-570. DOI 10.1007/s10038-003-007 387 Shinji Matsuzaki FT, Koshi Mimori, Kouichirou Tahara, Hiroshi Inoue, Masaki Mori. 2 388 Clinicopathologic significance of KIAA1199 overexpression in human gastric cancer. 389 Surg Oncol 16:2042-2051. DOI 10.1245/s10434-009-0469-6. 390 Xinyu Yang PQ, Bingbing Chen, Yaoyao Lin, Zhonghao Zhou, Renshan Ge, Hai Z 391 Jianmin Wang, Jianguang Wang. 2015. KIAA1199 as a potential diagnostic bioma 392 of rheumatoid arthritis related to angiogenesis. Arthritis Res Ther 17:140. 393 10.1186/s13075-015-0637-y. 394 Xuehua Jiao JY, Xiujie Wang, Xueyan Yin, Guodong Zhang, Xingbo Cheng. 2019. KIAA1 395 a Target of MicoRNA-486-5p, Promotes Papillary Thyroid Cancer Invasion by Influen 396 Epithelial-Mesenchymal Transition (EMT). Med Sci Monit 25:6788-6796. 397 10.12659/MSM.918682. 398 Yanyuan Wu CG, Juri Kim, Nicole Mosher, Seyung Chung, Dennis Slamon, Jaydu 399 Vadgama. 2012. Expression of Wnt3 activates Wnt/beta-catenin pathway and prom 400 EMT-like phenotype in trastuzumab-resistant HER2-overexpressing breast cancer c 401 Mol Cancer Res 10:1597-1606. DOI 10.1158/1541-7786.MCR-12-0155-T. 402 Yongsheng Zhang SJ, WEN G. JIANG. 2014. KIAA1199 and its biological role in human ca 403 and cancer cells (review). Oncol Rep 31:1503-1508. DOI 10.3892/or.2014.3038. 404 Zhengchen Jiang XZ, Binyao shi, Dan luo, Bin Jin. 2018. KIAA1199 overexpressio 405 associated with abnormal expression of EMT markers and is a novel indepen 406 prognostic biomarker for hepatocellular carcinoma. Onco Targets Ther 11:8341-8 407 DOI 10.2147/OTT.S187389. 408 Wei Li, Guolin Tan, Yanhong Ma, Heqing Li and Guangxiang He. 2012. Inhibition of a 409 folate receptor resulting in a reversal of taxol resistance in nasopharyngeal carcino 410 Otolaryngol Head Neck Surg 146:250-258. 10.1177/0194599811426260. 380 Rebecca L. Siegel KDM, Ahmedin Jemal, DVM. 2019. Cancer statistics, 2019. CA Cancer J 381 Clin 69:7-34. DOI 10.3322/caac.21551. 382 S. Michael Rothenberg LWE. 2012. The molecular pathogenesis of head and neck squamous 383 cell carcinoma. J Clin Invest 122:1951-1957. DOI 10.1172/jci59889. 384 Satoko Abe S-iU, Yusuke Nakamura. 2003. Manuscript to be reviewed 352 Kateryna Shostak XZ, Pascale Hubert, Serkan Ismail Go¨ktuna, Zheshen Jiang, Iva 353 Klevernic, Julien Hildebrand, Patrick Roncarati, Benoit Hennuy, Aure´lie Ladang, 354 Joan Somja, Andre´ Gothot, Pierre Close, Philippe Delvenne, Alain Chariot. 2014. 355 NF-kappaB-induced KIAA1199 promotes survival through EGFR signalling. Nat Commun 356 5:5232. DOI 10.1038/ncomms6232. 357 Lawrence C. LaPointe SKP, Robert Dunne, Glenn S. Brown, Letitia Pimlott,Snigdha Gaur, 358 Aidan McEvoy, Melissa Thomas, David Wattchow, Peter L. Molloy, Graeme P. 359 Young. 2012. Discovery and validation of molecular biomarkers for colorectal adenomas 360 and cancer with application to blood testing. PLoS One 7:e29059. 361 10.1371/journal.pone.0029059. 362 Mohammad-Saeid Jami JH, Miao Liu, Michelle L Varney, Hesham Hassan, Jixin 363 Dong,Liying Geng, Jing Wang, Fang Yu, Xin Huang, Hong Peng, Kai Fu1, Yan Li, 364 Rakesh K Singhand Shi-Jian Ding. 2014. Functional proteomic analysis reveals the 365 involvement of KIAA1199 in breast cancer growth, motility and invasiveness. BMC Cancer 366 14:194. DOI 10.1186/1471-2407-14-194. 367 Nikki A. Evensen CK, Hoang-Lan Nguyen, Kevin Zarrabi, Antoine Dufour, Pournima 368 Kadam, You-jun Hu, Ashleigh Pulkoski-Gross, Wadie F. Bahou, Stanley Zucker, Jian 369 Cao. 2013. Unraveling the role of KIAA1199, a novel endoplasmic reticulum protein, in 370 cancer cell migration. J Natl Cancer Inst 105:1402-1416. DOI 10.1093/jnci/djt224. 371 Peng Zhang YS, Yadong Sun, Xuechao Li, Lifeng Chen, Likun Yang, Yifei Xing. 2018. 372 AMPK/GSK3beta/beta-catenin cascade-triggered overexpression of CEMIP promotes 373 migration and invasion in anoikis-resistant prostate cancer cells by enhancing metabolic 374 reprogramming. FASEB J:fj201701078R. DOI 10.1096/fj.201701078R. PeerJ reviewing PDF \| (2020:01:44662:2:0:NEW 29 Jun 2020) Manuscript to be reviewed Manuscript to be reviewed Mutations in the gene encoding KIAA1199 protein, 385 an inner-ear protein expressed in Deiters' cells and the fibrocytes, as the cause of 386 nonsyndromic hearing loss. J Hum Genet 48:564-570. DOI 10.1007/s10038-003-0079-2. 387 Shinji Matsuzaki FT, Koshi Mimori, Kouichirou Tahara, Hiroshi Inoue, Masaki Mori. 2009. 388 Clinicopathologic significance of KIAA1199 overexpression in human gastric cancer. Ann 389 Surg Oncol 16:2042-2051. DOI 10.1245/s10434-009-0469-6. 390 Xinyu Yang PQ, Bingbing Chen, Yaoyao Lin, Zhonghao Zhou, Renshan Ge, Hai Zou, 391 Jianmin Wang, Jianguang Wang. 2015. KIAA1199 as a potential diagnostic biomarker 392 of rheumatoid arthritis related to angiogenesis. Arthritis Res Ther 17:140. DOI 393 10.1186/s13075-015-0637-y. 394 Xuehua Jiao JY, Xiujie Wang, Xueyan Yin, Guodong Zhang, Xingbo Cheng. 2019. KIAA1199, 395 a Target of MicoRNA-486-5p, Promotes Papillary Thyroid Cancer Invasion by Influencing 396 Epithelial-Mesenchymal Transition (EMT). Med Sci Monit 25:6788-6796. DOI 397 10.12659/MSM.918682. 398 Yanyuan Wu CG, Juri Kim, Nicole Mosher, Seyung Chung, Dennis Slamon, Jaydutt V. 399 Vadgama. 2012. Expression of Wnt3 activates Wnt/beta-catenin pathway and promotes 400 EMT-like phenotype in trastuzumab-resistant HER2-overexpressing breast cancer cells. 401 Mol Cancer Res 10:1597-1606. DOI 10.1158/1541-7786.MCR-12-0155-T. 402 Yongsheng Zhang SJ, WEN G. JIANG. 2014. KIAA1199 and its biological role in human cancer 403 and cancer cells (review). Oncol Rep 31:1503-1508. DOI 10.3892/or.2014.3038. 404 Zhengchen Jiang XZ, Binyao shi, Dan luo, Bin Jin. 2018. KIAA1199 overexpression is 405 associated with abnormal expression of EMT markers and is a novel independent 406 prognostic biomarker for hepatocellular carcinoma. Onco Targets Ther 11:8341-8348. 407 DOI 10.2147/OTT.S187389. 408 Wei Li, Guolin Tan, Yanhong Ma, Heqing Li and Guangxiang He. 2012. Inhibition of alpha 409 folate receptor resulting in a reversal of taxol resistance in nasopharyngeal carcinoma. 410 Otolaryngol Head Neck Surg 146:250-258. 10.1177/0194599811426260. PeerJ reviewing PDF \| (2020:01:44662:2:0:NEW 29 Jun 2020) Manuscript to be reviewed Manuscript to be reviewed 413 PeerJ reviewing PDF \| (2020:01:44662:2:0:NEW 29 Jun 2020) Manuscript to be reviewed Manuscript to be reviewed Figure 1 Figure 1 The protein of KIAA1199 was overexpression in LSCC tissue specimens. (A) (B) The protien expression of KIAA1199 in adjacent non cancerous tissue and LSCC tissue by Western bloting.P<0.001. (C) The mRNA expression of KIAA1199 in adjacent non cancerous tissue and LSCC tissue by RT-PCR. **P<0.001. PeerJ reviewing PDF \| (2020:01:44662:2:0:NEW 29 Jun 2020) PeerJ reviewing PDF \| (2020:01:44662:2:0:NEW 29 Jun 2020) Manuscript to be reviewed Manuscript to be reviewed Figure 2 Figure 2 Representative images of immunohistochemical staining for KIAA1199 expression in larynx specimens. (A) Negative expression of KIAA1199 in adjacent non cancerous specimens. (B) Low expression of KIAA1199 in LSCC pecimens. (C) High expression of KIAA1199 in LSCC specimens. Original magniﬁcation: 40X; scale bars: 20um. PeerJ reviewing PDF \| (2020:01:44662:2:0:NEW 29 Jun 2020) Figure 3 The expression of KIAA1199 in LSCC tissues and survival surve. Figure 3 The expression of KIAA1199 in LSCC tissues and survival surve. (A-F) KIAA1199 expression by immunohistochemical staining. (A) Adjacent nonc-ancerous tissue as the negative control. (B) Gastric cancer tissue as the positive control. (C) Ⅰ stage LSCC tissue. (D) Ⅱstage LSCC tissue. (E) Ⅲ stage LSCC tissue. (F) Ⅳ stage LSCC tissue. (G) Kaplan-Meier survival curves analysis of ov-erall survival for all patients with KIAA1199 negative and positive LSCC tissue. PeerJ reviewing PDF \| (2020:01:44662:2:0:NEW 29 Jun 2020) Manuscript to be reviewed Table 1(on next page) Table 1(on next page) Table 1(on next page) Table 1(on next page) Clinicopathological characteristics of patient samples and expression of KIAA1199 in LSCC PeerJ reviewing PDF \| (2020:01:44662:2:0:NEW 29 Jun 2020) Manuscript to be reviewed Manuscript to be reviewed 1 Table1: Clinicopathological characteristics of patient samples and expression of KIAA1199 in LSCC Parameters Case number / n (%) Gender Male Female 103 (98.1) 2 (1.9) Age(y) ≤60 ＞60 44 (41.9) 61 (58.1) Pathologic differentiation Poorly Moderately Highly 20 (19.05) 29 (27.62) 56 (53.33) Supraglottic type 10 (9.52) Trans glottic type 5 (4.76) Glottic type 87 (82.86) Clinic Region Subglottic type 3 (2.86) T stage T1-T2 70 (66.6) T3-T4 35 (33.4) N stage NO 85 (81) N1-N3 20 (19) Ⅰ 52 (49.5) Ⅱ 15 (14.3) Clinical Stages Ⅲ 11 (10.5) Table1: Clinicopathological characteristics of patient samples and expression of KIAA1199 PeerJ reviewing PDF \| (2020:01:44662:2:0:NEW 29 Jun 2020) Manuscript to be reviewed Ⅳ 27 (25.7) Total laryngectomy 28 (26.7) The partial laryngetomy 77 (73.3) No 40 (38.1) ection Radical cervical clearing 26 (24.8) Selective/functional neck cleanser 39 (37.1) No 30 (28.6) Yes 75 (71.4) No 53 (50.5) Yes 52 (49.5) AA1199 Low expression 50 (47.6) High expression 55 (52.4) Ⅳ Operation 2 2 PeerJ reviewing PDF \| (2020:01:44662:2:0:NEW 29 Jun 2020) Manuscript to be reviewed Manuscript to be reviewed Table 2(on next page) Table 2(on next page) Correlation between KIAA1199 expression and clinicopathologic characteristics of LSCC patients PeerJ reviewing PDF \| (2020:01:44662:2:0:NEW 29 Jun 2020) Manuscript to be reviewed Table 2: correlation between KIAA1199 expression and clinicopathologic characteristics of LSCC patients Expression of KIAA1199 （No.) Parameters Low High P Male 48 55 0.224 Gender Female 2 0 ≤60 21 23 1.000 Age(year) ＞60 29 32 Pathologic differentiation Poorly 1 19 <0.001 Moderately 18 11 Highly 31 25 Clinic Region Supraglottic type 3 7 0.072 Trans glottic type 0 5 Glottic type 46 41 Subglottic type 1 2 T stage T1-T2 48 22 <0.001 T3-T4 2 33 N stage N0 50 35 <0.001 N1-N3 2 18 Clinical Stage Ⅰ-Ⅱ 48 19 <0.001 Ⅲ-Ⅳ 2 36 Smoke No 18 12 0.132 Yes 32 43 1 Table 2: correlation between KIAA1199 expression and clinicopathologic characteristics of LSCC patients Expression of KIAA1199 （No.) Parameters Low High P Male 48 55 0.224 Gender Female 2 0 ≤60 21 23 1.000 Age(year) ＞60 29 32 Pathologic differentiation Poorly 1 19 <0.001 Moderately 18 11 Highly 31 25 Clinic Region Supraglottic type 3 7 0.072 Trans glottic type 0 5 Glottic type 46 41 Subglottic type 1 2 T stage T1-T2 48 22 <0.001 T3-T4 2 33 N stage N0 50 35 <0.001 N1-N3 2 18 Clinical Stage Ⅰ-Ⅱ 48 19 <0.001 Ⅲ-Ⅳ 2 36 Smoke No 18 12 0.132 Yes 32 43 PeerJ reviewing PDF \| (2020:01:44662:2:0:NEW 29 Jun 2020) Manuscript to be reviewed Manuscript to be reviewed Table 3(on next page) Manuscript to be reviewed Manuscript to be reviewed Drink No 26 27 0.846 Yes 24 28 Survival status survive 43 7 <0.001 death 7 48 Survival times（month） ≤12 2 7 0.008 ＞12，≤36 0 8 ＞36，≤60 23 22 ＞60 25 18 2 PeerJ reviewing PDF \| (2020:01:44662:2:0:NEW 29 Jun 2020) Table 3(on next page) Table 3(on next page) Univariate analyses of various prognostic parameters in patients with LSCC PeerJ reviewing PDF \| (2020:01:44662:2:0:NEW 29 Jun 2020) Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed Table 4(on next page) Table 4(on next page) Manuscript to be reviewed Manuscript to be reviewed 1 Table 3: Univariate analyses of various prognostic parameters in patients with LSCC 2 3 Univariate Cox Parameters Hazard ratio 95% CI p-Value Gender 0.048 0-25.791 0.422 Age(y) 1.032 1.001-1.063 0.040 Pathologic differentiation 0.643 0.524-0.789 <0.001 Clinic Region 0.068 0.49-1.026 0.068 T stage 1.402 1.139-1.724 0.001 N stage 1.679 1.148-2.457 0.008 Clinical Stages 1.445 1.180-1.769 <0.001 Operation 0.380 0.222-0.650 <0.001 Neck lymph dissection 0.957 0.7106-1.291 0. 774 Smoke 1.028 0.560-1.885 0.930 Drink 0.782 0.460-1.330 0.365 expression of KIAA1199 12.165 5.434-27.233 <0.001 1 Table 3: Univariate analyses of various prognostic parameters in patients with LSCC 1 Table 3: Univariate analyses of various prognostic parameters in patients with LSCC PeerJ reviewing PDF \| (2020:01:44662:2:0:NEW 29 Jun 2020) PeerJ reviewing PDF \| (2020:01:44662:2:0:NEW 29 Jun 2020) Table 4(on next page) Multivariate analyses of various prognostic parameters in patients with LSCC PeerJ reviewing PDF \| (2020:01:44662:2:0:NEW 29 Jun 2020) Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed 1 Table 4: Multivariate analyses of various prognostic parameters in patients with LSCC Multivariate Cox Parameters Hazard ratio 95% CI p-Value Age(y) 1.039 1.003-1.077 0.035 Clinic Stage 0.704 0.581-0.960 0.023 Operation 0.285 0.093-0.870 0.027 T stage 0.68 0.529-0.874 0.003 Smoke 0.400 0.204-0.785 0.008 expression of KIAA1199 27.937 10.600-73.632 0.001 1 Table 4: Multivariate analyses of various prognostic parameters in patients with LSCC 2 2 PeerJ reviewing PDF \| (2020:01:44662:2:0:NEW 29 Jun 2020)
https://openalex.org/W4327667688	https://ejournal.ipinternasional.com/index.php/ijphe/article/download/330/315	English	null	Antioxidant Activity Test of Ethanol Extract of Andaliman Fruit (Zanthoxylum Acanthopodium DC.) on Superoxide Dismutase (SOD) Levels in Rats	International Journal Of Public Health Excellence	2,022	cc-by	2,772	International Journal of Public Health Excellence (IJPHE) Vol. 01, No. 02, May 2022, pp. 237~240 Journal Homepage: https://ejournal.ipinternasional.com/index.php/ijphe ISSN: 2809-9826, DOI: 10.55299/ijphe.v1i2.330 International Journal of Public Health Excellence (IJPHE) Vol. 01, No. 02, May 2022, pp. 237~240 Journal Homepage: https://ejournal.ipinternasional.com/index.php/ijphe ISSN: 2809-9826, DOI: 10.55299/ijphe.v1i2.330 International Journal of Public Health Excellence (IJPHE) Vol. 01, No. 02, May 2022, pp. 237~240 Journal Homepage: https://ejournal.ipinternasional.com/index.php/ijphe ISSN: 2809-9826, DOI: 10.55299/ijphe.v1i2.330 Keywords: Antioxidant, Zanthoxylum Acanthopodium, Superoxide Dismutase Antioxidant, Zanthoxylum Acanthopodium, Superoxide Dismutase This article is licensed under a Creative Commons Attribution- ShareAlike 4.0 International License.. This article is licensed under a Creative Commons Attribution- ShareAlike 4.0 International License.. Antioxidant Activity Test of Ethanol Extract of Andaliman Fruit (Zanthoxylum Acanthopodium DC.) on Superoxide Dismutase (SOD) Levels in Rats Arsiaty 1 , Yulia Delfahedah 2, Andre Bastian Manik 3, Nurasima Kurniati Damanik 4 1, 2 Lecture of Universitas Efarina, Simalungun, Indonesia 3, 4 Student of Universitas Efarina, Simalungun, Indonesia Article Info Free radicals are compounds or molecules that contain one or more unpaired electrons in their outer orbitals. If the production of free radicals is excessive, it can cause oxidative damage which ends in cell death resulting in the acceleration of various degenerative diseases. To neutralize the work of free radicals, external antioxidants are needed. Andaliman (Zanthoxylum acanthopodium DC) is a source of natural antioxidants that contain flavonoids that can neutralize free radicals. This research included sample preparation, examination of simplicia characteristics, screening of simplicia phytochemicals, extract preparation, determination of SOD activity spectrophotometrically using reagents. A total of 30 rats were divided into 6 groups consisting of the control group, the stress-induced group, the comparison group, and the three stress-induced and EEBA groups with doses of 75 mg/kg, 150 mg/kg, and 300 mg respectively. /kg bw. Stress induction was carried out by administering EEBA for 7 days and continued by administering EEBA together with doxorubicin for the next 2 days. The results showed that the average SOD level in the control group was (4.626 0.2583), the doxorubicin group (1.956+ 0.0879), the EEBA group 75 mg kg body weight (2.444 0.0844), the EEBA group 150 mg/kg body weight ( 3.052 +0.1139), the EEBA 300 mg/kg body weight (3.646+0.1739) and the Routine 50 mg/kg body weight 5.594 + 0.2056), EEBA had higher SOD activity when compared to the doxorubicin group. Based on the statistical results, SOD activity increased with the increase in the dose of EEBA given and showed a significant difference (p<0.05) between the EPBA group and the doxorubicin group. Observations on liver tissue in the group given EEBA showed better conditions than the liver in the doxorubicin group. Simplicia Characteristics Examination Results Macroscopic examination Simplicia Characteristics Examination Results Macroscopic examination The results of macroscopic examination of andaliman fruit showed that young fruit was green in color, and when ripe it was dark red to brownish red. The shape of the fruit is round and small, smaller than pepper, when bitten it gives off a distinctive aroma and sharp taste, and can stimulate saliva production. The seeds are in the fruit and hard. Macroscopic examination was carried out on the andaliman fruit simplicia, namely the simplicia was black in color, had a characteristic odor, and the seeds were coming out of the fruit. Examination of characteristics of andaliman fruit macroscopically was carried out to obtain simplicia identity. Results of macroscopic examination of andaliman fruit and andaliman fruit simplicia. Microscopic examination Microscopically, there are covering hairs, endosperm vascular bundles with oil drops, oil drops, and reddish orange seed coat fragments. Data analysis The research data were analyzed using the SPSS version 17 program. The data were analyzed using the Kolmogorov Smirnov method to determine its homogeneity and normality. Then proceed to use the One Way ANOVA method to determine the average difference between groups. If there is a difference, it is continued by using the Post Hoc Tukey HSD test to see real differences between treatments. Formulation of the problem The formulation of the problem in this study is: a. Can the ethanol extract of Andaliman fruit increase the activity of the SOD enzyme? a. Can the ethanol extract of Andaliman fruit increase the activity of the SOD enzym b. Is the ethanol extract of Andaliman fruit able to prevent liver damage caused by doxor b. Is the ethanol extract of Andaliman fruit able to prevent liver damage caused by doxorubicin? b. Is the ethanol extract of Andaliman fruit able to prevent liver damage caused by doxorubicin? c. Does increasing the dose of Andaliman fruit ethanol extract increase SOD activity in rat blood? 2. METHOD The research method used was experimental research. The research included collection and preparation of plant materials, sample identification, sample processing, simplista characterization, phytochemical screening, extract preparation for experimental animals, testing the antidiarrheal activity of ethanol extract of srikaya leaf (Annona squamosa L.) in male mice, and processing. data Research results data were analyzed by ANOVA (Analysis of Variance) using SPSS (Statistical Product and Service Solution) version 17. 1. INTRODUCTION Free radicals are compounds or molecules that contain one or more unpaired electrons in their outer orbitals. The presence of unpaired electrons causes these compounds to be very reactive looking for partners by attacking and binding the electrons of the molecules around them [5]. Free radicals are generated normally in the body by cell metabolism, inflammation, or when the body is exposed to environmental pollution [3]. If the production of free radicals exceeds the ability of intracellular antioxidants to neutralize them, the excess free radicals have the potential to cause cell damage. Often this damage is referred to as oxidative damage, namely damage to the biomolecules that make up cells caused by reactions with free radicals. An increase in oxidative stress has a negative impact on several components of the cell membrane, namely damage to the membrane lipids to form malonaldehyde (MDA). damage to proteins, carbohydrates, and DNA [9].  23  237  Int Jou of PHE Oxidative damage caused by free radicals has implications for various pathological conditions, namely damage to cells, tissues, and organs such as liver, kidney, heart, both in humans and animals. This damage can end in cell death resulting in accelerated onset of various degenerative diseases [9]. Oxidative damage caused by free radicals has implications for various pathological conditions, namely damage to cells, tissues, and organs such as liver, kidney, heart, both in humans and animals. This damage can end in cell death resulting in accelerated onset of various degenerative diseases [9]. External antioxidants can be in the form of natural and synthetic antioxidants. However, various kinds of synthetic antioxidants such as butylated hydroxytoluene (BHT) have been reported to have several side effects such as liver damage and mutagenesis. Therefore, alternative antioxidants are needed that have better and safer activities, namely from natural or plant ingredients [13]. One of the plants that can be used as an antioxidant is andaliman. Research to determine the effect of andaliman fruit extract on superoxide dismutase (SOD) activity by spectrophotometry in rat blood has not been carried out, therefore researchers are interested in knowing the effect on SOD activity in rats through spectrophotometric blood measurements. Simplicity characteristics The results of the examination of water content, water soluble extract content, ethanol soluble extract content, total ash content and acid insoluble ash content. Based on the results of the examination, the Andaliman fruit simplicia had a water content of 7.58%, this result met the water content requirements of the fruit simplicia in the book How to Make Simplisia, namely not more than 8% (Ministry of Health RI, 1985). The smaller the water content of the simplicia, the smaller the possibility of microorganism growth and hydrolysis of chemical compounds contained in the simplicia. The water-soluble essence obtained was 10.30% and the ethanol-soluble extract was 12.62%. Determination of the extract content is very useful to give an idea of the amount of dissolved material from simplicia. While the total ash content of the simplicia obtained was 7.06% and the acid insoluble ash content was 0.23%. Simplicia powder extraction was carried out by maceration. Extracting 500 grams of Andaliman fruit simplicia using 96% ethanol produced 59.41 grams of extract with a yield percentage of 11.9%. 238  238 238 Int Jou of PHE  Phytochemical Screening Phytochemical screening of simplicia and Andaliman fruit extract was carried out to obtain information on the class of secondary metabolites contained therein. Results of phytochemical screening of simplicia and andaliman fruit extract. ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS Author thanks to all my team and I hope the research can be useful. Author thanks to all my team and I hope the research can be useful. Histology Examination of Rat Liver Tissue gy Hematoxylin Eosin (HE) staining . Hematoxylin is alkaline will color the tissue elements that are acidic (basophilic), namely the cell nucleus. Meanwhile, eosin is acidic so that it functions to color the cytoplasm which is alkaline (acidophilic[18]. Results of histological examination of the liver. Doxorubicin increases the apoptotic process in liver tissue, is induced by lipid peroxidation in microsomes and especially in mitochondria by the presence of Fe ions and includes damage to blood vessels and stenosis in bati cells [11]. Oxidative stress is the main pathogenetic event that occurs in several liver disorders, such as disturbances in cell metabolism to proliferate, and is the main cause of liver damage in ischemia [9]. High amounts of free radicals in the body attack biomacromolecules which are components of cell walls. As a result, the function of the cell wall decreases, causing cell damage in the form of degeneration as seen in the DOX- treated group [18] Free radicals do not have an electron pair, so these free radicals will be free in the body and try to achieve stability by binding to nearby molecules. Bonds between free radicals and nearby molecules result in damage to the molecular structure. Damage to cell membranes by free radicals occurs through a series of covalent bonding processes between free radicals and membrane components, oxidation of thiol groups on membrane components by free radicals and lipid peroxidation reactions. The results of peroxidation of membrane lipids by free radicals, have a direct effect on damage to important macromolecules such as lipids, proteins and DNA [15]. If observed microscopically, hydropic degeneration is characterized by the presence of vacuoles in the cytoplasm of the cells so that the liver cells are swollen and have a paler color. Hydropic degeneration can occur due to disruption of the sodium potassium pump in regulating the entry and exit of ions. Hydropic degeneration includes mild damage because it can heal and liver cells become normal again (reversible) [16]. The Effect of Andaliman Fruit Ethanol Extract on SOD Levels in Rats Examination of SOD levels was carried out quantitatively using the UV-Vis spectrophotometry method based on the Bioassay Systems procedure (EnzyChrom Superoxide dismutase Assay Kit) at a wavelength of 440 nm which can be seen in Appendix 10, page 70. This method is based on the colorimetric principle for determining SOD enzyme activity in quantitative biological samples. In the test, superoxide (O) is produced by a catalytic reaction of xanthine oxidase (XO). O2 reacts with WST-1 dye to form a colored product. SOD collects O so that reduced O is useful for chromogenic reactions. Color intensity (OD440nm) is used to determine SOD activity in the sample. The higher the absorbance obtained (AAOD), the higher the SOD activity of the sample [1][2]. 4. CONCLUSION The conclusions obtained based on the results and observations are: a. Andaliman fruit ethanol extract was able to increase the activity of the SOD enzyme, where the SOD activity in the group given EEBA showed a significant difference with the group given doxorubicin (oxidative stress). a. Andaliman fruit ethanol extract was able to increase the activity of the SOD enzyme, where the SOD activity in the group given EEBA showed a significant difference with the group given doxorubicin (oxidative stress). ( ) b. Andaliman fruit ethanol extract can prevent liver damage caused by doxorubicin c. An increase in SOD activity occurred along with an increase in the dose of EEBA given. Where the most effective dose was EEBA dose of 300 mg/kg body weight, with an average SOD level of 3.646 U/ml. REFERENCES [1] Anonymous. (2012). Sichuan Pepper and others (Zanthoxylum piperitum, simulans, bungeanum, rhetsa, acanthopodium). From http://www.gernot_katzers_spice_pages.com/engl/Zant_pip.html. Retrieved October 26, 2016. [2] Anonymous. (2012). EnzyChrom TM Super oxide Dismutase Assay Kit (ESOD-100). Accessed from www.bioassayssys.com. [3] Annapurna, A., Reddy, CS, Akondi, RB, and Rao, SR (2009). Cardioprotective Actions of Two Bioflavonoids, Quercetin, and Rutin, in experimental Myocardial Infarction in Both Normal and Streptozotocin-Induced Type I Diabetic Rats. JPharm Pharmacol, 61(10): 1365-1374. [3] Annapurna, A., Reddy, CS, Akondi, RB, and Rao, SR (2009). Cardioprotective Actions of Two Bioflavonoids, Quercetin, and Rutin, in experimental Myocardial Infarction in Both Normal and Streptozotocin-Induced Type I Diabetic Rats. JPharm Pharmacol, 61(10): 1365-1374. [3] Annapurna, A., Reddy, CS, Akondi, RB, and Rao, SR (2009). Cardioprotective Actions of Two Bioflavonoids, Quercetin, and Rutin, in experimental Myocardial Infarction in Both Normal and Streptozotocin-Induced Type I Diabetic Rats. 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https://openalex.org/W2765594473	https://biblio.ugent.be/publication/8612822/file/8612823.pdf	English	null	A damping circadian clock drives weak oscillations in metabolism and locomotor activity of aphids (Acyrthosiphon pisum)	Scientific reports	2,017	cc-by	8,340	A damping circadian clock drives weak oscillations in metabolism and locomotor activity of aphids (Acyrthosiphon pisum) Received: 12 July 2017 Accepted: 20 October 2017 Published: xx xx xxxx Received: 12 July 2017 Accepted: 20 October 2017 Published: xx xx xxxx Katharina Beer1, Jens Joschinski 2, Alazne Arrazola Sastre1, Jochen Krauss2 & Charlotte Helfrich-Förster 1 Katharina Beer1, Jens Joschinski 2, Alazne Arrazola Sastre1, Jochen Krauss2 & Charlotte Helfrich-Förster 1 Timing seasonal events, like reproduction or diapause, is crucial for the survival of many species. Global change causes phenologies worldwide to shift, which requires a mechanistic explanation of seasonal time measurement. Day length (photoperiod) is a reliable indicator of winter arrival, but it remains unclear how exactly species measure day length. A reference for time of day could be provided by a circadian clock, by an hourglass clock, or, as some newer models suggest, by a damped circadian clock. However, damping of clock outputs has so far been rarely observed. To study putative clock outputs of Acyrthosiphon pisum aphids, we raised individual nymphs on coloured artificial diet, and measured rhythms in metabolic activity in light-dark illumination cycles of 16:08 hours (LD) and constant conditions (DD). In addition, we kept individuals in a novel monitoring setup and measured locomotor activity. We found that A. pisum is day-active in LD, potentially with a bimodal distribution. In constant darkness rhythmicity of locomotor behaviour persisted in some individuals, but patterns were mostly complex with several predominant periods. Metabolic activity, on the other hand, damped quickly. A damped circadian clock, potentially driven by multiple oscillator populations, is the most likely explanation of our results. The environment cycles in a daily manner due to the earth’s rotation around its own axis. In order to cope with these rhythmic changes, organisms rely on endogenous circadian clocks. Clocks drive behavioural and phys- iological rhythms with periods of approximately 24 h, and thus align the organism with the environmental rhythm1,2. These rhythms persist even without environmental time cues (Zeitgebers) such as light or tempera- ture3. Therefore, circadian clocks enable organisms not only to react to, but also to predict environmental oscilla- tions, which may be adaptive4. Hence, circadian clocks play an important role in coping with daily environmental changes. Circadian timekeeping might also be involved in the photoperiodic calendar and control of diapause induc- tion, although this hypothesis is still under discussion5,6. Bünning proposed that the circadian clock forms the basis of photoperiodism7, and this idea was later formalized as external8 and internal9 coincidence models. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports SCiENTifiC REPOrTS \| 7: 14906 \| DOI:10.1038/s41598-017-15014-3 Received: 12 July 2017 Accepted: 20 October 2017 Published: xx xx xxxx A damping circadian clock drives weak oscillations in metabolism and locomotor activity of aphids (Acyrthosiphon pisum) In contrast, Lees did not observe any circadian pattern in the photoperiodic reaction of aphids and excluded there- fore the involvement of a circadian oscillator. He proposed an hourglass mechanism instead, in which some biochemical product accumulates during night, so that only sufficiently long nights can trigger a photoperiodic response10,11. But some phenomena of long night experiments cannot be explained by an hour-glass mecha- nism, and a model with a damping circadian oscillator describes diapause induction better12. By rephrasing the hour-glass mechanism as a damping oscillator, the model unites these apparently contrary views13. However empirical data demonstrating the actual damping of circadian oscillations is largely lacking. Aphids are central to the discussion about photoperiodic clock involvement. During summer the insect pro- duces offspring via parthenogenesis, thereby ensuring quick population growth. In autumn sexual morphs are induced, which produce cold resisting eggs able to survive winter14. The switch in reproductive modes is induced 1Neurobiology and Genetics, Biocenter, University of Würzburg, Würzburg, Germany. 2Animal Ecology and Tropical Biology, Biocenter, University of Würzburg, Würzburg, Germany. Beer Katharina and Joschinski Jens contributed equally to this work. Correspondence and requests for materials should be addressed to B.K. (email: katharina. beer@uni-wuerzburg.de) or J.J. (email: jens.joschinski@uni-wuerzburg.de) 1 www.nature.com/scientificreports/ by shortening day length15,16 and, to a lesser extent, drops in temperature. This extraordinary phenotypic plasticity allows aphids to cope with seasonal changes, and is probably one reason for their global distribution. Due to the rapid population growth in summer, various species are classified as crop pests17, and climate change with asso- ciated shifts in phenology (seasonal timing) will likely exacerbate the pest pressure of aphids18. Thus, aphids are well-suited models for circadian and seasonal time-keeping, not only due to their long research history, but also because the timing matters for applied pest management.h by shortening day length15,16 and, to a lesser extent, drops in temperature. This extraordinary phenotypic plasticity allows aphids to cope with seasonal changes, and is probably one reason for their global distribution. Due to the rapid population growth in summer, various species are classified as crop pests17, and climate change with asso- ciated shifts in phenology (seasonal timing) will likely exacerbate the pest pressure of aphids18. Material and Methods The time schedule was allocated in a non-random order, so that each observer occupied a different time slot every day. We always measured the replicates in the same order and timeframe. Measurements in the light phase were conducted under room light while measure- ments in the dark phase were made under red LED light. The excreted blue honeydew drops were equally visible under both light conditions.hi The first half of the replicates in the LD treatment was removed from the experiment, because they were acci- dentally taken into room-light during lights-out in the first night. Hence, sample sizes were 30 in LD and 60 in DD. Because we did not renew the diet during the experiment in order to reduce disturbance, petri dishes became contaminated during the experiment and were subsequently removed. Thus, sample sizes decreased over time, with 80% of the samples remaining for at least 5 days. In LD sample size decreased in total from 30 to 14, and in DD from 60 to 17. Only 3.4% of the aphids produced more than one honeydew drop in the observed time interval of three hours, so we treated the response as binomial. To test for metabolic rhythmicity, we applied a generalized linear mixed-effects model with binomial error distribution28 with the fixed factors “time of day” and “day”, and with the random term (“time of day” \| “ID”). For the DD treatment, we expected the effect to dampen over the course of the experiment, so we incorporated a change in effect size over time. Hence, we used the fixed factor “time of day”, interacting with the continuous variable “time since start”, and the random term (“time of day” \| ID). We checked all models for overdispersion. Because p-values are not reliable for GLMMs29, we report only confidence intervals30 for all models.h Honeydew production was infrequently interrupted by moulting, usually three times per individual. Thus, the aphids were in the final larval stage at the end of the experiment. To test whether moulting individuals pro- duce less honeydew, we pooled all time points and applied a chi-square test of goodness of fit (2 × 2 contingency table: drop production vs moulting). Because moulting individuals indeed produced less honeydew (see results), we repeated the analysis with a reduced dataset that included only non-moulting individuals (6% of all meas- urements removed). Material and Methods In order to investigate the circadian behaviour of aphids, we performed two experiments: First, we supplied a coloured diet and tested for rhythms in honeydew excretion. Secondly, we applied a novel method that allows constant locomotor activity monitoring of sap-sucking insects. All experiments were performed independently of the host plant on an artificial diet (based on Febvay, et al.24, 20% (w/v) sugar). The aphids were reared in a small containment and separated from the food source by a parafilm M membrane (BEMIS COMPANY INC., USA)25, so they could access the diet from below by piercing the parafilm. The artificial diet was sterile filtrated through a 0.45 µm Minisart® syringe filter (Sartorius, Germany), and all materials that came in contact with the diet were sterilized before use. For the metabolic activity experiments we reduced the diet by eight ingredients, reordered the ingredients list, and supplemented it with 1.25 mg/ml Brilliant blue FCF26. For all experiments we used an asexual Acyrthosiphon pisum line (LL01), a green alfalfa biotype from the Lusignan area that was kindly provided by G. Febvay (INRA Lyon, France). Stock cultures were kept on Pisum sativum (L.) var. Fuego plants in climate chambers (Sanyo/Panasonic MLR-H series; 18 ± 0.5 °C, 80% ± 10% RH, LD 16:08). All statistical tests were performed in R version 3.1.127. Metabolic activity. We measured metabolic activity simultaneously under LD 16:08 and constant darkness for eight days. We conducted the experiment in climate chambers (Sanyo MLR-352H) at 18 °C and 70% humidity under a 15000 lux fluorescent light source. We placed 100 adult aphids on freshly cut broad bean leaves. On the next day, we discarded the adults, placed 120 nymphs individually in petri dishes (∅35 mm) and fed each with 500 µl coloured artificial diet. 60 aphids were moved into DD after five hours (Zeitgeber time (ZT) 12 = 12 hours after lights on), whereas the other 60 nymphs remained in LD, but at a reduced light intensity (7200 lux). At ZT 21 (i.e., three hours before lights-on), we counted and marked all honeydew drops which have been produced so far. Thus, the aphids in DD were given 9 hours to locate the food source before the start of measurements. We then counted honeydew every 3 hours (8 measurements per day). In addition, we counted all exuviae, as moulting individuals are not expected to produce honeydew.h Four observers have been involved in taking measurements. A damping circadian clock drives weak oscillations in metabolism and locomotor activity of aphids (Acyrthosiphon pisum) Thus, aphids are well-suited models for circadian and seasonal time-keeping, not only due to their long research history, but also because the timing matters for applied pest management.h g pp p g There is ongoing interest in the circadian clock of aphids. Several behavioural studies suggest the existence of a functional circadian clock in sexual as well as in parthenogenetic aphid forms of various aphid species19,20, although the experimental protocols did not allow to investigate damping of the clock. More recently, two studies identified putative clock genes and the location of clock gene expressing neurons in the pea aphid Acyrthosiphon pisum (Harris)21,22. Unfortunately the challenge to uncouple the aphid’s activity from the plant’s influence remained unattended in most studies. Recently we described the first daily rhythms of aphids completely inde- pendent of their host plant23, but have so far not monitored aphid rhythms in constant darkness for methodolog- ical reasons.f In the present study we investigate two different outputs of the circadian clock, namely locomotor activity and metabolic activity independently of the host plant. We show that the aphid clock drives weak, but stable, circadian output rhythms in locomotion, whereas oscillations in metabolic activity dampen quickly in constant conditions. SCiENTifiC REPOrTS \| 7: 14906 \| DOI:10.1038/s41598-017-15014-3 Material and Methods In addition, we tested whether moulting itself was rhythmic, using the same model as for honeydew production in LD. SCiENTifiC REPOrTS \| 7: 14906 \| DOI:10.1038/s41598-017-15014-3 2 www.nature.com/scientificreports/ Figure 1. Locomotor activity monitoring set up. (A) Monitor (DAM2, Trikinetics) placed horizontally with illumination from the sides. 32 IR-beams on the monitor detect movements of individual aphids in glass tubes (B) with blue food tubes on top. Aphid (blue arrow) in a monitoring glass tube hanging from the food tube. The roaming space was limited by a cotton plug (yellow arrow). IR-beam (IR-beam level defined by the position of IR-beam emitter and sensor in the monitor is indicated by the red arrow and beam line crossing the monitoring tube is indicated by the dotted line) is positioned on the mid plane of the monitor and detects moving aphids. Figure 1. Locomotor activity monitoring set up. (A) Monitor (DAM2, Trikinetics) placed horizontally with illumination from the sides. 32 IR-beams on the monitor detect movements of individual aphids in glass tubes (B) with blue food tubes on top. Aphid (blue arrow) in a monitoring glass tube hanging from the food tube. The roaming space was limited by a cotton plug (yellow arrow). IR-beam (IR-beam level defined by the position of IR-beam emitter and sensor in the monitor is indicated by the red arrow and beam line crossing the monitoring tube is indicated by the dotted line) is positioned on the mid plane of the monitor and detects moving aphids. Locomotor activity. For the locomotor activity experiments we used one day old offspring of aphids that were raised on plants and then kept on artificial diet for one day in order to control for age. We adapted the DAM2 (Drosophila Activity Monitor) (Trikinetics, USA), which monitors locomotor activity via an infrared-light barrier (IR-beam), to aphid specific requirements (Fig. 1). Aphids were provided with food ad libitum in a shortened micropipette tip (volume 1000 µl) on top of the monitor tube (∅7 mm) while the activity monitors were located horizontally. We positioned the IR-beam directly under the micropipette tip and limited the animals roaming space to 1 cm. The setup allowed monitoring 32 aphids simultaneously. All animals were entrained as nymph to an LD 16:08 regime (16 hours light and 8 hours darkness) for at least 12 days. Material and Methods A light intensity of 200–400 lux was produced by white light LEDs (depending on the position of the monitor tube) in the light phase and 0 lux in the dark phase while temperature and humidity were kept constant (18 ± 0.5 °C, 80% ± 10% RH) in the climate chamber (Percival INTELLUS, CLF Plant Climatics GmbH, 86637 Wertingen, Germany). On day 13 one treat- ment group (32 aphids) was released into constant DD conditions whereas another group of 32 aphids received LD conditions throughout their life. g Activity data was recorded in beam crosses per minute and evaluated with the ImageJ software plugin ActogramJ31 (Fiji ImageJ Version 1.49, © Wayne Rasband, National Institutes of Health, USA). We calculated the average day activity profile and mean activity in light and dark phase with the activity data of several consec- utive days in Microsoft Excel (2013 Microsoft Office) and day activity was tested with Wilcoxon rank sum test. We excluded days 8–12, as earlier trials have shown that aphids undergo their last moulting in this time. It was impossible to appoint the aphids to either nymph or adult status reliably during this period without interrupting data acquisition. q We tested for rhythmicity in activity of individual adult aphids across at least five consecutive days in entrain- ing (LD) and free-running (DD) conditions with Lomb-Scargle (LS) and chi-square periodogram (CS) analysis (period 1140–1740 minutes; smoothing factor 10 (only in chi-square method); p-level 0.05). In case of several narrow spikes that barely exceeded the significance level, we appointed the periodogram as false positive and the animals as arrhythmic (see definition in32). Differences in the groups “arrhythmic”, “rhythmic” (with simple circa- dian rhythms) and “complex” (with complex rhythms) between LD and DD conditions were tested with Fisher’s exact test. Individuals that did not undergo all four moultings were excluded from analysis. Data availability. All data generated or analysed during this study are included in this published article (and its Supplementary Information files). This includes R - scripts for analysis and figure reproduction of the metabolic activity experiment. Results M t b l Metabolic activity: Bimodal rhythmicity profile under LD and DD. We monitored metabolic activity of individual aphids over a period of eight days, by counting the number of exuviae and of visibly coloured honey- dew drops every three hours. The aphids moulted three times during the eight days of measurement, on average after 24 (±3.25), 71 (±3.21) and 127 (±4.19) hours (days 1, 3 and 6). In total, we observed 252 of the expected 270 occurrences of moulting, because some aphids died during the course of the experiment. These occurrences of moulting suppressed honeydew excretion, because only 13.75% of the moulting individuals in LD produced honeydew drops, compared to 33.76% of the non-moulting individuals (χ² = 13.75, p < 0.001). In DD 18.13% of the moulting, and 29.41% of the non-moulting individuals excreted honeydew (χ² = 9.38, p < 0.01). We therefore excluded moulting individuals from the analysis of honeydew excretion. Moulting itself was not rhythmic, neither in LD nor in DD (not shown).h The aphids which did not moult produced on average 2.7 drops per day in LD and 2.4 drops per day in DD (1230 drops in total). The data for LD conditions was best described with a model that only incorporates ‘time of day’ and ‘day’ (Fig. 2A; AIC = 1736), because the inclusion of a damping component (‘time of day’ * ‘time’ interac- tion) estimated more parameters but did not significantly improve the model fit (AIC = 1752). Using the ‘Effects’ SCiENTifiC REPOrTS \| 7: 14906 \| DOI:10.1038/s41598-017-15014-3 3 www.nature.com/scientificreports/ Figure 2. Rhythms in honeydew excretion. (A) Honeydew production of 30 aphid nymphs, kept under LD 16:08 for seven consecutive days. Red dots mark the mean number of produced honeydew drops during a time interval of three hours. The bold black line describes model fit to the data. (B) Double plotted model results for the “Time” factor (average day for an average individual) of metabolic activity (mean of 7 days in LD 16:08) with confidence intervals (red). (C) Red marked values are the mean number of honeydew drops produced during seven consecutive days in DD. Under these conditions a model that includes damping (“time of day” and “day” interaction) yielded the best fit to the data (bold black line). (D) Effect plot of metabolic rhythm during seven days in DD with confidence intervals (red). Results M t b l Lighting conditions during the experiment are indicated above and below in the environmental bars. White: light, black: darkness, grey: the light phase that was present before switching to DD. Figure 2. Rhythms in honeydew excretion. (A) Honeydew production of 30 aphid nymphs, kept under LD Figure 2. Rhythms in honeydew excretion. (A) Honeydew production of 30 aphid nymphs, kept under LD 16:08 for seven consecutive days. Red dots mark the mean number of produced honeydew drops during a time interval of three hours. The bold black line describes model fit to the data. (B) Double plotted model results for the “Time” factor (average day for an average individual) of metabolic activity (mean of 7 days in LD 16:08) with confidence intervals (red). (C) Red marked values are the mean number of honeydew drops produced during seven consecutive days in DD. Under these conditions a model that includes damping (“time of day” and “day” interaction) yielded the best fit to the data (bold black line). (D) Effect plot of metabolic rhythm during seven days in DD with confidence intervals (red). Lighting conditions during the experiment are indicated above and below in the environmental bars. White: light, black: darkness, grey: the light phase that was present before switching to DD. package, we isolated the factor of interest, ‘time of day’, while keeping the other elements (individual variance and day of experiment) constant (Fig. 2B). This procedure is akin to calculating an average day for an average individ- ual, and allows calculating confidence intervals (CI). This plot shows that honeydew production was markedly rhythmic under LD conditions and peaked twice during day-time (ZT 0: mean 0.42, CI 0.34–0.51; ZT 9: mean 0.45, CI 0.37–0.54) and lowest activity at night time (ZT 18: mean 0.19, CI 0.13–0.27 drops). Activity appeared to be bimodal, though the confidence intervals overlap. i Under constant darkness the model with an interaction term (damping) was better suited (AIC = 3219) than the simpler model without damping (AIC = 3222). Honeydew excretion was rhythmic during the first days (day 1, peak at 20h after lights-off: mean 0.63, CI 0.51–0.75 drops; trough at 29h after lights-off: mean 0.24, CI 0.15– 0.35 drops), but the only discernible peak damped quickly and disappeared after three days (Fig. 2C,D). SCiENTifiC REPOrTS \| 7: 14906 \| DOI:10.1038/s41598-017-15014-3 Results M t b l t We decided to measure moulting mainly because it causes aphids to stop moving for longer time periods33, and thus disrupts the activity in the young aphids. By removing moulting individuals from the analysis of metab- olism, we corrected for these gaps in activity. The disruption by moulting was more severe in our measures of locomotor activity. Therefore we considered metabolic activity of nymphs, but locomotor activity of adults for analysis of circadian rhythmicity. Locomotor activity: diurnal rhythms in LD and complex rhythms in DD. We monitored locomotor activity rhythms of aphids during their whole live from nymphal stage L1 onward in a novel monitoring set up (Fig. 1). Both nymphs (age: 1–7 days; Fig. 3A) and adult aphids (age: 13–23 days; Fig. 3B) showed significantly higher average activity during the light phase than during the dark phase (p(nymphs) < 0.05, p(adult) < 0.05, paired Wilcoxon signed rank test). The activity patterns of the aphids were, however, disrupted by activity breaks lasting entire days during the first 9 days of their life (Fig. 4A/B), leading to lower activity levels in nymphs (0.08 ± 0.02 counts/min) than in adult aphids (0.31 ± 0.07 counts/min). The latter displayed two main activity bouts that were best visible in the average day, one after lights-on the other in the late light phase (Fig. 3B). In between the two activity bouts activity levels were lower. y y While we detected diurnal rhythms in activity of adult aphids in LD, they appeared largely arrhythmic under constant conditions, at least in the average actogram (Fig. 4A/B). Nevertheless, averaging concealed the temporal and inter-individual variation, so we supplement our analysis with an individual-based approach. We found that SCiENTifiC REPOrTS \| 7: 14906 \| DOI:10.1038/s41598-017-15014-3 4 www.nature.com/scientificreports/ Figure 3. Diurnal activity profile in aphids. Average daily activity profile of nymphs (A) and adult (B) aphids measured by counting IR-beam passages (black line = mean activity, red line = standard error). Average day is calculated for day 1–7 for nymphs and day 13–23 for adult aphids. The light regime (LD 16:08) is indicated by the white (light phase) and grey (dark phase) bars above the graphs. Panels on the right: Mean activity of nymphs (1–7 days) (A) and adult (13–23 days) (B) aphids during light and dark phase. Figure 3. Diurnal activity profile in aphids. Results M t b l Average daily activity profile of nymphs (A) and adult (B) aphids measured by counting IR-beam passages (black line = mean activity, red line = standard error). Average day is calculated for day 1–7 for nymphs and day 13–23 for adult aphids. The light regime (LD 16:08) is indicated by the white (light phase) and grey (dark phase) bars above the graphs. Panels on the right: Mean activity of nymphs (1–7 days) (A) and adult (13–23 days) (B) aphids during light and dark phase. many of the adult subjects (67%) showed no activity at all during the first three days after switching to constant conditions of DD at day 13 (Fig. 4B). We therefore selected periods of at least 5 continuous days with activity, and determined the percentage of rhythmic adults (from day 13 on) in LD and DD conditions via periodogram analysis (Lomb-Scargle (LS) method in Fig. 4C and chi-square (CS) method in Fig. 4D). In DD (N = 15) 0% (CS) to 13% (LS) of the aphids were rhythmic with a single peak in the periodogram and further 80% (CS: 53%) displayed complex rhythms with more than one significant predominant period. In LD (N = 15) 60% (67% with CS method) were rhythmic with one periodogram peak and 33% (both methods) showed complex rhythmicity. Although the percentage of arrhythmic individuals was significantly higher with the CS method in DD condi- tions (p < 0.01, Fisher’s exact test), the results of both analysis methods are similar, as the difference in rhythmic individuals between light treatments was significant in both tests (LS: p < 0.05; CS: p < 0.01, Fisher’s exact test). SCiENTifiC REPOrTS \| 7: 14906 \| DOI:10.1038/s41598-017-15014-3 Discussion By combining measurements of aphid honeydew production and locomotor activity, we find that aphid activity is bimodal under LD conditions. In constant darkness the rhythm persists initially, but then dampens quickly and individuals display complex rhythmicity.l By combining measurements of aphid honeydew production and locomotor activity, we find that aphid activity is bimodal under LD conditions. In constant darkness the rhythm persists initially, but then dampens quickly and individuals display complex rhythmicity.l We adapted a commercially available measurement system for locomotor activity of flies to monitor aphid rhythms with high data throughput. This novel monitoring method provides the first measurements of aphid locomotor activity rhythms independent of the host plant. It was sensitive enough to monitor adult aphids as well as nymphs, though the general activity level was lower than in most other insect species. For example Drosophila melanogaster34, Musca domestica35 and Apis mellifera36 are approximately 10 times more active than the aphids in our locomotion setup. This finding can partly be explained by the unusual food source, which is known to com- promise performance17. The aphids produced on average 2.5 honeydew drops per day, which is lower than the 4–8 drops/day reported in studies on plants37. On the other hand, different linden bug (Pyrrhocoris apterous) strains, which are of the same order (Hemiptera) and also feed by sucking, show similarly low activity levels38. Thus, the low locomotor activity levels might be related to the sap-sucking feeding behaviour.h Despite the low activity levels, aphids were clearly day active in both metabolism and locomotion. These results are in line with various studies that detected diurnal activity of aphids on plants20,39,40 as well as inde- pendently of the host plant23. Furthermore, aphids were shown to find their host plants more effectively in day light conditions41,42, indicating adaptation to a diurnal lifestyle.h g g p y The activity appeared bimodal in both behaviours with one peak around the time of lights-on and another one in the late light phase. Bimodal patterns of activity were described in a variety of different animals, for example fruit flies43, mosquitoes44, birds and rodents45, and have been linked to multi-oscillator systems46. 5 www.nature.com/scientificreports/ Figure 4. Locomotor activity rhythms of aphids under LD cycles and constant conditions. (A) Aphid activity plotted in average actograms (double plots of two consecutive days). Discussion Animals are monitored from nymphal stage L1 onwards for several days in LD 16:08 (light regime is indicated by the white (light phase) and grey (dark phase) background in the actograms). Activity levels day 20–23 are lower because a few aphids died in this period (in A: 4, in B: 1) (B) Like in A for the first 12 days in the setup, then aphids are released into constant darkness (DD). Percentage of rhythmic individuals analysed from day 13 onward for at least 5 consecutive days with activity (marked in A and B) in periodogram analysis with either Lomb-Scargle (C) or Chi-Square (D) method of testing rhythmicity. Figure 4. Locomotor activity rhythms of aphids under LD cycles and constant conditions. (A) Aphid activity plotted in average actograms (double plots of two consecutive days). Animals are monitored from nymphal stage L1 onwards for several days in LD 16:08 (light regime is indicated by the white (light phase) and grey (dark phase) background in the actograms). Activity levels day 20–23 are lower because a few aphids died in this period (in A: 4, in B: 1) (B) Like in A for the first 12 days in the setup, then aphids are released into constant darkness (DD). Percentage of rhythmic individuals analysed from day 13 onward for at least 5 consecutive days with activity (marked in A and B) in periodogram analysis with either Lomb-Scargle (C) or Chi-Square (D) method of testing rhythmicity. In the metabolic activity profile there is only one activity peak left in DD, which is probably due to a melting of the separate peaks like it has been observed in Drosophila melanogaster activity43. This peak damped quickly during the first 3–4 days in constant conditions. The observed damping of circadian rhythms could either be due to desynchronization of individual persisting rhythms or damping of individual rhythms, but the low number of honeydew drops and the resulting binomial data make it difficult to assess damping on the individual level. We thus incorporated inter-individual variation in a mixed-effects model to exclude population-level damping. SCiENTifiC REPOrTS \| 7: 14906 \| DOI:10.1038/s41598-017-15014-3 www.nature.com/scientificreports/ occur at the individual level. Overall we found complex circadian rhythms in locomotion and quick damping in metabolism, and we argue that the aphid circadian clocks cannot drive strong activity rhythms. occur at the individual level. Overall we found complex circadian rhythms in locomotion and quick damping in metabolism, and we argue that the aphid circadian clocks cannot drive strong activity rhythms. g p g y y Aphids played a central role in the development of the hourglass mechanism of the endogenous clock, which Lees postulated for the diapause induction of aphids10,11. Lees found no circadian involvement in diapause induc- tion, causing him to question the coincidence models. g q However, various aphid behaviours were found to be controlled by a circadian oscillator. For example clock gene homologues have recently been characterized, and their mRNA was shown to cycle21,22. On the behavioural level the circadian clock was shown to govern the release of aphid sex pheromone19, and fresh weight-gain and larviposition20. In the latter experiment, aphids were disrupted by shifting the light phase and the rhythms needed more than 3–4 days to re-entrain to the new conditions, which led the authors to the conclusion that this behav- iour is governed by a circadian oscillator. g y As an alternative model to the hour-glass model, the circadian oscillator model with damping rhythms48 can simulate the “hour-glass reaction” observed in diapause induction of aphids. However, although this model fits to the data of Lees, only recently actual damping of circadian rhythms in aphids has been demonstrated21. In this study the cycling of mRNA levels extracted from whole mount aphid brains appeared to rapidly dampen in con- stant conditions. Although whole mount extracts provide only a population perspective and the animals were not kept independently of the host plant, these results point to a damped circadian clock in aphids. Our study adds the analysis of output rhythms independently of the host plant and supports the model of an oscillator driving damping circadian rhythms in two points. p g y p Firstly we observe circadian oscillations in behaviour and metabolic activity in DD, which would not be the case if the aphid endogenous clock would have an hourglass mechanism. Secondly, we see a clear damping of activity rhythms in the first few days in constant conditions, at least in metabolic activity. References 1. Allada, R. & Chung, B. Y. Circadian organization of behavior and physiology in. Drosophila. Annu. Rev. Physiol. 72, 605–624, https:// doi.org/10.1146/annurev-physiol-021909-135815 (2010). g y 2. Mohawk, J. A., Green, C. B. & Takahashi, J. S. Central and peripheral circadian clocks in mammals. Annu. Rev. Neurosci. 35 445–462, https://doi.org/10.1146/annurev-neuro-060909-153128 (2012). p g 3. Roenneberg, T., Daan, S. & Merrow, M. The art of entrainment. J. Biol. Rhythms 18, 183–194, https://doi. org/10.1177/0748730403253393 (2003).i g 4. Vaze, K. M. & Sharma, V. K. On the adaptive significance of circadian clocks for their owners. Chronobiol. Int. 30, 413–433, https:/ doi.org/10.3109/07420528.2012.754457 (2013). 5. Danks, H. V. How similar are daily and seasonal biological clocks? J. Insect Physiol. 51, 609–619, https://doi.org/10.1016/j jinsphys.2005.01.005 (2005). j p y 6. Kostal, V. Insect photoperiodic calendar and circadian clock: Independence, cooperation, or unity? J. Insect Physiol. 57, 538–556 https://doi.org/10.1016/j.jinsphys.2010.10.006 (2011). j p y 6. Kostal, V. Insect photoperiodic calendar and circadian clock: Independence, cooperation, or unity? J. Insect Physiol. 57, 538–556, https://doi.org/10.1016/j.jinsphys.2010.10.006 (2011). 7. Bünning, E. Die endogene Tagesrhythmik als Grundlage der photoperiodischen Reaktion. Ber. Deut. Bot. Ges. 54, 590–607 (1936). 8. Pittendrigh, C. S. & Minis, D. H. The entrainment of circadian oscillations by light and their role as photoperiodic clocks. Am. Nat. 98, 261–294, https://doi.org/10.1086/282327 (1964). 7. Bünning, E. Die endogene Tagesrhythmik als Grundlage der photoperiodischen Reaktion. Ber. Deut. Bot. Ges. 54, 590–607 (1936). 8. Pittendrigh, C. S. & Minis, D. H. The entrainment of circadian oscillations by light and their role as photoperiodic clocks. Am. Nat. 98, 261–294, https://doi.org/10.1086/282327 (1964). p g 9. Pittendrigh, C. S. Circadian surfaces and diversity of possible roles of circadian organization in photoperiodic induction. Proc. Natl Acad. Sci. USA 69, 2734–2737, https://doi.org/10.1073/pnas.69.9.2734 (1972). , , p // g/ /p ( ) 10. Lees, A. D. Photoperiodic time measurement in the aphid Megoura viciae. J. Insect Physiol. 19, 2279–2316, https://doi. org/10.1016/0022-1910(73)90237-0 (1973).f p g p 0. Lees, A. D. Photoperiodic time measurement in the aphid Megoura viciae. J. Insect Physiol. 19, 2279–2316, https://doi org/10.1016/0022-1910(73)90237-0 (1973).f g ( ) ( ) 11. Lees, A. D. Some effects of temperature on the hour glass photoperiod timer in the aphid Megoura viciae. J. Insect Physiol. 32, 79–89, https://doi.org/10.1016/0022-1910(86)90160-5 (1986). g 1. Lees, A. D. Some effects of temperature on the hour glass photoperiod timer in the aphid Megoura viciae. J. Insect Physiol. 32, 79–89 https://doi.org/10.1016/0022-1910(86)90160-5 (1986). p g 2. Vaz Nunes, M. & Hardie, J. www.nature.com/scientificreports/ Hence, our study pro- vides a first empirical evidence for a damping oscillation in aphid activity. i Though anticipating daily reoccurring events is an advantage, an oscillator that drives strong circadian rhythms over a long time is potentially less flexible in resetting to seasonal changes. There are environmental sit- uations on earth that require organisms to adapt to extreme deviations from daily rhythms. For example reindeer living in polar regions have been found to be arrhythmic during extreme illumination conditions of the arctic summer and winter49–51. This adaptation enables those animals to optimally use the short season for gaining resources and hence show a strong seasonal rhythm in locomotor and metabolic activity. A similar seasonal adaptation of the circadian system has been found in Drosophila species, D. montana, D. ezoana and D. littoralis, living in Northern Europe52–54. Unlike D. melanogaster, which has shallow photoperiodic diapause induction55, these flies are exposed to extreme photoperiods and evolved a very robust diapause induction, while they have poor circadian activity rhythms in constant conditions. We can certainly draw here parallels to our results on aphid rhythmicity. Like the polar reindeer or the Northern Drosophila species, aphids exhibit very weak circadian rhythms, while the seasonal response to shortening photoperiod is highly robust.h y p g p p g y The weak circadian response in aphids might be the reason why most interest so far was put on the investiga- tion of photoperiodism while the circadian clock remained widely unattended. But this way a complete picture of clock output characteristics was missing. Our study demonstrates that the investigation of different outputs, by combining expert knowledge of different disciplines is important to gain a better understanding about properties of the aphid circadian clock. There is an ongoing discussion whether the circadian clock and the annual photoper- iodic clock are two independent timing systems or if they are at least partially the same5,6. While our study offers no direct causal link between damping activity rhythms and photoperiodism, it provides the tools of measuring two different clock outputs that could be used in future experiments with varying photoperiods. Further investi- gation of the circadian clock in aphids, also on the molecular level, might provide us with a deeper understanding of the potential involvement of the circadian system in seasonal timing. Discussion The model is constrained in that the period length is fixed at 24 h, but we find it unlikely to affect our conclusions, given the 3-hour intervals of the data.h The results from locomotor activity are similar: As in the metabolic activity assay, the aphids showed clearly rhythmic behaviour in LD, and rhythmicity also persisted in DD, as the majority of individuals (LS: 93%, CS: 53%) remained rhythmic. This indicates that the behaviour is governed by the circadian clock. In contrast to the metabolic activity essay we found, however, no damping of rhythms, but instead the activity patterns transformed into complex rhythms in DD. Admittedly, most individuals in the locomotor assay were not active at all in the first few days in constant conditions, so we cannot exclude that the rhythmicity damped to some extent during the first days. i At the moment we can only speculate about the mechanisms that give rise to complex rhythms and individual level damping in DD. Complex rhythms are not unusual, and have been described before for example in mosqui- toes44 and New Zealand Weta47. Lewis and co-authors proposed a two population model of circadian oscillators, driving different output rhythms. In this model different clock neuron populations in the oscillator are weakly coupled and oscillations driven by them become asynchronous in DD. Therefore different free running periods (FRPs) for the oscillator subgroups are detected, which creates complex rhythms as well as a damping in one circadian output rhythm, and eventually arrhythmicity on the individual and/or population level. We find this model appealing for aphid clocks, because it simultaneously explains the damping in one output and complex rhythms in the other output. Moreover, the model provides an explanation why some studies found clear circa- dian patterns19, whereas other studies with different outputs indicate hour-glass mechanisms15. Further studies on the molecular organization of the clock are needed to verify this model. g y Moreover, since aphids produce honeydew during feeding and therefore metabolic and locomotor activity is directly connected, it is likely that similar mechanisms operate for these two outputs. Like the complex rhythms in locomotor activity might lead to dampened rhythmicity of the individual, damping of metabolic activity could SCiENTifiC REPOrTS \| 7: 14906 \| DOI:10.1038/s41598-017-15014-3 6 www.nature.com/scientificreports/ References Circadian rhythmicity is involved in photoperiodic time measurement in the aphid Megoura viciae Experientia 49, 711–713, https://doi.org/10.1007/BF01923957 (1993). p , , p g ( ) 13. Saunders, D. S., Bünning, E. & Lees, T. Two giants of chronobiology, and the problem of time measurement in insect photoperiodism. J. Insect Physiol. 51, 599–608, https://doi.org/10.1016/j.jinsphys.2004.12.002 (2005). p p g 13. Saunders, D. S., Bünning, E. & Lees, T. Two giants of chronobiology, and the problem of time measurement in insect photoperiodism. J. Insect Physiol. 51, 599–608, https://doi.org/10.1016/j.jinsphys.2004.12.002 (2005). SCiENTifiC REPOrTS \| 7: 14906 \| DOI:10.1038/s41598-017-15014-3 7 www.nature.com/scientificreports/ 4. Simon, J.-C., Rispe, C. & Sunnucks, P. Ecology and evolution of sex in aphids. Trends Ecol. Evol. 17, 34–39, https://doi.org/10.1016 s0169-5347(01)02331-x (2002).h s0169-5347(01)02331-x (2002). 15. Lees, A. D. The role of photoperiod and temperature in the determination of parthenogenetic and sexual forms in the aphid Megoura viciae Buckton—I. J. Insect Physiol. 3, 92–117, https://doi.org/10.1016/0022-1910(59)90024-1 (1959). ( ) ( ) 15. Lees, A. D. The role of photoperiod and temperature in the determination of parthenogenetic and sexual forms in the aphid Me viciae Buckton—I. J. Insect Physiol. 3, 92–117, https://doi.org/10.1016/0022-1910(59)90024-1 (1959). y p g 16. Marcovitch, S. 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Environ. Exp. Bot. 55, 77–86, https://doi.org/10.1016/j.envexpbot.2004.10.001 (2006) p y gy p p g j p 40. Taylor, S. H., Parker, W. E. & Douglas, A. E. Patterns in aphid honeydew production parallel diurnal s composition. Entomol. Exp. Appl. 142, 121–129, https://doi.org/10.1111/j.1570-7458.2011.01206.x (2012). d l fi f d fl h h bl k b h d h f b h l p y gy p p g j p 40. Taylor, S. H., Parker, W. E. & Douglas, A. E. Patterns in aphid honeydew production parallel diurnal s iti E t l E A l 142 121 129 htt //d i /10 1111/j 1570 7458 2011 01206 (2012) 40. Taylor, S. H., Parker, W. SCiENTifiC REPOrTS \| 7: 14906 \| DOI:10.1038/s41598-017-15014-3 Author Contributions Author Contributions K.B. and J.J. designed the study, collected and analysed data (K.B.: locomotor activity, J.J.: metabolic activity) and wrote the manuscript. A.A.S. contributed to study design and data collection (locomotor activity). J.K. and C.H.-F. conceived the project, contributed funding, guided research and revised the manuscript. SCiENTifiC REPOrTS \| 7: 14906 \| DOI:10.1038/s41598-017-15014-3 Acknowledgementsh g This research was supported by funds from the German Research Foundation CRC 1047 projects A1 and C3. g This research was supported by funds from the German Research Foundation CRC 1047 projects A1 and C3. References E. H., Tyler, N. J. C., Gerkema, M. P., Folkow, L. & Stokkan, K. A. Where clocks are redundant: weak circadian mechanisms in reindeer living under polar photic conditions. Naturwissenschaften 94, 183–194, https://doi.org/10.1007/s00114- 006-0174-2 (2007). 2. Kauranen, H. et al. Flies in the north: locomotor behavior and clock neuron organization of Drosophila montana. J. Biol. Rhythm 27, 377–387, https://doi.org/10.1177/0748730412455916 (2012). p g 3. Menegazzi, P. et al. Adaptation of circadian neuronal network to photoperiod in high-latitude european Drosophilids. Curr. Biol. 27 833–839, https://doi.org/10.1016/j.cub.2017.01.036 (2017). 54. Vaze, K. M. & Helfrich‐Förster, C. Drosophila ezoana uses an hour‐glass or highly damped circadian clock for measuring night length and inducing diapause. Physiol. Entomol. 41, 378–389, https://doi.org/10.1111/phen.12165 (2016). g g p y p g p ( ) 55. Saunders, D. S., Henrich, V. C. & Gilbert, L. I. Induction of diapause in Drosophila melanogaster: photoperiodic regulation an impact of arrhythmic clock mutations on time measurement. Proc. Natl. Acad. Sci. USA 86, 3748–3752 (1989). SCiENTifiC REPOrTS \| 7: 14906 \| DOI:10.1038/s41598-017-15014-3 8 www.nature.com/scientificreports/ Acknowledgementsh Additional Information Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-017-15014-3.h Competing Interests: The authors declare that they have no competing interests. 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https://openalex.org/W4242430272	https://hal-meteofrance.archives-ouvertes.fr/meteo-03657896/document	English	null	Saharan dust events in the European Alps: role on snowmelt and geochemical characterization	null	2,018	cc-by	17,717	To cite this version: Biagio Di Mauro, Roberto Garzonio, Micol Rossini, Gianluca Filippa, Paolo Pogliotti, et al.. Saharan dust events in the European Alps: role in snowmelt and geochemical characterization. The Cryosphere, 2019, 13 (4), pp.1147-1165. 10.5194/tc-13-1147-2019. meteo-03657896 Saharan dust events in the European Alps: role in snowmelt and geochemical characterization Biagio Di Mauro, Roberto Garzonio, Micol Rossini, Gianluca Filippa, Paolo Pogliotti, Marta Galvagno, Umberto Morra Di Cella, Mirco Migliavacca, Giovanni Baccolo, Massimiliano Clemenza, et al. snowmelt and geochemical characterization Biagio Di Mauro, Roberto Garzonio, Micol Rossini, Gianluca Filippa, Paolo Pogliotti, Marta Galvagno, Umberto Morra Di Cella, Mirco Migliavacca, Giovanni Baccolo, Massimiliano Clemenza, et al. Saharan dust events in the European Alps: role in snowmelt and geochemical characterization Biagio Di Mauro, Roberto Garzonio, Micol Rossini, Gianluca Filippa, Paolo Pogliotti, Marta Galvagno, Umberto Morra Di Cella, Mirco Migliavacca, Giovanni Baccolo, Massimiliano Clemenza, et al. Distributed under a Creative Commons Attribution 4.0 International License Correspondence: Biagio Di Mauro (biagio.dimauro@unimib.it) Correspondence: Biagio Di Mauro (biagio.dimauro@unimib.it) Correspondence: Biagio Di Mauro (biagio.dimauro@unimib.it) Received: 7 November 2018 – Discussion started: 15 November 2018 Revised: 5 March 2019 – Accepted: 19 March 2019 – Published: 8 April 2019 origin and compared with Saharan sources. A strong enrich- ment in Fe was observed in snow containing Saharan dust. In our case study, the comparison between modelling results and observations showed that impurities deposited in snow anticipated the disappearance of snow up to 38 d a out of a to- tal 7 months of typical snow duration. This happened for the season 2015–2016 that was characterized by a strong dust deposition event. During the other seasons considered here (2013–2014 and 2014–2015), the snow melt-out date was 18 and 11 d earlier, respectively. We conclude that the effect of the Saharan dust is expected to reduce snow cover duration through the snow-albedo feedback. This process is known to have a series of further hydrological and phenological feed- back effects that should be characterized in future research. Abstract. The input of mineral dust from arid regions im- pacts snow optical properties. The induced albedo reduc- tion generally alters the melting dynamics of the snowpack, resulting in earlier snowmelt. In this paper, we evaluate the impact of dust depositions on the melting dynamics of snowpack at a high-elevation site (2160 m) in the European Alps (Torgnon, Aosta Valley, Italy) during three hydrological years (2013–2016). These years were characterized by sev- eral Saharan dust events that deposited signiﬁcant amounts of mineral dust in the European Alps. We quantify the short- ening of the snow season due to dust deposition by com- paring observed snow depths and those simulated with the Crocus model accounting, or not, for the impact of impu- rities. The model was run and tested using meteorological data from an automated weather station. We propose the use of repeated digital images for tracking dust deposition and resurfacing in the snowpack. The good agreement between model prediction and digital images allowed us to propose the use of an RGB index (i.e. snow darkening index – SDI) for monitoring dust on snow using images from a digital camera. We also present a geochemical characterization of dust reaching the Alpine chain during spring in 2014. El- ements found in dust were classiﬁed as a function of their 1 Introduction Mineral dust (hereafter referred as dust) plays an important role in Earth’s climate and in biogeochemical cycles (Ma- howald et al., 2010, 2013; Thornton et al., 2009). It provides nutrients such as iron, nitrogen, and phosphorous to ma- rine and terrestrial ecosystems (Aciego et al., 2017; Jickells, HAL Id: meteo-03657896 https://meteofrance.hal.science/meteo-03657896v1 Submitted on 3 May 2022 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License The Cryosphere, 13, 1147–1165, 2019 https://doi.org/10.5194/tc-13-1147-2019 © Author(s) 2019. This work is distributed under the Creative Commons Attribution 4.0 License. Saharan dust events in the European Alps: role in snowmelt and geochemical characterization Biagio Di Mauro1, Roberto Garzonio1, Micol Rossini1, Gianluca Filippa2, Paolo Pogliotti2, Marta Galvagno2, Umberto Morra di Cella2, Mirco Migliavacca3, Giovanni Baccolo1,4, Massimiliano Clemenza4,5, Barbara Delmonte1, Valter Maggi1, Marie Dumont6, François Tuzet6,7, Matthieu Lafaysse6, Samuel Morin6, Edoardo Cremonese2, and Roberto Colombo1 1Earth and Environmental Sciences Department, University of Milano-Bicocca, 20126 Milan, Italy 2Environmental Protection Agency of Aosta Valley, Aosta, Italy 3 3Max Planck Institute for Biogeochemistry, Jena, Germany 4National Institute of Nuclear Physics (INFN), University of Milano-Bicocca, 20126 Milan, Italy 5Department of Physics “Giuseppe Occhialini”, University of Milano-Bicocca, 20126 Milan, Italy 6Université Grenoble Alpes, Université de Toulouse, Météo-France, CNRS, CNRM, Centre d’Etudes de la Neige, Grenoble, France 7UGA/CNRS, Institut des Géosciences de l’Environnement (IGE), Saint Martin d’Hères, France A/CNRS, Institut des Géosciences de l’Environnement (IGE), Saint Martin d’Hères, France B. Di Mauro et al.: Saharan dust events in the European Alps (2010) presented a comprehensive character- ization of the mineralogical and geochemical properties of dust deposited from the atmosphere in the San Juan Moun- tains (Colorado, US). In this area, dust is dominated by silt and clay particles, indicating a regional source area. In the European Alps, a large fraction of dust reaching high moun- tains and glaciers originate from the Sahara (Haeberli, 1977; Kandler et al., 2007; Krueger et al., 2004; Schwikowski et al., 1995; Thevenon et al., 2009), but inputs from local sources cannot be excluded. Even though the Alps are located at a distance of about 3000 km from the largest desert of the planet, they are frequently affected by dust depositions. Due to their considerable elevation, the Alps act as an orographic barrier, enhancing cloud formation, precipitation, and hence dust scavenging from the atmosphere to the ground (De An- gelis and Gaudichet, 1991; Prodi and Fea, 1979). Dust de- position in the Alps is a well-known process, and its fre- quency is studied using ice cores from mountain glaciers (De Angelis and Gaudichet, 1991; Thevenon et al., 2009). Each year, the Sahara provides up to 760 million tons of dust to the atmosphere (Callot et al., 2000). Dust reaching Eu- rope is dominated by silicates and aluminium oxide (Goudie and Middleton, 2001); other contributions come from quartz, calcium-rich particles, sulfates, hematite, and soot (Kandler et al., 2007). The optical properties of particles are directly related to dust composition (Linke et al., 2006), and hence the latter is expected to modify the dust radiative effect on snow (Reynolds et al., 2013). Saharan dust can provide nutrients to many alpine ecosys- tems (Field et al., 2010; Okin et al., 2004). Aciego et al. (2017) recently showed that dust transported from Asia to the western US provides nutrients to montane forest ecosys- tems. This aspect has never been evaluated for mountain ecosystems in the European Alps, where dust may com- pete with ﬁne debris from local rocks in providing nutrients to soils. Conversely, the direct deposition of dust on plants can limit the photosynthetic capacity (Neves et al., 2009). Steltzer et al. (2009) reported results from a manipulation experiment conducted in the western US to study the depen- dence of vegetation phenology on snowmelt. They measured an advancement of 7 d in snowmelt when dust was manu- ally added to the snowpack. B. Di Mauro et al.: Saharan dust events in the European Alps Because of its peculiar optical properties, dust ef- ﬁciently scatters incoming solar radiation and exerts a direct climate forcing in the atmosphere (Tegen and Lacis, 1996). As a function of key variables (e.g. imaginary part of the refractive index, height of the dust layer, dust particle size, and dust optical depth), the net radiative forcing of dust can be either negative or positive at the top of the atmosphere (Liao and Seinfeld, 1998; Tegen et al., 1996), representing a signiﬁcant uncertainty in current climate models (Potenza et al., 2016). The main sources of dust are arid and hyper- arid regions of the planet. Under speciﬁc atmospheric condi- tions, ﬁne and coarse particles of dust can be suspended in the troposphere, generating characteristic dust storms (Fran- cis et al., 2018; Goudie and Middleton, 2001). Finer dust (< 5 µm) has a prolonged atmospheric lifetime, of the order of days, allowing for its long-range transport (Mahowald et al., 2013; Tegen and Lacis, 1996). When dust is deposited on snow- and ice-covered regions, its radiative impact at the surface results in a positive radiative forcing (Painter et al., 2012; Skiles et al., 2018). Snow optical properties largely de- pend on its microstructure and on the presence of impurities (also referred as light-absorbing particles – LAPs), such as carbonaceous or mineral particles (Warren and Wiscombe, 1980). Indeed, dust lowers the snow albedo in the visible wavelengths, enhancing the absorption of solar radiation (Di Mauro et al., 2015; Painter et al., 2007) and thus triggering the snow-albedo feedback (Hansen and Nazarenko, 2004). The alterations of the optical properties of snow are known to accelerate the melting processes (Drake, 1981; Painter et al., 2012). First estimations of the impact of dust on snow date back to the beginning of the last century; Jones (1913) estimated 1 month of earlier snowmelt due to dust deposition in the US. Drake (1981) estimated 4 d of advancement in the snowmelt. cal properties and snowpack dynamics. Impacts on glaciers optical properties and mass balance were also reported in the literature (Gabbi et al., 2015; Di Mauro et al., 2017; Oerle- mans et al., 2009). ) The composition of dust varies as a function of its ori- gin (Krueger et al., 2004) and timing (Kumar et al., 2018), with an effect on its optical properties (Caponi et al., 2017). Lawrence et al. B. Di Mauro et al.: Saharan dust events in the European Alps B. Di Mauro et al.: Saharan dust events in the European Alps 1148 2005; Yu et al., 2015), and it inﬂuences the shortwave radia- tion balance of the atmosphere (Ginoux, 2017; Mahowald et al., 2013). Because of its peculiar optical properties, dust ef- ﬁciently scatters incoming solar radiation and exerts a direct climate forcing in the atmosphere (Tegen and Lacis, 1996). As a function of key variables (e.g. imaginary part of the refractive index, height of the dust layer, dust particle size, and dust optical depth), the net radiative forcing of dust can be either negative or positive at the top of the atmosphere (Liao and Seinfeld, 1998; Tegen et al., 1996), representing a signiﬁcant uncertainty in current climate models (Potenza et al., 2016). The main sources of dust are arid and hyper- arid regions of the planet. Under speciﬁc atmospheric condi- tions, ﬁne and coarse particles of dust can be suspended in the troposphere, generating characteristic dust storms (Fran- cis et al., 2018; Goudie and Middleton, 2001). Finer dust (< 5 µm) has a prolonged atmospheric lifetime, of the order of days, allowing for its long-range transport (Mahowald et al., 2013; Tegen and Lacis, 1996). When dust is deposited on snow- and ice-covered regions, its radiative impact at the surface results in a positive radiative forcing (Painter et al., 2012; Skiles et al., 2018). Snow optical properties largely de- pend on its microstructure and on the presence of impurities (also referred as light-absorbing particles – LAPs), such as carbonaceous or mineral particles (Warren and Wiscombe, 1980). Indeed, dust lowers the snow albedo in the visible wavelengths, enhancing the absorption of solar radiation (Di Mauro et al., 2015; Painter et al., 2007) and thus triggering the snow-albedo feedback (Hansen and Nazarenko, 2004). The alterations of the optical properties of snow are known to accelerate the melting processes (Drake, 1981; Painter et al., 2012). First estimations of the impact of dust on snow date back to the beginning of the last century; Jones (1913) estimated 1 month of earlier snowmelt due to dust deposition in the US. Drake (1981) estimated 4 d of advancement in the snowmelt 2005; Yu et al., 2015), and it inﬂuences the shortwave radia- tion balance of the atmosphere (Ginoux, 2017; Mahowald et al., 2013). 2.2 Digital image analysis In recent years, digital images analysis was applied to mon- itor vegetation phenology (Julitta et al., 2014; Migliavacca et al., 2011; Richardson et al., 2007), landslides, glaciers (Jung et al., 2010), and snow (Corripio, 2004; Dumont et al., 2011; Hinkler et al., 2002; Parajka et al., 2012). Regarding the latter two, snow albedo and snow cover were success- fully estimated using digital cameras in alpine areas. For this study, digital RGB images were collected using a Nikon dig- ital camera (model d5000, also referred as “Phenocam”) in- stalled at the experimental site in 2013 in the vicinity of the AWS. Following Richardson et al. (2007), the camera was pointed north and set at an angle of about 20◦below horizon- tal. The camera focal length is 33 mm, and the ﬁeld of view is 79.8◦. The camera was ﬁxed at 2.5 m above the ground, and the same scene was repeatedly photographed. Digital im- ages were collected in the Joint Photographic Experts Group (JPEG) format with a resolution of 12 megapixels and three- colour channels (namely red, green, and blue) featuring eight bits of radiometric resolution. The images were collected from 10:00 to 17:00 LT (local time: UTC+1), with an hourly temporal resolution. Exposure mode and white balance were set to automatic. p g In this paper, we quantitatively estimate the impact of dust from the Sahara on snow dynamics. As a test area, we use the experimental site in Torgnon (Aosta Valley, western Ital- ian Alps) equipped with several sensors for measuring snow properties. Snow dynamics were simulated with a multilayer, physically based energy balance model (Crocus, Vionnet et al., 2012), which can incorporate the effect of LAPs (min- eral dust and black carbon) in snow and estimate their im- pact on snowmelt (Tuzet et al., 2017). The timing and inten- sity of Saharan dust depositions were simulated using two independent models (ALADIN-Climate and NMMB/BSC- Dust). Observed and simulated snow variables are compared, and the role of impurities on snowmelt is discussed. We also made use of repeated images from a digital camera to track the deposition and resurfacing of impurities. Finally, we present a geochemical characterization of dust reaching the Alps, and thus we discuss the possible biogeochemical and hydrological role of dust in the Alps. A region of interest (ROI) was ﬁrstly identiﬁed in an ap- proximately ﬂat area to analyse snow evolution. B. Di Mauro et al.: Saharan dust events in the European Alps (45◦50′40′′ N, 7◦34′41′′ E). The experimental site belongs to the Phenocam (Torgnon-nd, https://phenocam.sr.unh. edu/webcam/, last access: 2 April 2019), ICOS (IT-Tor; https://www.icos-ri.eu/, last access: 2 April 2019), and LTER (lter_eu_it_077; https://deims.org/site, last access: 2 April 2019) networks. The area is a subalpine unmanaged pasture classiﬁed as intra-alpine with a semi-continental cli- mate. The site is generally covered by snow from the end of October to late May. Further information regarding the site can be found in Galvagno et al. (2013). An AWS was in- stalled in 2009 at the experimental site of Torgnon. Air tem- perature is measured by a HMP45 (Vaisala Inc.); snow depth is measured with a SR50A sonic sensor (Campbell Scientiﬁc, Inc.). Albedo is measured with a Kipp & Zonen CNR4 net radiometer. The snow water equivalent (SWE) is measured with a gamma monitor (GMON; Campbell Scientiﬁc, Inc.) sensor. Solid and liquid precipitations were measured with a Pluvio2 OTT instrument. Wind speed and direction were measured with a CSAT3 three-dimensional sonic anemome- ter (Campbell Scientiﬁc, Inc.). Data are available at hourly time resolution. (45◦50′40′′ N, 7◦34′41′′ E). The experimental site belongs to the Phenocam (Torgnon-nd, https://phenocam.sr.unh. edu/webcam/, last access: 2 April 2019), ICOS (IT-Tor; https://www.icos-ri.eu/, last access: 2 April 2019), and LTER (lter_eu_it_077; https://deims.org/site, last access: 2 April 2019) networks. The area is a subalpine unmanaged pasture classiﬁed as intra-alpine with a semi-continental cli- mate. The site is generally covered by snow from the end of October to late May. Further information regarding the site can be found in Galvagno et al. (2013). An AWS was in- stalled in 2009 at the experimental site of Torgnon. Air tem- perature is measured by a HMP45 (Vaisala Inc.); snow depth is measured with a SR50A sonic sensor (Campbell Scientiﬁc, Inc.). Albedo is measured with a Kipp & Zonen CNR4 net radiometer. The snow water equivalent (SWE) is measured with a gamma monitor (GMON; Campbell Scientiﬁc, Inc.) sensor. Solid and liquid precipitations were measured with a Pluvio2 OTT instrument. Wind speed and direction were measured with a CSAT3 three-dimensional sonic anemome- ter (Campbell Scientiﬁc, Inc.). Data are available at hourly time resolution. dust may exert a stronger effect with respect to dry depo- sitions. Shifts in vegetation phenology also affect the timing of migration, breeding, and asynchronies between interacting animal species (Cohen et al., 2018; Thackeray et al., 2016). B. Di Mauro et al.: Saharan dust events in the European Alps Dust-induced snowmelt can anticipate the beginning of the growing season, and this can result in an earlier start of the seasonal cycle of both animals and plants. Changes in snow- falls and dust depositions are likely to occur more frequently in a warming climate. At the moment, the impact of Saharan dust events on the biogeochemistry of ecosystems in the Eu- ropean Alps has been poorly analysed (Avila and Peñuelas, 1999). Seasonal snow represents an important reservoir of fresh water in mountain ranges and polar regions. Recent climate changes showed that this exerts a strong impact on the du- ration of snow cover (Vaughan et al., 2013), in particular in the European Alps (Beniston, 2005; Beniston et al., 2018). It has been observed that, especially in spring, snow cover extent has decreased in the Northern Hemisphere (Brown and Robinson, 2011; Brown et al., 2009). Earlier snowmelt can have an impact on vegetation phenology (Steltzer et al., 2009) and water availability (Beniston et al., 2003), and it is expected to alter hydrologic regimes in the future. Accel- erated snowmelt due to dust can alter also surface hydrol- ogy in large mountain chains like the European Alps. In the Po Plain, for example, the most important renewable energy source is represented by hydropower. Meltwater from sea- sonal snow is a fundamental resource for agriculture during spring and summer (Huss et al., 2017). 2.2 Digital image analysis Images were acquired during the hydrological years 2013–2016. Red, green, and blue chromatic coordinates were extracted from the selected ROI using the Phenopix R package (Filippa et al., 2016). Then, the snow darkening index (SDI; Di Mauro et al., 2015) was calculated from a red and green digital num- bers (DNs) as follows: 2.1 Torgnon experimental site The study area is located in the north-western Italian Alps (Aosta Valley, Italy) at an elevation of 2160 m a.s.l. The Cryosphere, 13, 1147–1165, 2019 B. Di Mauro et al.: Saharan dust events in the European Alps This process can simulate a dry deposition from the atmosphere. In the Alps, most dust de- positions occur by wet deposition (mainly snowfalls; Sode- mann et al., 2006), so dust is expected to be included within ice grains. Flanner et al. (2012) showed that when black car- bon is internally mixed in ice grains, its radiative effect is stronger. If this also holds true for dust, wet deposition of The impact of dust on snow melting has been largely in- vestigated in the western US, where both radiative and hy- drological effects have been assessed using aerial, satellite, and automatic weather station (AWS) data (Painter et al., 2012a, b, 2013a, 2018; Reynolds et al., 2013; Skiles et al., 2012). In this area, the proximity of arid regions to the moun- tain ranges determines massive dust depositions on snow- covered mountain ranges. Dust depositions caused an earlier snowmelt that ranged from 35 d (Painter et al., 2007) to a maximum of 51 d (Skiles et al., 2012), strongly impacting water supplies around the area (Painter et al., 2012, 2018). Increases in dust deposition have been recently observed in this area, and they were linked to human activity and climate change (Neff et al., 2008). Other studies, conducted in Ice- land (Dagsson-Waldhauserova et al., 2015; Wittmann et al., 2017), in the Himalayas (Gautam et al., 2013), in Norway (Matt et al., 2018), and in the European Alps (Dumont et al., 2017; Di Mauro et al., 2015; Tuzet et al., 2017; Greilinger et al., 2018) reported signiﬁcant impacts of dust on snow opti- The Cryosphere, 13, 1147–1165, 2019 www.the-cryosphere.net/13/1147/2019/ 1149 B. Di Mauro et al.: Saharan dust events in the European Alps B. Di Mauro et al.: Saharan dust events in the European Alps B. Di Mauro et al.: Saharan dust events in the European Alps Figure 1. (a) Location of Torgnon (Aosta) and Artavaggio plains (Lecco) in the European Alps. (b) A picture of the experimental site of Torgnon (2160 m a.s.l.). (c) Aerial view of the site in Torgnon with the location of different instruments installed. The ﬁeld of view of the Phenocam is represented with a blue shaded area. Figure 1. (a) Location of Torgnon (Aosta) and Artavaggio plains (Lecco) in the European Alps. (b) A picture of the experimental site of Torgnon (2160 m a.s.l.). (c) Aerial view of the site in Torgnon with the location of different instruments installed. The ﬁeld of view of the Phenocam is represented with a blue shaded area. obtain simulations of snow spectral albedo as a function of snow properties, LAP concentrations, and LAP optical properties. The TARTES model is based on the asymp- totic approximation of the radiative transfer theory (AART; Kokhanovsky and Zege, 2004) and accounts for the effect of snow microstructure and impurities, such as dust and black carbon. The snow spectral albedo simulated with TARTES was used to calculate the SDI (using the formulation pro- posed in Di Mauro et al., 2015), and it was compared with the SDI calculated from the digital camera. A complete de- scription of this speciﬁc Crocus model version can be found in Tuzet et al. (2017). Crocus is embedded in the SURFEX surface scheme and is permanently coupled with the ISBA- DIF soil model. SDI = DNRed −DNGreen DNRed + DNGreen . (1) SDI = DNRed −DNGreen DNRed + DNGreen . (1) The SDI was correlated with the concentration of dust in snow (Di Mauro et al., 2015) and was used to represent the spatial distribution of impurities from space (Ganey et al., 2017; Di Mauro et al., 2017) and from hyperspectral images of ice cores (Garzonio et al., 2018). The SDI calculated from RGB data collected from an unmanned aerial vehicle (UAV) was found to be correlated with the SDI calculated from ﬁeld spectroscopy data (Di Mauro et al., 2015). This motivated the idea to monitor dust deposition and resurfacing dynam- ics using repeated digital images from the camera installed in Torgnon. In this work, the SDI was calculated for each avail- able image, then a daily average was calculated. B. Di Mauro et al.: Saharan dust events in the European Alps Days with an SDI > 0 were considered to be markers of the presence of dust on snow. Variables needed for running Crocus simulations are the following: air temperature, direct and diffuse shortwave in- coming radiation, longwave radiation, wind speed and direc- tion, speciﬁc humidity, surface pressure, and solid and liq- uid precipitation. The model was forced using meteorolog- ical data from the station in Torgnon for the seasons 2013 and 2016 at an hourly time step. All variables were mea- sured at the station of Torgnon, except for diffuse short- wave incoming radiation, which was measured (with a BF3 sensor; Delta-T Devices Ltd., Cambridge, UK) in another station located 2 km from Torgnon. The instrument used for precipitation measurements (Pluvio2 OTT) does not fea- ture a windshield. This can be problematic, since underesti- mations of snowfall can occur during intense wind events. For this reason, we corrected the data following the pre- scriptions proposed in Kochendorfer et al. (2017). Some manual adjustments to solid precipitations were needed in the case of strong wind events. In addition to the above- mentioned meteorological data, the Crocus version of Tuzet et al. (2017) needs dust and black carbon deposition ﬂuxes. www.the-cryosphere.net/13/1147/2019/ 1150 www.the-cryosphere.net/13/1147/2019/ B. Di Mauro et al.: Saharan dust events in the European Alps retrieve 202 ± 11 µg of particulate matter. For both samples, in addition to absolute concentration (mass fraction), normal- ized ones were also calculated. The average upper continen- tal crust composition (UCC; Rudnick and Gao, 2003) was selected as a normalizing reference to highlight the inﬂu- ence played by crust-derived material and the possible role of non-crustal sources for speciﬁc elements. Neutron irradi- ation was performed at the LENA laboratories at the Univer- sity of Pavia (Borio di Tigliole et al., 2010), where a TRIGA Mark II research nuclear reactor is installed (250 kW). Acti- vated samples were successively analysed using high-purity germanium detector available at the Radioactivity laboratory of the Milano-Bicocca University. Two irradiations and sev- eral acquisitions of the γ spectra were necessary to detect the largest number of radionuclides, ranging from the short-lived species to the long-lived ones. For a complete description of the method, including the estimation of errors, see Baccolo et al. (2015, 2016). In this study, these ﬂuxes were taken from the atmospheric model ALADIN-Climate (Nabat et al., 2015). For evaluat- ing the impact of dust depositions on the snowpack dynam- ics, key variables (e.g. albedo, snow depth, and snow wa- ter equivalent) measured from the AWS were compared with Crocus simulations with and without impurities (dust and black carbon) in snow. In addition, soil temperature was ini- tialized using a spin-up simulation of 4 years. 2.4 Dust concentration, size distribution, and geochemistry On 6 April 2016, a ﬁeld campaign was organized to col- lect snow samples at the experimental site of Torgnon. Six snow pits were dug in different locations placed a few me- tres away from the AWS. For each snow pit, we collected a surface sample at 0 cm and three samples at depths equal to 20, 40, and 60 cm from the surface. Samples were collected using sterilized Corning tubes (50 mL) and kept frozen until measurements. Dust concentration and size distribution were measured using a Coulter counter technique. Samples were melted in a clean room (class 1000 clean room at EuroCold Laboratory Facilities, University of Milano-Bicocca) and analysed with a Multisizer™4e COULTER COUNTER®. The instrument was set with a 100 µm oriﬁce, allowing for the detection of particles with a diameter (equivalent spheri- cal) between 2 and 60 µm, divided into 400 size channels. To obtain dust mass from particle volume, a crustal density of 2.5 g cm−3 was adopted. Total dust concentration was calcu- lated considering the integral of the concentration between 2 and 60 µm. Details about the technique can be found in Ruth et al. (2008). 2.5 Dust transport and deposition modelling In addition to the ALADIN-Climate model, dust transport and deposition were monitored using the NMMB/BSC-Dust model. This is an online multi-scale atmospheric dust model (Pérez et al., 2011); it was used here to provide dust fore- casts from the Sahara to the European Alps. NMMB/BSC- Dust provides both atmospheric concentration and depo- sition ﬂuxes of dust with a 0.3◦× 0.3◦horizontal resolu- tion. During the three seasons considered here, we classi- ﬁed dust events as “strong” events with dust deposition ﬂuxes larger than 800 mg m−2 and “weak” events with lower con- centrations. The timings of the events simulated with the NMMB/BSC-Dust model were qualitatively compared to those simulated with the ALADIN-Climate model during the period analysed here (2013–2016). In addition, dust samples collected in the Alps at 150 km from Torgnon (Artavaggio, Lecco, Italy, 1650 m a.s.l.) in March 2014 are used here to characterize the bulk com- position of dust events and the elemental input to alpine ecosystems. Snow samples were transported before melting in a cold facility, where they were stored until the prepara- tion for the successive analyses. At ﬁrst, they were melted, and an aliquot (5–10 mL) was measured through Coulter counter technique (CC) for the determination of dust size distribution. These data were already published (Di Mauro et al., 2015). A second aliquot consisting in few millilitres of melted snow was dedicated to instrumental neutron acti- vation analysis (INAA) for the analysis of elemental com- position (Greenberg et al., 2011). To this aim, dust was ex- tracted and separated using a ﬁltration system equipped with polycarbonate membranes (pore size 0.4 µm, well below the typical volume mode grain size of Saharan dust deposited on the Alps). Two distinct samples were prepared. One sample (SH1) was extracted from the reddish snow corresponding to the snow deposited during the Saharan event; it consisted of 7.2 ± 0.2 mg of dust. A second sample (SH2) was pre- pared for comparison, ﬁltering clean white snow. In this case, given the low concentration of impurities, it was possible to 2.3 Snowpack modelling Snow dynamics in Torgnon were simulated using the SUR- FEX and ISBA-Crocus model, hereinafter referred as Cro- cus. Crocus is a snow model initially developed for avalanche forecasting and used for hydrological estimation as well as numerical prediction (Brun et al., 1989). Crocus is a one- dimensional multilayer model that simulates the evolution of the snowpack based on input meteorological driving con- ditions (Brun et al., 1989, 1992). Snow dynamics are rep- resented as a function of energy and mass transfer within the snowpack, between both the snowpack and the atmo- sphere, and the snowpack and the ground below (Vionnet et al., 2012). In this study, we used a speciﬁc Crocus version using the Two-stream Analytical Radiative TransfEr in Snow (TARTES) radiative transfer model (Libois et al., 2013) to www.the-cryosphere.net/13/1147/2019/ The Cryosphere, 13, 1147–1165, 2019 1151 3.1 Modelled dust deposition events The period between 2013 and 2016 was characterized by two strong events (dust ﬂuxes > 800 mg m−2) and several weak events (dust ﬂuxes < 800 mg m−2) distributed during the sea- sons. The strong events that occurred on February 2014 and on April 2016. The event of February 2014 was already analysed in the scientiﬁc literature (Di Mauro et al., 2015; Tuzet et al., 2017; Dumont et al., 2017). The event of April 2016 lasted several days and transported a considerable dust amount to the western sector of the European Alps (Fig. 2; Greilinger et al., 2018). According to the NMMB/BSC-Dust model, during these two strong events, dust was primarily deposited in the Alpine chain by wet deposition. In Fig. 2, we show an example (5 April 2016) of the concentration of dust deposited according to NMMB/BSC-Dust and a longi- tudinal and latitudinal transect. NMMB/BSC-Dust predicted 3.2 Observed and simulated snow dynamics In Fig. 3, a comparison of variables observed at the Torgnon station and simulated with the Crocus model using impu- rity ﬂuxes is presented. We show time series of the snow albedo, SWE, and snow depth (SD). In general, the Crocus model represented snow dynamics well during the hydro- logical seasons 2013–2016. In Fig. 4, we present a quanti- tative comparison (coefﬁcient of determination – R2 – and root-mean-square error – RMSE) between snow variables observed and simulated, including and excluding the effect of LAPs. Crocus simulations accounting for the impact of LAPs showed a better agreement with observations than Cro- cus simulations that not account for the effect of LAPs. The snow albedo was underestimated from Crocus during the ac- cumulation period (see Fig. 3a). Instead, during the melt- ing period, the decreasing trend observed in snow albedo was reproduced well by the Crocus model accounting for the role of impurities. During the accumulation period, the albedo modelled by both Crocus simulations was always lower than the observed albedo. During the melting season, a clear divergence is observed between the Crocus simula- tion with LAPs and that without LAPs. Instead, the Cro- cus simulation with LAPs is more correlated with the ob- served snow depth. The observed and simulated SWE and snow depth show a large interannual variability. The SWE is strongly overestimated in the season 2013–2014; while dur- ing the accumulation period snow depth is represented well in the model, the melting rate is higher in the observed snow depth. This results in a delay of snow melt-out dates in both Crocus simulations (with and without impurities). A similar pattern in snow depth is also observed in the season 2014– 2015. Unfortunately, the measured SWE was not available for this season. During the season 2015–2016, the corre- lation between the observed and simulated snow depth ac- counting for the impact of impurities was very high, both for snow depth (R2 = 0.96; RMSE = 0.05 m) and the SWE (R2 = 0.97; RMSE = 13 mm). The difference in snow melt- out dates between observed and simulated data accounting for LAPs was 12, 10, and 11 d, respectively, for the sea- sons 2013–2014, 2014–2015, and 2015–2016. Instead, the comparison between snow melt-out dates simulated with and without impurities was 18, 11, and 38 d for the seasons 2013– 2014, 2014–2015, and 2015–2016, respectively. For snow depth (Fig. B. Di Mauro et al.: Saharan dust events in the European Alps 1152 Figure 2. NMMB/BSC-Dust forecast for the event of April 2016. In the top panel is the estimated surface concentration by wet deposition. The lower panels represent, respectively, a latitudinal and longitudinal transect centred on the city of Aosta (45.74◦N; 7.36◦E). Images are from the NMMB/BSC-Dust model, operated by the Barcelona Supercomputing Center (http://www.bsc.es/ess/ bsc-dust-daily-forecast/, last access: 2 April 2019). www.the-cryosphere.net/13/1147/2019/ The Cryosphere, 13, 1147–1165, 2019 1152 B. Di Mauro et al.: Saharan dust events in the European Alps This can be due to the longer duration of the dust event in April 2016 and may also explain the large change (38 d) in the snow melt-out dates observed in the data. Results from samples collected in Torgnon showed that signiﬁcant concentrations of dust were present in the snow- pack in April 2016 (Fig. 5). It is interesting to note that the mode of the dust size distribution is 7.9 µm for surface snow and 8.5 µm for snow samples collected at 20 and 40 cm depth. Instead, dust particles found in snow at the bottom of the snowpack (60 cm depth) feature a mode of 3.2 µm. This deeper layer can be probably due to the scavenging of small dust particles by meltwater or to other undetected pro- cesses. The ﬁrst three distributions can be due to the weak depositions that happened in February and March and were then buried by new snow. Dust size distributions are com- patible with other measurements of dust enclosed in snow and ice in the Alps (3–5 µm; Maggi et al., 2006) and in the Caucasus (1.98–4.16 µm; Kutuzov et al., 2013). Differences between our samples and these studies may be ascribed to the different elevation of the samplings. Samples shown in Fig. 5 feature a signiﬁcant noise in the tail of the distribution. This can be ascribed to the aggregation of ﬁne particles or to an input of local particles with larger diameters. A contri- bution of large particles of local origin cannot be excluded, and it may have a strong inﬂuence on snowmelt. At the mo- ment, we do not have enough data to decouple the effect of large and small particles on the snow albedo. Total concentra- tion of dust in Torgnon was estimated by adding up different channels from the size distributions. Among the six differ- ent snow proﬁles measured, surface concentrations reached a maximum of 65 µgdust g−1 snow, with a mean of 45.6 µgdust g−1 snow and a standard deviation of 15.8 µgdust g−1 snow. Hereafter we focus on the season 2015–2016, since Cro- cus simulations with impurities resulted in a 38 d advance- ment of the snow melt-out date compared to the correspond- ing simulations without impurities. This season was charac- terized by dust surface concentration in snow being almost double with respect to the other two seasons considered in this study (see Fig. 3f). B. Di Mauro et al.: Saharan dust events in the European Alps the most concentrated surface sample and 65.1 µgdust g−1 snow for the mean of the six snow pits. This variability can be ex- plained by the strong spatial mismatch between the spatial resolution of ALADIN-Climate model (50 km) and the point measurement of dust concentration. Differences can also de- pend on snow sampling, vertical resolution, and Crocus layer thickness. Model improvements are needed to downscale the spatial resolution of LAPs ﬂuxes. The installation of a wet and dry sampler (e.g. deposimeter) at experimental sites may help to drive the Crocus model with measured deposition ﬂuxes. It is important to notice that ALADIN-Climate pre- dicted also depositions of black carbon. At the moment, we do not have measurements to validate this estimation, but the presence of black carbon in snow may have ampliﬁed the snow-albedo feedback in the snowpack. The role of black carbon in Alpine snow still represents a great uncertainty in snow modelling and climate prediction in the Alps. While the role of industrial black carbon on post-industrial glacier retreat has been debated (Painter et al., 2013b; Sigl et al., 2018), its role in seasonal snow melting has not been studied in the European Alps. In Fig. 3d, we show also a qualitative comparison between the dust ﬂuxes simulated with ALADIN-Climate and with the NMMB/BSC-Dust model. In general, a good agreement between the two models was observed. The two most intense events (February 2014 and April 2016) are identiﬁed by both models. Smaller events are also reproduced, whereas some- times small events are seen only by ALADIN-Climate. Once dust ﬂuxes are deposited on the snowpack, they are buried by subsequent snowfalls. In Fig. 3e, we show the mul- tilayer concentration of dust in snow simulated with Crocus. It is clear that dust is resurfacing towards the end of the sea- son, when the snow albedo feedback intensiﬁes and promotes the melting. The surface concentration of dust (average of the ﬁrst 10 cm of snow) in the three seasons considered in this study show an important interannual variability (Fig. 3f). In fact, whereas the ﬁrst two seasons show surface concentra- tions of dust lower than 150 µg g−1, the last season (2015– 2016) shows concentrations up to 350 µg g−1 at the end of the season. 3.2 Observed and simulated snow dynamics 4a) and the SWE (Fig. 4b), Crocus simulations with LAPs generally resulted in a lower RMSE and higher R2 with respect to Crocus simulations without LAPs. In- stead, for snow albedo (Fig. 4c), the resulting RMSE was smaller for Crocus simulations without LAPs. We under- line that these RMSE values are associated with very low explained variance (R2 ∼0.2 for the seasons 2014–2015, 2015–2016, and all years, and R2 = 0.43 for the season 2013–2014). Thus, Crocus simulations with LAPs perform Figure 2. NMMB/BSC-Dust forecast for the event of April 2016. In the top panel is the estimated surface concentration by wet deposition. The lower panels represent, respectively, a latitudinal and longitudinal transect centred on the city of Aosta (45.74◦N; 7.36◦E). Images are from the NMMB/BSC-Dust model, operated by the Barcelona Supercomputing Center (http://www.bsc.es/ess/ bsc-dust-daily-forecast/, last access: 2 April 2019). up to 1600 mg m−2 of dust deposition in the western Alps. In the latitudinal and longitudinal proﬁles, it is clearly visible that the plume reached almost 6 km in altitude. The highest concentrations in the atmosphere were reached in southern France and in the north-west of Italy. The experimental site of Torgnon is located in the Italian western Alps, and it repre- sents a good candidate for analysing the effect of this strong dust event on snow dynamics. The Cryosphere, 13, 1147–1165, 2019 www.the-cryosphere.net/13/1147/2019/ 1153 B. Di Mauro et al.: Saharan dust events in the European Alps In Fig. 6, we show the comparison of the snow depth simulated with Crocus, including and exclud- ing the impact of impurities. During the season 2015–2016, about 1 m of snow was on the ground in Torgnon. The model without impurities predicted a longer duration of snow on the ground than the model with impurities. Two late snow- falls occurred in May, and this probably increased the differ- ence between the simulations. Since air temperatures were still close to 0◦(data not shown), snow was preserved at the ground in the simulations without impurities, and this fur- ther prolonged the snow season duration. Considering that the ﬁrst signiﬁcant snowfalls occurred in January, the snow season was shortened by about 20 % of the total because of impurities. In Fig. 6, we plot the SDI calculated from the radiative transfer model (TARTES) included in Crocus (SDI-Crocus hereafter) and from the RGB camera (SDI-Phenocam here- after). Regarding the digital camera data, days with SDI > 0 are represented as shaded green bands. We observed an agreement between the two datasets. SDI-Crocus increased during April. In particular, at the beginning of April two peaks in SDI-Crocus are also seen by SDI-Phenocam. A peak then is not clearly seen by the digital camera. This could be due to the occurrence of two small snowfalls during the resur- facing of dust layers. At the end of April, the concentration of dust on the surface of snow is represented well, both by Crocus and digital images. During this last period, a marked change in snowmelt rate is observed from the snow depth Measurements of dust concentrations are available only for 6 April 2016. On this day, the dust concentration pro- ﬁle simulated by Crocus spans from 11 (bottom) to 108.7 (top) µgdust g−1 snow. Modelled and measured surface concen- trations of dust showed some difference: 43.7 µgdust g−1 snow for www.the-cryosphere.net/13/1147/2019/ The Cryosphere, 13, 1147–1165, 2019 B. Di Mauro et al.: Saharan dust events in the European Alps 1154 B. Di Mauro et al.: Saharan dust events in the European Alps Figure 3. (a, b, c) Time series of albedo, snow water equivalent (SWE), and snow depth (SD) measured with the AWS and simulated with Crocus model including and excluding the impact of LAPs. SWE data are missing in December 2013 because of problems with the power supply. (d) Dust ﬂuxes simulated with ALADIN (maroon bars, note that the scale is inverted), and strong (large stars) and weak (small stars) dust events simulated with NMMB/BSC-Dust. (e) Dust concentration (µg g−1) in the snowpack (yellow to black palette) simulated with Crocus and superimposed on the snow depth proﬁle (grey shaded area). (f) Surface concentration (averaged over the ﬁrst 10 cm) of dust simulated with Crocus. Figure 3. (a, b, c) Time series of albedo, snow water equivalent (SWE), and snow depth (SD) measured with the AWS and simulated with Crocus model including and excluding the impact of LAPs. SWE data are missing in December 2013 because of problems with the power supply. (d) Dust ﬂuxes simulated with ALADIN (maroon bars, note that the scale is inverted), and strong (large stars) and weak (small stars) dust events simulated with NMMB/BSC-Dust. (e) Dust concentration (µg g−1) in the snowpack (yellow to black palette) simulated with Crocus and superimposed on the snow depth proﬁle (grey shaded area). (f) Surface concentration (averaged over the ﬁrst 10 cm) of dust simulated with Crocus. Figure 4. Comparison between (a) snow depth, (b) snow water equivalent (SWE), and (c) albedo observed from the AWS in Torgnon and simulated with Crocus accounting (red dots) and not accounting (cyan dots) for the impact of LAPs on snow. Figure 4. Comparison between (a) snow depth, (b) snow water equivalent (SWE), and (c) albedo observed from the AWS in Torgnon and simulated with Crocus accounting (red dots) and not accounting (cyan dots) for the impact of LAPs on snow. series around the 20 April (Fig. 6). The agreement between SDI-Crocus and SDI-Phenocam suggests that low-cost digi- tal RGB data can be used for monitoring the resurfacing of dust in snowﬁelds, useful for satellite and model validation. In order to use these RGB data quantitatively, further com- parisons with ﬁeld spectroscopy and ground data are needed. posure. B. Di Mauro et al.: Saharan dust events in the European Alps 1155 Figure 5. Dust particle distribution (expressed in µgdust kg−1 snow) for a snow proﬁle sampled at Torgnon on 6 April at different depths (0, 20, 40, and 60 cm). Blue lines are experimental data, and black lines are moving averages (kernel: 25 points). Numbers in the plots represent the peak of the size distributions. Please note that the scale changes within different plots. Figure 6. Comparison between snow depth simulated using Cro- cus with impurities (grey area) and without impurities (purple line). Observed data are also shown (black line). Dust concentration in snow is represented. Shaded green bands represent days with SDI- Phenocam > 0. SDI-Crocus is represented as a continuous green line. Figure 6. Comparison between snow depth simulated using Cro- cus with impurities (grey area) and without impurities (purple line). Observed data are also shown (black line). Dust concentration in snow is represented. Shaded green bands represent days with SDI- Phenocam > 0. SDI-Crocus is represented as a continuous green line. we inverted the non-linear (rational) model developed in Di Mauro et al. (2015) that links mineral dust concentration and SDI values, and we obtained an estimated dust concentra- tion equal to 56 µgdust g−1 snow. This value is very close to the concentrations measured with the Coulter counter integrating particles smaller than 60 µm, which reached a maximum of 65 µgdust g−1 snow. snow Our estimations of shifts in the melt-out day are compa- rable to previous ﬁndings in the western US estimating a reduction of snow cover up to 51 days due to the presence of mineral dust in snow (Painter et al., 2007; Skiles et al., 2012). Despite the different deposition rates in the Alps, the advancement of the snowmelt due to dust is comparable to published results regarding the western US. This is true at least for one season (2015–2016) characterized by a major Saharan dust deposition. Tuzet et al. (2017) estimated up to 9 d of advanced snowmelt during 2014 in a lower eleva- tion site located in the European Alps as well. In this pa- per, we estimate an advance in snow melt-out days of 18, 11, and 38 d for the three seasons considered. The estima- tion for the season 2015–2016 is very high, also considering that snow cover normally lasts about 7 months at this ele- vation (2160 m a.s.l.). www.the-cryosphere.net/13/1147/2019/ Surface runoff may also represent an important pro- cess in shaping snow surface and in distributing dust in snow- ﬁelds. This may explain the variability observed in SDI and also the differences between the measured dust concentration in snow samples and Crocus modelled concentrations. Dust redistribution on snowﬁelds might strongly affect its radia- tive impact. In Fig. 7c and d, we present two SDI maps ac- quired before (10 April) and after (20 April) the resurfacing of dust layers. The transition from cold to warm colours re- ﬂects the increase in the values of the index. Positive values of the SDI are associated here with the presence of dust on snow (Di Mauro et al., 2015). On the right of both images, a snow pit is visible. It is interesting to note that in the SDI map series around the 20 April (Fig. 6). The agreement between SDI-Crocus and SDI-Phenocam suggests that low-cost digi- tal RGB data can be used for monitoring the resurfacing of dust in snowﬁelds, useful for satellite and model validation. In order to use these RGB data quantitatively, further com- parisons with ﬁeld spectroscopy and ground data are needed. In Fig. 7a and b, we show two examples of digital im- ages collected from the digital camera. The spatial variabil- ity of SDI can be explained by local topography. The exper- imental site is located in a plain area, with a gentle slope (∼5◦). Microtopography created by snow melting and re- freezing cycles can locally concentrate and dilute impurities in the snowﬁeld, also in relation to the differential sun ex- In Fig. 7a and b, we show two examples of digital im- ages collected from the digital camera. The spatial variabil- ity of SDI can be explained by local topography. The exper- imental site is located in a plain area, with a gentle slope (∼5◦). Microtopography created by snow melting and re- freezing cycles can locally concentrate and dilute impurities in the snowﬁeld, also in relation to the differential sun ex- The Cryosphere, 13, 1147–1165, 2019 www.the-cryosphere.net/13/1147/2019/ 3.3 Geochemical characterization of dust in snow Dust composition is strictly tied to its optical characteris- tics and hence to its radiative effect on snow (Caponi et al., 2017; Reynolds et al., 2013). Iron oxides contained in dust are particularly absorptive in the visible wavelengths (Alfaro et al., 2004; Linke et al., 2006), and this further enhances the albedo feedback when dust is deposited on snow. The com- position of dust is also important for the correct representa- tion of dust in radiative transfer models and global climate models (Albani et al., 2014). Saharan dust events provide an input of nutrients to alpine ecosystems (Goudie and Middle- ton, 2001), and this has been poorly studied in the scientiﬁc literature (Arvin et al., 2017). Figure 7. (a, b) Examples of digital images acquired from the Phe- nocam installed at Torgnon before and after the resurfacing of dust layers. (c, d) Snow darkening index (SDI) calculated using the red and green channels of the images. A region of interest (ROI, a) was used to create SDI time series. The asterisk in (a) indicates the po- sition of the snow pit. The main source area of Saharan dust events reaching the Alps is represented by northern Algeria (potential source area in Northern Africa 1 – PSANAF-1 – in Formenti et al., 2011). For this reason, the dataset presented in this study can be considered representative for the main composition of long-range dust deposition on snow in the Alps. Saharan dust events are regional episodes that move large quantities of mineral dust from arid regions to different latitudes and longitudes. There are two main pathways for the transport of dust: it can reach Europe overpassing the Mediterranean and also by looping back over the Atlantic (Israelevich et al., 2012; Sodemann et al., 2006). For this reason, we can assume that the bulk geochemical composition of dust events that oc- curred in different locations in the Alps and at different times is comparable. Although no trends were found in the annual number of Saharan dust days since 1997 (Flentje et al., 2015), further re- search is needed to assess the role of impurities on snow dy- namics in the Alps. Measurements of surface concentrations of dust and black carbon in snow are very scarce in the whole Alpine chain. At the Jungfraujoch station (3454 m a.s.l.), dust concentration in the atmosphere is measured continuously (Collaud Coen et al., 2004). 3.3 Geochemical characterization of dust in snow A comparison with these data will be fundamental in validating Saharan dust ﬂuxes in the Alps and quantifying their effect on snow dynamics. Between 18 and 20 February 2014 a relevant event was observed, involving not only southern Europe and the Alps but also a large fraction of Europe. It was described as one of the most intense events of this kind that was observed in previous years. The event was associated to a particularly favourable atmospheric setting which could uplift a massive amount of Saharan dust from northern Africa and transport it toward Europe in association to south-westerly winds driven by an anticyclonic structure located on the central Mediter- ranean. Given the magnitude of the event, many studies re- ported it, spanning from microbiology (Meola et al., 2015; Weil et al., 2017) to remote and proximal sensing (Dumont et al., 2017; Di Mauro et al., 2015; Tuzet et al., 2017) and at- mospheric chemistry and physics (Belosi et al., 2017; Telloli et al., 2018). p q y g y Snow duration was very short during the 2015–2016 hy- drologic year. Usually, grassland in Torgnon is covered by a thick snow cover from the end of October to late May (aver- age 1928–2010). During 2015–2016 snow arrived in January and disappeared at the beginning of May. It is known that ear- lier snowmelt impacts the carbon uptake period (Galvagno et al., 2013), altering carbon exchange with the atmosphere during spring. Shifts in phenological dates, such as the be- ginning and end of season, may impact ecosystem function- ing related to net and gross ecosystem productivity in alpine grasslands and might lead to early depletion of soil mois- ture and early senescence related to summer water stress. Extreme events like heatwaves have impacts on phenology of mountain grasslands (Cremonese et al., 2017). With future climate change, these extreme events are likely to increase. With the intensiﬁcation of climate change, snow is expected to occur later in autumn and to be depleted earlier in spring (Frei et al., 2018; Verfaillie et al., 2018), with signiﬁcant con- sequences for the hydrological cycle. The effect of Saharan dust in the European Alps is to accelerate the melt via the di- rect and indirect effect on snow albedo, thus enhancing snow season shortening. Hereafter, we provide results from a geochemical charac- terization of dust sampled in snow in the Alps (Artavaggio, Lecco, Italy). B. Di Mauro et al.: Saharan dust events in the European Alps 1156 Figure 7. (a, b) Examples of digital images acquired from the Phe- nocam installed at Torgnon before and after the resurfacing of dust layers. (c, d) Snow darkening index (SDI) calculated using the red and green channels of the images. A region of interest (ROI, a) was used to create SDI time series. The asterisk in (a) indicates the po- sition of the snow pit. B. Di Mauro et al.: Saharan dust events in the European Alps In the future, impurity concentration estimated with the atmospheric model should be evaluated using ground observations. In this sense, data from in situ spectrometers (e.g. Dumont et al., 2017; Picard et al., 2016) and repeated digital images can be very helpful. In fact, the concentration of different impurities may be retrieved from spectral reﬂectance using both inversion of radiative transfer models and spectral indices. Figure 5. Dust particle distribution (expressed in µgdust kg−1 snow) for a snow proﬁle sampled at Torgnon on 6 April at different depths (0, 20, 40, and 60 cm). Blue lines are experimental data, and black lines are moving averages (kernel: 25 points). Numbers in the plots represent the peak of the size distributions. Please note that the scale changes within different plots. from 20 April, a red layer is visible in the snow pit. This can be possibly associated with the precedent weak depositions from February and March, which were concentrated in a thin snow layer by melting during early spring. At the end of the season, weak and strong depositions are concentrated by sur- face melting. This process ampliﬁes the feedback mechanism of dust on snow. In fact, while the melting of snow concen- trates the dust on the surface, higher concentrations of dust intensify the melting. This feedback is expected to act for each day with sufﬁcient solar radiation during the melting period. The feedback is also expected to be enhanced until the total disappearing of the snow cover. The SDI is also sensitive to other impurities, such as black carbon (Di Mauro et al., 2017) and organic material (Ganey et al., 2017). We cannot exclude that other impu- rities were present on snow surface, but at present we do not have enough data to evaluate these aspects. We interpret SDI variability only in relation to dust deposition and resur- facing. In the selected ROI (Fig. 7a), frequency distribution of the SDI shows a peak at 0.005. Using this information, Dust transport is a natural phenomenon, but it can be inten- siﬁed by anthropogenic activities (Neff et al., 2008). Further research is needed to assess possible inputs of local dust to mountain environments. Recently, dust was found to be more important than temperature in determining snowmelt in the western US (Painter et al., 2018). The Cryosphere, 13, 1147–1165, 2019 www.the-cryosphere.net/13/1147/2019/ www.the-cryosphere.net/13/1147/2019/ B. Di Mauro et al.: Saharan dust events in the European Alps (a) Major elements, (b) anthropogenic elements, and (c) incompatible elements (with respect to Fe), listed following the order proposed by Sun and McDonough (1989). Figure 8. The elemental composition of the Saharan dust extracted from the snow precipitated on the Alps in February 2014 (red line, SH1), and the composition of the particulate matter retrieved from the clean snow deposited few days later (blue line, SH2). Grey lines refer to samples collected from the Sahara–Sahel dust corridor (Moreno et al., 2006). Data are expressed in terms of concentration normalized to the average upper continental crust (UCC; Rudnick and Gao, 2003). (a) Major elements, (b) anthropogenic elements, and (c) incompatible elements (with respect to Fe), listed following the order proposed by Sun and McDonough (1989). One of the main differences between SH1 and SH2 is re- garding iron (Fe). With respect to this element, SH1 presents absolute concentrations that are more than 2 orders of mag- nitude higher than in SH2. This suggests that Saharan dust could be important for supplying this essential element to high-elevation alpine ecosystems where other nutrient sources could be limited, as already pointed out to in rela- tion to other species and to other environments (Avila et al., 1998; Greilinger et al., 2018; Rizzolo et al., 2017). Another issue related to Fe concentration in atmospheric dust is re- lated to its optical properties, since iron oxide concentration and mineralogy strongly inﬂuence them (Alfaro et al., 2004; Caponi et al., 2017; Formenti et al., 2014; Linke et al., 2006). The large abundance of Fe is thus expected to affect the ra- diative effects of dust on snow (Reynolds et al., 2013). mobilization of dust from the central sector of the Sahara– Sahel dust corridor, i.e. the Hoggar, Chad, and Niger basins (Moreno et al., 2006). The elemental composition of dust might also have an im- portant effect on the biogeochemical cycles of the alpine grasslands. Among the elements listed in Table 1 there are elements such as K and Ca that are known to be rele- vant to ecosystem functioning (Sardans and Peñuelas, 2015; Schaffner et al., 2012). For both elements, SH1 shows no- tably higher concentrations (see Table 1). This requires more attention and further studies to understand the feedback of Saharan dust deposition on the biogeochemistry of high- elevation ecosystems. B. Di Mauro et al.: Saharan dust events in the European Alps 1157 Figure 8. The elemental composition of the Saharan dust extracted from the snow precipitated on the Alps in February 2014 (red line, SH1), and the composition of the particulate matter retrieved from the clean snow deposited few days later (blue line, SH2). Grey lines refer to samples collected from the Sahara–Sahel dust corridor (Moreno et al., 2006). Data are expressed in terms of concentration normalized to the average upper continental crust (UCC; Rudnick and Gao, 2003). (a) Major elements, (b) anthropogenic elements, and (c) incompatible elements (with respect to Fe), listed following the order proposed by Sun and McDonough (1989). may pave the way for a more exhaustive characterization of dust composition in the future. The concentrations of major elements normalized to the upper continental crust composition are shown in Fig. 8a. It can be easily appreciated that SH1 and SH2 display a very different composition. SH1, corresponding to the dusty snow deposited during the Saharan advection episode of Febru- ary 2014, presents a typical crustal signature, with UCC nor- malized values close to 1. On the contrary, SH2 shows very low normalized concentrations, suggesting that in this case, the crustal fraction is not the dominant one. Since all the considered major elements are strongly depleted (normal- ized concentrations span from 0.17 in the case of Na to 0.38 for Fe), it can be inferred that its composition is probably dominated by the only major element which is not consid- ered here: carbon. Unfortunately INAA is not suited for its detection, but it is known that the carbonaceous fraction is an important component of snow impurities (Li et al., 2016; Wang et al., 2015). Comparing SH1 to Sahel and Saharan dust source composition, a substantial correspondence can be appreciated, as illustrated in Fig. 8. This is not unexpected, but direct observations linking the geochemical properties of Saharan dust to the dust deposited in the Alps are quite scarce. Figure 8. The elemental composition of the Saharan dust extracted from the snow precipitated on the Alps in February 2014 (red line, SH1), and the composition of the particulate matter retrieved from the clean snow deposited few days later (blue line, SH2). Grey lines refer to samples collected from the Sahara–Sahel dust corridor (Moreno et al., 2006). Data are expressed in terms of concentration normalized to the average upper continental crust (UCC; Rudnick and Gao, 2003). 3.3 Geochemical characterization of dust in snow The analysis of the elemental composition al- lowed detecting 36 elements, spanning from the so-called major elements (the ones whose oxides constitute more than 1 % of the average composition of Earth’s crust) to many mi- nor and trace ones. Data of interest are shown in Fig. 8, the full list of elemental concentrations is reported in Table 1. We acknowledge that only one snow sample containing dust is not enough to provide a complete overview on the compo- sition of Saharan dust in snow in the Alps, but our analysis The Cryosphere, 13, 1147–1165, 2019 www.the-cryosphere.net/13/1147/2019/ www.the-cryosphere.net/13/1147/2019/ B. Di Mauro et al.: Saharan dust events in the European Alps Looking at Ca and Ti, further information can be inferred about the most likely provenance of SH1. Northern African sources (grey lines in Fig. 8a) can be clearly distinguished in relation to the content of these two elements, and indeed two groups are recognized. A ﬁrst one is characterized by high Ca concentration and low Ti content, and the second group shows an opposite composition, with a larger amount of Ti and a lower amount of Ca (see Fig. 8a). Carbonates are rich in Ca (a constituent of these rocks) and poor in Ti, in relation to their limited content in accessory and heavy minerals. The ﬁrst group (high Ca and low Ti) corresponds to the samples collected in Western Sahara, where carbonate rocks are com- mon. On the contrary, samples from northern Africa display an opposed composition. Comparing SH1 to these groups, it is clear that its composition is in accordance with the sec- ond group. Its provenance is more probably related to the Anthropogenic elements are presented in Fig. 8b. They are W, Hg, Sb, Se, and Zn. This group of elements con- cerns those elements presenting important positive deviations with respect to UCC composition. They are deﬁned “anthro- pogenic” to highlight that the their biogeochemical cycles have been strongly impacted by human activities in the last decades and that their mobilization in the environment re- lated to anthropogenic activities exceeds the natural one (Sen and Peucker-Ehrenbrink, 2012). Again, the signature of SH1 is completely different from that of SH2. Unlike the case of major and incompatible elements, for anthropogenic el- ements the sample presenting the higher relative concentra- B. Di Mauro et al.: Saharan dust events in the European Alps Table 1. The elemental composition of SH1 (snow sample containing mineral dust) and SH2 (clean snow sample). Data are expressed (in terms of µg g−1) and are referred to the mass of the extracted material, not to the considered snow volume. Values in brackets are measurement uncertainties. Normalized concentrations were calculated considering the upper continental crust as a reference (Rudnick and Gao, 2003). Element SH1 SH2 Conc. Conc. Conc. Conc. (µg g−1) (UCC norm.) (µg g−1) (UCC norm.) Na 5600(500) 0.47(0.04) 2000(300) 0.17(0.03) Mg 12 000(2000) 0.8(0.1) 3600(900) 0.24(0.06) Al 63 000(14 000) 0.8(0.2) 13 500(3000) 0.17(0.04) Si 200 000(35 000) 0.6(0.1) 48 000(25 000) 0.15(0.08) K 17 000(2000) 0.75(0.09) 4500(400) 0.19(0.02) Ca 16 000(5000) 0.6(0.2) 6000(2000) 0.25(0.09) Ti 6000(500) 1.6(0.1) 900(200) 0.25(0.06) Mn 470(50) 0.61(0.07) 180(33) 0.23(0.04) Fe 40 000(4000) 1.0(0.1) 180(30) 0.38(0.05) Sc 13(1) 0.91(0.08) 3.1(0.4) 0.22(0.03) V 100(10) 1.0(0.1) 26(5) 0.27(0.05) Cr 123(27) 1.3(0.03) 84(22) 0.9(0.2) Co 14(1) 0.82(0.06) 6.6(0.7) 0.38(0.05) Ni 35(8) 0.7(0.2) 38(11) 0.8(0.2) Zn 132(13) 2.0(0.2) 233(30) 3.5(0.4) As 6(1) 1.3(0.2) 4(1) 0.9(0.2) Se < 0.002 – 0.5(0.1) 5(1) Rb 82(8) 0.98(0.09) 28(5) 0.34(0.06) Sr 118(19) 0.37(0.06) 86(23) 0.27(0.07) Sb 1.3(0.2) 3.1(0.4) 12(2) 30(5) Cs 3.7(0.4) 0.75(0.08) 1.5(0.2) 0.30(0.05) Ba 500(100) 0.8(0.2) 300(200) 0.5(0.4) La 43(5) 1.4(0.1) 9(2) 0.29(0.05) Ce 87(4) 1.39(0.06) 29(2) 0.46(0.04) Nd 38(8) 1.4(0.3) 10(6) 0.4(0.2) Sm 7.2(0.9) 1.5(0.2) 1.5(0.2) 0.32(0.05) Eu 1.5(0.2) 1.5(0.2) 0.24(0.07) 0.24(0.07) Tb 1.05(0.08) 1.5(0.1) 0.22(0.05) 0.31(0.06) Ho 1.05(0.08) 1.3(0.1) 0.24(0.05) 0.29(0.07) Yb 3.9(0.6) 1.9(0.3) 0.7(0.2) 0.3(0.1) Hf 7.6(0.7) 1.4(0.1) 1.5(0.2) 0.28(0.04) Ta 1.8(0.5) 2.0(0.6) 0.4(0.1) 0.4(0.1) W 3.1(0.7) 16(3) 19(3) 100(15) Hg 0.4(0.1) 8(2) 2.9(0.9) 60(20) Th 12(1) 1.2(0.1) 2.6(0.6) 0.24(0.05) U 2.5(0.6) 0.9(0.2) 2.2(0.9) 0.8(0.3) tion is SH2. SH1 shows values near 1, suggesting that its composition is also mainly crustal for these elements. On the contrary, sample SH2 presents extremely high enrichments, near 100 in the case of W. Such values are not compatible with a crustal origin. Contributions from other sources must be involved. Atmospheric emissions related to human activ- ities are the best candidate for explaining the enrichment of almost all of these elements. Hg, Sb, Se, and Zn are all quite volatile elements, are easily mobilized in the atmosphere, and are related to industrial processes. Indeed, the sampling site is located less than 100 km far from the Po Valley, one of the most industrialized and densely inhabited regions of Eu- rope. www.the-cryosphere.net/13/1147/2019/ www.the-cryosphere.net/13/1147/2019/ The Cryosphere, 13, 1147–1165, 2019 1158 www.the-cryosphere.net/13/1147/2019/ B. Di Mauro et al.: Saharan dust events in the European Alps depth) simulated with Crocus model were compared with ob- served variables from an AWS in the Aosta Valley (western Alps). Good agreement between observations and simula- tions accounting for the role of impurities was observed. The size distribution of dust found in snow conﬁrms the Saha- ran origin of the event during April 2016 (Baumann-Stanzer et al., 2018). The geochemical characterization of dust and particulate matter samples distinguished the snow associated with Saharan dust from clean snow. Dusty snow showed a composition compatible with the geochemistry of the dust sources located in the central sector of the Sahara–Sahel dust corridor, i.e. the Hoggar, Chad, and Niger basin northern African sources. On the contrary, clean snow was character- ized by strong contaminations related to anthropogenic ele- ments. These results demonstrate that an accurate geochemi- cal characterization of dust deposited on the Alps allows the identiﬁcation of the different Saharan sources involved in the single transport events, but the ﬁngerprint of the local sources may also play an important role. al., 2008), refractory and non-volatile elements are instead more easily transported directly as airborne particles gener- ated by industrial processes (Sheppard et al., 2007). y p ( pp , ) The composition of a suite of elements found in trace is presented in Fig. 8c. The elements displayed there were or- dered following their incompatibility degree with respect to Fe (Sun and McDonough, 1989). This is a useful geochem- ical feature for understanding the provenance and the geo- chemical signature of rock-related samples. Focussing on SH1, it can be appreciated that there is a slight enrichment of poorly incompatible elements (the ones on the right side of Fig. 8c). The same feature is also recognized in the African dust sources, as it was extensively discussed by Moreno et al. (2006), which related the point to the geochemical and mineralogical properties of the sources. Sr and Ta are the two elements presenting the most evident anomalies: a depletion in the ﬁrst case and an enrichment in the second case. The concentration of Sr is generally related to the presence or ab- sence of carbonates, since Sr is a well-known substituent for Ca in carbonate lattice. In Fig. 8c, it is possible to appreciate that SH1 and most of the African sources are signiﬁcantly de- pleted in Sr, conﬁrming what was already suggested by ma- jor elements. B. Di Mauro et al.: Saharan dust events in the European Alps Indeed, the samples with low Sr content are the same samples presenting low Ca concentrations, pointing to a limited presence of carbonates and conﬁrming that sources from Western Sahara were not involved in this episode. In the paper, we also made use of repeated digital images for monitoring dust deposition and resurfacing in the snow- pack of Torgnon. Dust deposition and resurfacing agreed well with modelling predictions. This allowed us to propose the use of an RGB index (i.e. snow darkening index – SDI) for tracking dust on snow using repeated digital images from digital cameras. The good agreement between dust deposi- tion and the SDI suggests that data from this experimen- tal site can be used as a possible calibration and validation for satellite imagery (e.g. MODIS, Landsat, and Sentinel) and for regional and global climate model (WFR-Chem and CLM) validation. p The case of Ta is completely different, given the analyt- ical difﬁculties related to its detection; its behaviour in the environment is not yet well constrained, but it seems quite common to deal with samples that present an enrichment, in particular when atmosphere-related samples are considered (Filella, 2017). Looking at Fig. 8c, it can be seen that both the African sources, and to a lesser extent SH1, present a positive anomaly for Ta. Recent studies suggested that the Ta enrichment in rocks, sediments, and atmospheric particulate matter could be attributed to the effect of chemical weather- ing. Being extremely stable from a chemical and geochem- ical perspective, the loss of mobile fractions during weath- ering, enhanced by atmospheric transport, could explain the enrichment of Ta (Baccolo et al., 2016; Vlastelic et al., 2015). Several questions are still open regarding the role of dust in the Alps. For example, the spatial distribution of dust concen- tration on snow at alpine scale has never been quantitatively estimated. Possible differences between eastern and western Alps may arise as a function of distance from the sources. Another unresolved issue is the input from local sources: coarser dust particles can be suspended from snow-free ar- eas and deposited on snow. Regarding the geochemical and mineralogical characteristics of dust, future research should explore, in detail, the relation between dust characteristics and their radiative effect on snow. In addition to the well- known snow-albedo feedback, other complex mechanisms can inﬂuence the impact of dust on snow. B. Di Mauro et al.: Saharan dust events in the European Alps For example, the presence of dissolved carbonates may accelerate the melt of snow lowering the melting point of snow and ice crystals. The role of carbonaceous particles on snow optical properties in the Alps is also an open question. Measurements of black carbon, brown carbon, organic carbon, and elemental carbon concentration in snow are virtually absent in surface snow in the Alps. The Po Plain is one of the most polluted areas of the planet. At lower elevations, black carbon emissions from fossil fuel combustion and biomass burning may reach snow- covered areas and exert an impact on snow optical proper- ties. Future research efforts should aim at providing spatially B. Di Mauro et al.: Saharan dust events in the European Alps The same interpretation is not sufﬁcient for explain- ing the considerably high amount of W in SH2. There is no previous available information about its occurrence in snow, and in general its behaviour in the environment is quite ob- scure (Koutsospyros et al., 2006). It is traditionally consid- ered a non-volatile element, given its refractory properties. The high concentration found in SH2 could be related to the anthropogenic activities, since W is used in many indus- trial and manufacturing activities (Koutsospyros et al., 2006). Thus, a different transport mechanism is probably involved. Volatile elements can easily be scavenged from the atmo- sphere after being adsorbed on particulate matter (Marx et The Cryosphere, 13, 1147–1165, 2019 www.the-cryosphere.net/13/1147/2019/ www.the-cryosphere.net/13/1147/2019/ 1159 4 Conclusions In this paper, we investigated the role of impurity depositions in snow dynamics. In particular, we analysed the role of Sa- haran dust events on snowmelt in a high-elevation site of the European Alps. We estimated that impurities induced an ad- vance in snow melt-out dates of 38 d for the season 2015– 2016. During the other seasons considered here (2013–2014 and 2014–2015), the advancement in snow melt-out dates was 18 and 11 d, respectively. The season 2015–2016 was characterized by dust depositions that were almost double with respect to the other years considered in this study. Snow key variables (snow water equivalent, snow albedo, and snow The Cryosphere, 13, 1147–1165, 2019 The Cryosphere, 13, 1147–1165, 2019 Competing interests. The authors declare that they have no conﬂict of interest. Competing interests. The authors declare that they have no conﬂict of interest. Acknowledgements. We acknowledge the ARPA (Environmental Protection Agency) of the Aosta Valley region for maintaining the AWS in Torgnon and for providing the dataset. RGB im- ages were analysed using the Phenopix R package (https://r-forge. r-project.org/projects/phenopix/, last access: 2 April 2019). The complete set of Phenocam images is available at the following web- site: https://phenocam.sr.unh.edu/webcam/sites/torgnon-nd/ (last access: 2 April 2019). Edoardo Cremonese acknowledges the sup- port of the NextData Data-LTER-Mountain project CNRM. CEN and IGE are part of Labex OSUG@2020. The modelling work was funded by the ANRJCJC EBONI grant no. 16-CE01-0006. We thank the editor and the two reviewers for the constructive com- ments on a previous version of the paper. 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https://openalex.org/W4283724740	https://ejurnal.unikarta.ac.id/index.php/jemi/article/download/1056/944	Indonesian	null	Analisis Efektivitas Jalur Antrian Pada Sistem Transaksi PT. Pos Indonesia Di Tenggarong	Jurnal Ekonomi dan Manajemen Indonesia	2,022	cc-by	5,384	ANALISIS EFEKTIVITAS JALUR ANTRIAN PADA SISTEM TRANSAKSI PT. POS INDONESIA DI TENGGARONG Oleh : Intan Juwita, Iskandar, Ilham Penulis adalah Mahasiswa dan Dosen Pada Fakultas Ekonomi dan Bisnis Universitas Kutai Kartanegara Abstract: The purpose of this study was to determine the effectiveness of the application of the queuing system at the payment counter at PT. Pos Indonesia Tenggarong Branch. In this study, the analysis of the multiple line queuing system (M/M/S) was used. The problem that exists in this study is the fact that the queue conditions at the payment counter at PT. Pos Indonesia Tenggarong Branch at 08.30-10.30 is quite busy and at 11.30-12.30 it is not busy, so the application of a queuing system is very necessary to obtain an efficient number of teller services. The results of the analysis with two service servers, the time spent by a consumer in the system (Ws) the fastest is 1.39 minutes and the longest is 4.62 minutes. Meanwhile, by using three service servers, the time spent by a consumer in the fastest (Ws) system is 1.07 minutes and the longest is 3.51 minutes compared to the standard time of the post office company for 4 minutes of service. Operations become larger, with the addition of one service teller to three service tellers at the Post Office, it is declared ineffective because the Post Office must add service fees, while the number of people queuing in the system is still in the range of 1 or people queuing in the system and the time difference service is only 30 seconds to 1 minute apart. PENDAHULUAN Salah satu cara konsumen menilai kualitas operasional sebuah Kantor Pos adalah melihat kualitas pelayanannya. Pelayanan sendiri menurut Tjiptono (2007;26) meliputi suatu kecepatan kompetensi, kenyamanan, keramahan serta penanganan keluhan yang memuaskan. Kantor Pos Indonesia adalah Salah satu perusahaan yang bergerak dibidang jasa yang merupakan salah satu sektor penting dalam perekonomian suatu negara. Beberapa layanan Kantor Pos selain untuk mengirimkan paket, surat dan dokumen diantaranya adalah layanan pembayaran pajak, pembayaran tagihan telepon, air, listrik pengiriman dan penerimaan uang. Bentuk usaha Pos Indonesia ini berdasarkan peraturan Pemerintah Republik Indonesia Nomor 5 tahun 1995. Peraturan pemerintah tersebut berisi tentang pengalihan bentuk awal pos Indonesia yang beruapa perusahaan umum (PERUM) menjadi sebuah perusahaan persero. Transaksi pada loket pembayaran merupakan aktivitas operasi pada pos, dimana setiap transaksi pada loket pembayaran merupakan aktivitas operasi pada pos , dimana setiap transaksi akan dilayani oleh karyawan diloket transaksi dan untuk mendapatkan pelayanan tersebut para konsumen harus mengantri. JEMI Vol.22/No.1/JUNI/2022 16 JEMI Vol.22/No.1/JUNI/2022 Antrian terjadi disebabkan oleh kebutuhan akan layanan melebihi kemampuan (kapasitas) pelayanan, sehingga nasabah yang tiba tidak bisa segera mendapatkan pelayanan, hal ini disebabkan karena kesibukkan yang terjadi pada loket pos. kesibukkan yang terjadi pada transaksi ini bisa berkurang dengan menambah fasilitas pelayanan, akan tetapi dengan menambah fasilitas pelayanan maka akan menimbulkan biaya, sebaliknya apabila antrian terlalu panjang maka akan mengakibatkan nasabah akhirnya keluar dari sistem antrian. loket, sehingga terlihat adanya waktu dimana teller menganggur. Adanya permasalahan dalam penentuan jumlah teller yang tepat pada loket pembayaran PT. Pos Indonesia Cabang Tenggarong ini menarik perhatian peneliti untuk mengadakan penelitian guna menganalisis masalah antrian tersebut. Berdasarkan uraian diatas, maka tujuan penelitian ini adalah untuk mengetahui efektivitas penerapan sistem antrian pada loket pembayaran dan mengetahui jumlah teller yang efektif pada loket pelayanan di PT. Pos Indonesia Cabang Tenggarong. Antrian dapat dihindari apabila pihak- pihak yang terlibatkan mengetahui sampai dimana proses mengantri tersebut menguntungkan atau dapat merugikan, antrian yang lama tentu tidak di inginkan oleh berbagai pihak yang berkepentingan. Oleh karena itu, suatu perusahaan dibidang jasa maupun manufaktur harus mampu memberikan pelayanan yang cepat serta terbaik sesuai dengan keinginan pelanggan untuk memenuhi kebutuhan nasabah untuk memenuhi kebutuhan dan keperluannya dengan keinginan pelanggan untuk memenuhi kebutuhan dan keperluan mengingat akan jumlah populasi yang banyak. Pengertian Teori Antrian (Prawirosentoso, 2005;148 ) Antrian adalah ilmu pengetahuan tentang bentuk antrian dan merupakan orang – orang atau barang dalam barisan yang sedang menunggu untuk dilayani atau meliputi bagaimana perusahaan dapat menentukan waktu dan fasilitas yang sebaik-baiknya agar dapat melayani pelanggan dengan efesien. Heizier dan Render (2014;852) Dimyati, (2010;349) mengatakan bahwa teori antrian adalah teori yang menyangkut studi matematis dari antrian – antrian atau baris-baris penunggu. Formasi baris-baris penungguan ini terjadi apabila kebutuhan akan suatu pelayanan melebihi kapasitas yang tersedia untuk menyelenggarakan pelayanan itu. Sebagian besar konsumen yang datang ke Kantor Pos Indonesia Cabang Tenggarong adalah untuk melakukan pembayaran tagihan seperti; tagihan air, listrik, telepon, pembayaran iuran BPJS, tagihan angsuran kredit, pengiriman paket dan surat, serta pengiriman uang melalui wesel pos. Karena kemudahan membayar semua tagihan pada satu tempat membuat konsumen kebanyakan memilih membayar melalui kantor Pos, hal ini menyebabkan antrian yang cukup panjang terutama di jam sibuk pada pukul 08.00-09.00. Rata – rata kedatangan nasabah pada pukul 11.00 - 12.00 mulai berkurang dan loket pembayaran yang tersedia berjumlah 3 Tujuan dari model-model antrian adalah untuk meminimumkan total dua biaya, yaitu biaya langsung penyediaan fasilitas pelayanan dan biaya tidak langsung yang timbul karena individu harus menunggu untuk dilayani. Bila suatu sistem mempunyai fasilitas pelayanan lebih dari jumlah optimal, ini berarti membutuhkan investasi modal yang berlebihan, tetapi bila jumlahnya kurang dari optimal hasilnya adalah JEMI Vol.22/No.1/JUNI/2022 17 tertundanya pelayanan. ( Subagyo, dkk 2000 ). Dimyati (2010;349) mengatakan bahwa tujuan dalam teori antrian ialah mencapai kesimbangan antara ongkos yang disebabkan oleh adanya waktu menuggu tersebut. menyesuaikan situasi dan kondisi masing – masing. Dengan mengoptimalkan sistem pelayanan, dapat ditemukan waktu pelayanan, jumlah saluran antrian, dan jumlah pelayanan yang tepat dengan menggunakan model – model antrian (Heizer dan Render, 2014) empat model antrian tersebut terdapat pada tabel 1. Formula Model Antrian Ada empat model yang paling sering digunakan oleh perusahaan dengan Tabel 1. Model Antrian Model Nama Jumlah jalur Jumlah Tahapan Pola Tingkat Kedatangan Pola Waktu Pelayanan Ukuran Antrian Aturan A Jalur Tunggal (M/M/1) Tunggal Tunggal Poisson Eksponensial Tidak Terbatas FCFS B Jalur Berganda (M/M/s) Jalur Berganda Tunggal Poisson Eksponensial Tidak Terbatas FCFS C Pelayanan Konstan (M/D/1) Tunggal Tunggal Poisson Konstan Tidak Terbatas FCFS D Populasi Terbatas Tunggal Tunggal Poisson Eksponensial Terbatas FCFS Tabel 1. Model Antrian Hubungan Manajemen Operasi dan Antrian Hubungan antara manajemen operasional terhadap penerapan teori antrian yang dimana analisis antrian dalam hal panjangnya lini tunggu, waktu tunggu rata – rata, dan faktor – faktor lainnya yang membantu memahami sistem jasa seperti mesin bor tekan yang rusak (yang sedang menunggu difasilitas perbaikan) memiliki banyak perasamaan dari sudut pandang manajemen opersional. Baik penggunaan sumber daya manusia maupun perlengkapan untuk memperbaiki asset produksi yang berharga ( orang dan mesin) pada kondisi yang lebih baik agar terciptanya efesiensi. Model Nama Jumlah jalur Jumlah Tahapan Pola Tingkat Kedatangan Pola Waktu Pelayanan Ukuran Antrian Aturan A Jalur Tunggal (M/M/1) Tunggal Tunggal Poisson Eksponensial Tidak Terbatas FCFS B Jalur Berganda (M/M/s) Jalur Berganda Tunggal Poisson Eksponensial Tidak Terbatas FCFS C Pelayanan Konstan (M/D/1) Tunggal Tunggal Poisson Konstan Tidak Terbatas FCFS D Populasi Terbatas Tunggal Tunggal Poisson Eksponensial Terbatas FCFS Tabel 1. Model Antrian Hubungan Manajemen Operasi dan Antrian Hubungan Manajemen Operasi dan Antrian perbaikan) memiliki banyak perasamaan dari sudut pandang manajemen opersional. Baik penggunaan sumber daya manusia maupun perlengkapan untuk memperbaiki asset produksi yang berharga ( orang dan mesin) pada kondisi yang lebih baik agar terciptanya efesiensi. Hubungan antara manajemen operasional terhadap penerapan teori antrian yang dimana analisis antrian dalam hal panjangnya lini tunggu, waktu tunggu rata – rata, dan faktor – faktor lainnya yang membantu memahami sistem jasa seperti mesin bor tekan yang rusak (yang sedang menunggu difasilitas JEMI Vol.22/No.1/JUNI/2022 18 Kerangka Pikir Gambar 1. Kerangka Pikir Gambar 1. Kerangka Pikir Gambar 1. Kerangka Pikir Hipotesis sampling ini adalah salah satu teknik pengambilan sampel secara sengaja atau berdasarkan penilaian. Maka sampel yang digunakan dalam penelitian ini adalah konsumen yang mengantri dengan antrian teller, dengan asumsi kinerja dan waktu pelayanan bervariasi menyusaikan dengan keperluan transaksi dari masing – masing konsumen. Alat Analisis Penentuan sistem antrian dengan menggunakan struktur antrian Multi channel – Single phase akan memberikan efektivitas yang baik pada sitem transaksi kantor Pos. oleh karena itu jawaban sementara dalam penelitian ini adalah: 1. Jika sistem transaksi Pos Indonesia menggunakan struktur antrian Multi channel – Single Phase, maka efektivitas pada sistem transaksi PT. Pos Indonesia Cabang Tenggarong akan tercapai. Dalam proses transaksi untuk melayani nasabah, PT. Pos Indonesia Cabang Tenggarong menggunakan model antrian jalur berganda artinya terdapat lebih dari satu loket dan hanya ada satu tahapan pelayanan yang harus dilalui oleh konsumen untuk menyelasikan pemabayaran. Oleh karena itu, untuk mengoptimalkan proses pembayaran dapat digunakan rumus antrian untuk model B/M/M/S (Heizer dan Render, 2014;859) 2. Sebaliknya jika system transaksi PT. Pos Indonesia Cabang Tenggarong menggunakan Multi Channel – Multi Single Phase maka efektivitas tidak akan tercapai. METODE PENELITIAN Dimana : Dimana : S : Jumlah fasilitas pelayanan Ct : Biaya per periode waktu per fasilitas pelayanan e. Jumlah pelanggan rata – rata dalam sistem Dari kedua biaya diatas maka rumusan total excepted cost per periode waktu adalah sebagai berikut: ( ) ( ) ( ) E(Ct) = E(Cw)= SCs + ntCw E(Ct) = E(Cw)= SCs + ntCw Keterangan : : Jumlah kedatangan rata – rata per satuan waktu Karena parameter nt valid hanya untuk sistem dengan tiga fasilitas pelayanan, maka bila S ditambah atau dikurangi, nt baru harus dihitung kembali. Perhitungan biaya pelayanan menggunakan biaya rill atau gaji yang diterima pada bagian teller. µ : Jumlah orang yang dilayani per satuan waktu µ : Jumlah orang yang dilayani per satuan waktu Ls : Jumlah pelanggan rata – rata dalam sistem Ls : Jumlah pelanggan rata – rata dalam sistem Ws : Jumlah waktu rata – rata yang dihabiskan dalam sistem (waktu menunggu ditambah waktu pelayanan) Ws : Jumlah waktu rata – rata yang dihabiskan dalam sistem (waktu menunggu ditambah waktu pelayanan) Dimana : nt = rata – rata individu yang menunggu dalam suatu sistem Cw = biaya total per unit waktu yang melekat pada individu Cw = biaya total per unit waktu yang melekat pada individu Dalam hal ini penentuan biaya menunggu menggunakan asumsi upah minimum Kabupaten/Kota yang berlaku. c. Jumlah unit rata – rata yang menunggu dalam antrian Dalam hal ini penentuan biaya menunggu menggunakan asumsi upah minimum Kabupaten/Kota yang berlaku. d. Waktu rata – rata yang dihabiskan untuk menunggu dalam antrian METODE PENELITIAN a. Probabilitas terdapat 0 unit dalam sistem (unit pelayanan kosong) Populasi dan Sampel Populasi pada penelitian ini adalah konsumen yang bertransaksi di kantor Pos Indonesia Cabang Tenggarong, yang mengambil nomor antrian populasinya bersifat tak terbatas. Teknik pengambilan sampel yang digunakan adalah purposive sampling, yang dimana purposive [∑ ( ) ] ( ) untuk Mµ > [∑ ( ) ] ( ) untuk Mµ > JEMI Vol.22/No.1/JUNI/2022 19 E(Cw) = ntCw b. Jumlah waktu rata – rata yang dihabiskan dalam sistem (waktu menunggu ditambah waktu pelayanan) b. Jumlah waktu rata – rata yang dihabiskan dalam sistem (waktu menunggu ditambah waktu pelayanan) ANALISIS DAN PEMBAHASAN Analisis Sistem Antrian a) Efektifitas Jalur Antrian Analisis Sistem Antrian ANALISIS DAN PEMBAHASAN Lq : Jumlah unit rata – rata yang menunggu dalam antrian Analisis Sistem Antrian a) Efektifitas Jalur Antrian Wq: Waktu rata – rata yang dihabiskan untuk menunggu dalam antrian Wq: Waktu rata – rata yang dihabiskan untuk menunggu dalam antrian M : Jumlah Fasilitas Pelayanan Sistem antrian yang diterapkan oleh Kantor Pos Cabang Tenggarong adalah sistem model Multi Chanel- Single Phase atau M/M/S dimana pada sistem antrian ini terdapat beberapa loket pembayaran yang betugas melayani konsumen namun fase yang dilalui hanya satu tahap saja. gg M : Jumlah Fasilitas Pelayanan P0 : Probabilitas terdapat 0 unit dalam sistem (unit pelayanan kosong) Dalam menghitung minimasi biaya terdapat dua komponen, menurut Subagyo, dkk. (2000) komponen – komponen kedua biaya itu dapat dihitung dengan formula sebagai berikut: a. Biaya menunggu (Cost of waiting) JEMI Vol.22/No.1/JUNI/2022 20 Disiplin pelayanan yang diterapkan Kantor Pos Cabang Tenggarong adalah First Come, First Served (FCFS), konsumen yang terlebih dahulu datang mengambil antrian adalah konsumen yang akan mendapatkan pelayanan terlebih dahulu. Tingkat kedatangan per jamnya dapat dicari dengan cara menjumlahkan kedatangan per jam yang sama dibagi dengan 7 hari kerja. Rata-rata tingkat kedatangan perjam (ƛ) dapat dicari dengan cara : = Jumlah kedatangan pada jam tertentu Jumlah hari kerja / selama penelitian Tabel 2. Rata-rata Tingkat Kedatangan Periode Waktu (Jam) Rata – Rata Kedatangan 08.00 - 09.00 61,29 61 09.00 - 10.00 30,24 30 10.00 - 11.00 21,71 22 11.00 - 12.00 21,14 21 12.00 - 13.00 19,14 19 13.00 - 14.00 21,86 22 14.00 - 15.00 20,86 21 Total Kedatangan 196 Orang S b Di l h P li i Tabel 2. Rata-rata Tingkat Kedatangan Tabel 2. Rata-rata Tingkat Kedatangan Sumber : Diolah Peneliti Tingkat kemampuan untuk melayani kebutuhan atau kepuasan konsumen dalam setiap kedatangan disebut sebagai kemampuan pelayanan. Tingkat pelayanan (µ) per jamnya di Kantor Pos Cabang Tenggarong dapat dicari dengan cara : Pada tabel diatas menunjukan bahwa tingkat kedatangan paling tinggi terletak pada jam 08.00 - 09.00 dengan jumlah rata-rata 61 orang, sedangkan tingkat kedatangan konsumen yang paling rendah terletak pada jam 12.00 - 13.00 dengan jumlah rata-rata 19 orang saja. Jam 08.00 – 09.00 lebih banyak, karena pada saat jam 08.00 – 09.00 mudah bertransaksi, dan mungkin akan menghindari antrian yang panjang. = Jumlah Pelayanan pada jam tertentu Jumlah hari kerja /selama penelitian Tabel 3. Rata –rata Tingkat Pelayanan Fasilitas Tabel 3. Sumber: Diolah Peneliti Sumber: Diolah Peneliti konsumen yang menunggu untuk dilayani oleh fasilitas dan termasuk pelanggan yang sedang dilayani. Jumlah pelanggan yang dihitung adalah pelanggan yang menunggu mendapatkan giliran untuk melakukan transaksi pada pelanggan yang sedang mendapatkan pelayanan dari Teller. konsumen yang menunggu untuk dilayani oleh fasilitas dan termasuk pelanggan yang sedang dilayani. Jumlah pelanggan yang dihitung adalah pelanggan yang menunggu mendapatkan giliran untuk melakukan transaksi pada pelanggan yang sedang mendapatkan pelayanan dari Teller. Berdasarkan hasil pengolahan data antrian yang telah diperoleh selama penelitian akan menganalisis sistem antrian yang diterapkan pada sistem Kantor Pos Cabang Tenggarong yaitu sistem (Multi Chanel – Single Phace) dengan menggunakan alat analisis yang dijelaskan pada BAB III. Untuk dapat melihat kinerja sistem antrian yang ada dengan langkah – langkah sebagai berikut Jumlah pelanggan rata – rata dalam sistem merupakan petunjuk tentang beberapa jumlah pelanggan yang dilayani oleh teller Kantor Pos selama jam kerja beserta jumlah antrian pelanggan yang sedang menunggu untuk mendapatkan pelayanan. Informasi ini dapat dipergunakan sebagai dasar penentuan jumlah jalur pelayanan yang tersedia, yang bertujuan untuk meminimalisirkan antrian yang terjadi agar tidak menumpuk dan pelanggan dapat segera mendapatkan pelayanan. M :Jumlah Jalur yang terbuka (Server) µ :Jumlah Orang yang dilayani per satuan waktu (Mu) Satu konsumen yang belum mendapatkan pelayanan dan harus menunggu beberapa saat agar bisa mendapatkan pelayanan. Analisis Sistem Antrian a) Efektifitas Jalur Antrian Rata –rata Tingkat Pelayanan Fasilitas Periode Waktu (Jam) Rata – rata Pelayanan 08.00 - 09.00 59,43 59 09.00 - 10.00 27,86 28 10.00 - 11.00 19,57 20 11.00 - 12.00 20,29 20 12.00 - 13.00 18,14 18 13.00 - 14.00 23,71 24 Total Pelayanan 196 Orang Sumber : Diolah Peneliti JEMI Vol.22/No.1/JUNI/2022 21 dari fasilitas yang tersedia dalam memeberikan pelayanan selama jam operasional maka perlu dicari rata – rata kapasitasnya sistem pelayanan selama satu jam dengan cara sebagai berikut: = Jumlah rata – rata kedatangan Total jam kerja Pada tabel diatas menunjukan tingkat pelayanan paling tinggi terletak pada jam 08.00 - 09.00 dengan jumlah rata – rata 59 orang, sedangkan tingkat pelayanan yang paling rendah terletak pada jam 12.00 – 13.00 dengan jumlah rata – rata Cuma 18 orang. Untuk mengetahui kapasitas sistem pelayanan Tabel 4. Kapasitas Pelayanan Sistem Per Jam Periode waktu (Jam) Jumlah Kedatangan Jam Oprasional Kapasitas Pelayanan Per Jam 08.00 - 09.00 61 7 Jam 28 Orang 09.00 - 10.00 30 10.00 - 11.00 22 11.00 - 12.00 21 12.00 - 13.00 19 13.00 - 14..00 22 14.00 - 15.00 21 196 Orang Sumber: Diolah Peneliti Tabel 4. Kapasitas Pelayanan Sistem Per Jam Periode waktu (Jam) Jumlah Kedatangan Jam Oprasional Kapasitas Pelayanan Per Jam 08.00 - 09.00 61 7 Jam 28 Orang 09.00 - 10.00 30 10.00 - 11.00 22 11.00 - 12.00 21 12.00 - 13.00 19 13.00 - 14..00 22 14.00 - 15.00 21 196 Orang Sumber: Diolah Peneliti Tabel 4. Kapasitas Pelayanan Sistem Per Jam b) Jumlah Pelanggan Rata – Rata Dalam Sistem Jumlah nasabah rata – rata dalam seluruh sistem merupakan jumlah rata – rata JEMI Vol.22/No.1/JUNI/2022 22 Tabel 5. Jumlah Konsumen Rata – Rata Dalam Sistem Periode Waktu (Jam) Jumlah Pelanggan Rata – Rata Dalam Sistem/ Ls 08.00 - 09.00 1,41 09.00 - 10.00 1,50 10.00 - 11.00 1,58 11.00 - 12.00 1,45 12.00 - 13.00 1,46 13.00 - 14.00 1,16 14.00 - 15.00 0,92 Tabel 5. Jumlah Konsumen Rata – Rata Dalam Sistem Sumber : Diolah Peneliti Sumber : Diolah Peneliti dari pelanggan yang menunggu pelayanan dan waktu rata – rata fasilitas dalam menyelesaikan pelayanan. Waktu total dalam sistem dihitung ketika pelanggan mulai mengantri, menunggu untuk dilayani, saat dilayani sampai pelanggan selesai dilayani. Rata – rata total waktu dalam sistem merupakan petunjuk tengtang tingkat pelayanan yang diberikan oleh pihak Kantor Pos didalam menyelesaikan transaksi pelanggan. Dari tabel diatas dapat diketahui bahwa jumlah pelanggan rata – rata dalam sistem (Ls) terkecil adalah 0,92 terjadi pada jam 14.00 – 15.00 hal ini menunjukan bahwa banyaknya pelanggan dalam sistem (Pelanggan dalam antrian dan pelanggan dalam pelayanan atau yang sedang dilayani) pada jam tersebut adalah sebanyak 1 orang. c) Waktu Rata – Rata Dalam sistem Rata – rata waktu dalam sistem merupakan rata – rata keseluruhan waktu c) Waktu Rata – Rata Dalam sistem Rata – rata waktu dalam sistem merupakan rata – rata keseluruhan waktu Tabel 6. Jumlah Rata – Rata Dalam Sistem Priode Waktu (Jam) Waktu Rata – Rata Dalam Sistem (Menit)/ Ws 08.00 - 09.00 1,39 09.00 - 10.00 3 10.00 - 11.00 4,3 11.00 - 12.00 4,14 12.00 - 13.00 4,62 13.00 - 14.00 3,16 14.00 - 15.00 2,62 Sumber: Diolah Peneliti Tabel 6. Jumlah Rata – Rata Dalam Sistem Priode Waktu (Jam) Waktu Rata – Rata Dalam Sistem (Menit)/ Ws 08.00 - 09.00 1,39 09.00 - 10.00 3 10.00 - 11.00 4,3 11.00 - 12.00 4,14 12.00 - 13.00 4,62 13.00 - 14.00 3,16 14.00 - 15.00 2,62 Sumber: Diolah Peneliti Tabel 6. Jumlah Rata – Rata Dalam Sistem jam tersebut yang paling tinggi dan tingkat pelayanan tersebut adalah yang paling cepat dibandingkan jam lain. Dapat dilihat jumlah waktu rata – rata dalam sistem (Ws) pada tabel diatas yang terkecil adalah 1,39 menit atau 83 detik. Hal ini menunjukan bahwa tingkat kedatangan dan tingkat pelayanan pada JEMI Vol.22/No.1/JUNI/2022 23 10.00 - 11.00 1,3 11.00 - 12.00 1,14 12.00 - 13.00 1,29 13.00 - 14.00 0,66 14.00 - 15.00 0,4 S b Di l h li i Keterangan : Satuan nilai pada tabel diatas adalah perjam kecuali Ws dan Wq Sumber Data : Hasil Penelitian Setelah Diolah d) Waktu Rata – Rata Yang Dihabiskan Dalam Antrian Dihabiskan Dalam Antrian Rata – rata waktu menunggu merupakan lamanya waktu yang diperlukan oleh pelanggan yang datang dan antri untuk mendapatkan pelayanan. Waktu tunggu dihitung mulai dari pelanggan mengantri sampai dilayani oleh fasilitas. Waktu tunggu timbul disebabkan oleh beberapa hal, antara lain : tingat pelayanan yang ada pada teller kurang memenuhi disbanding dengan jumlah pelanggan yang datang untuk mendapatkan pelayanan dan pola kedatangan para pelanggan hanya pada saat – saat tertentu. Sumber Data : Hasil Penelitian Setelah Diolah PEMBAHASAN menunjukan bahwa peluang tidak ada pelanggan dalam sistem adalah sebesar 29% yang artinya probalitas ada yang mengantri (orang) untuk mendapatkan pelayanan cukup besar, sedangkan pada jam 13.00 sampai jam 15.00 probalitasnya 37% sampai 44%. 1. Penerapan Sistem Antrian p Disiplin pelayanan yang diterapkan Kantor Pos Cabang Tenggarong Kota ialah First Come, First Served (FCFS), sistem pemanggilan nomor antrian yang diterapkan teller akan dipanggil secara berurutan dari nomor pertama hingga selanjutnya. Jika ada pelanggan yang mengambil nomor antrian keluar dari sistem (terlewat), maka akan dilayani setelah pelanggan yang sedang dilayani oleh teller selesai mendapatkan pelayanan dan jika telah keluar (terlewat) lebih dari lima nomor urut sistem antrian maka dianjurkan untuk mengambil nomor antrian baru. p y p Jumlah pelanggan dalam sistem (Ls) pada jam 08.00 sampai jm 13.00 adalah sebanyak 1 sampai 2 orang. Sedangka terlama adalah 4,62 menit. Hal ini menunjukan bahwa seorang pelanggan menghabiskan waktu sebanyak 1,39 dengan 4,62 menit dalam sistem. Jumlah pelanggan dalam antrian (Lq) adalah 0,14 sampai dengan 0,48 atau sebanyak 1 orang. Dan waktu yang dihabiskan oleh seorang pelanggan untuk menunggu dalam antrian (Wq) yang tercepat adalah 0,37 menit sedangkan waktu terlama pelanggan menunggu dalam antrian adalah 1,3 menit. Berdasarkan pengamatan selama satu minggu (7 hari kerja) dapat dianalisa bahwa Kantor Pos Cabang Tenggarong mempunyai tiga (3) server tersedia dan hanya dua yang beroprasi dengan nilai rata – rata kedatangan konsumen per hari 196 orang dengan kapasitas pelayanan sebanyak 28 orang per jamnya dari hasil analisis sistem pada tabel 5.8 mempunyai tingkat utilitas/kesibukan yang tinggi dari jam 08.00 sampai dengan jam 12.00 yang berada pada kisaran 52% - 55% dari waktu kerjanya. Nilai tersebut masih jauh dari angka 1 atau 100% hal tersebut menunjukan bahwa antrian yang terjadi pada Kantor Pos Cabang Tenggarong tidak terlalu panjang. Sedangkan utulitas yang rendah terjadi pada jam 13.00 sampai dengan jam 15.00 dengan nilai 46% - 39%. Hal tersebut menunjukan bahwa tingkat kedatangan jauh lebih rendah dari sebelumnya. Sumber : Diolah peneliti Dari tabel diatas dapat dilihat bahwa jumlah waktu rata – rata yang dihabiskan pelanggan dalam antrian (Wq) yang terkecil adalah 0,37 menit atau 22 detik pada jam 08.00 – 09.00. Hal ini menunjukan bahwa waktu yang dihabiskan pelanggan menunggu dalam antrian pada jam tersebut tidak lama hal ini dikarekan kecepatan pelayanan dari server jauh lebih cepat dibandingkan jam lainnya. Tabel 7.Waktu Rata – Rata Yang Dihabiskan Dalam antrian Tabel 7.Waktu Rata – Rata Yang Dihabiskan Dalam antrian Periode Waktu (Jam) Jumlah Rata – Rata Yang Menunggu Dalam Antrian 08.00 - 09.00 0,37 09.00 - 10.00 0,86 Berdasarkan hasil anlisa tersebut, guna memudahkan dalalm mengadakan pemahaman dan pembahasan, maka perhitungan hasil analisis dimasukan kedalam tabel yang sama kemudian dibandingkan dengan priode waktu lainnya. Tabel 8 . Hasil Analisis Dengan 2 Server Pelayanan Periode Waktu ƛ µ M Po P Ls Wq Minutes Ws Minutes Cq Cs 08.00 - 09.00 61 59 2 0.32 0.52 1.41 1.39 0.38 48.025 62.198 09.00 - 10.00 30 28 2 0.3 0.54 1.50 3 0.86 48.768 63.456 10.00 - 11.00 22 20 2 0.29 0.55 1.58 4.3 1.3 49.396 64.475 11.00 - 12.00 21 20 2 0.31 0.53 1.45 4.14 1.14 48.333 62.727 12.00 - 13.00 19 18 2 0.31 0.53 1.46 4.62 1.29 48.443 62.913 13.00 - 14.00 22 24 2 0.37 0.46 1.16 3.16 0.66 46.197 58.764 14.00 – 15.00 21 27 2 0,44 0.39 0.92 2.62 0.4 44.755 55.418 b D H il P li i S l h Di l h Tabel 8 . Hasil Analisis Dengan 2 Server Pelayanan Sumber Data : Hasil Penelitian Setelah Diolah JEMI Vol.22/No.1/JUNI/2022 24 2. Evaluasi Sistem Antrian Berdasarkan hasil dari pembahasan sebelumnya, dan dari salah satu tujuan pada penelitian ini yaitu untuk mengetahui jumlah teller yang efektif pada sistem transaksi teller disaat sibuk dan tidak sibuk. Maka diperlukan evaluasi sistem terhadap jumlag fasilitas teller yang tersedia, seperti yang diketahui bahwa Kantor Pos Cabang Tenggarong memili 3 (tiga) dari 2 (dua) server yang beroprasi, dengan demikian diperlukan analisis terhadap waktu pelayanan, jumlah antrian dan biaya yang ditimbulkan apabila menambah satu orang pekerja teller lagi, dengan perbandingan pada Tabel 9. Pada waktu sibuk yaitu jam 08.00 sampai jam 12.00 berdasarka rata – rata kedatanga per hari menunjukan bahwa probalitas tidak ada konsumen dalam sistem (Po) ada kecil 0,32% sampai dengan 0,29 atau 29%. Hal ini JEMI Vol.22/No.1/JUNI/2022 25 Tabel 9. Analisis Antrian Dengan Penambahan 3 Fasilitas Tabel 9. Analisis Antrian Dengan Penambahan 3 Fasilitas Periode Waktu ƛ µ M Po P Ls Wq Minutes Ws Minutes Cq Cs 08.00 - 09.00 61 59 3 0.35 0.34 1.09 1.07 0.05 64.995 79.168 09.00 - 10.00 30 28 3 0.34 0.36 1.13 2.26 0.12 65.103 79.791 10.00 - 11.00 22 20 3 0.33 0.37 1.17 3.18 0.18 65.193 80.273 11.00 - 12.00 21 20 3 0.35 0.35 1.11 3.16 0.16 65.040 79.434 12.00 - 13.00 19 18 3 0.34 0.35 1.11 3.51 0.18 65.056 79.526 13.00 - 14.00 22 24 3 0.4 0.31 0.95 2.59 0.09 64.726 77.292 14.00 – 15.00 21 27 3 0,46 0.26 0.79 2.27 0.05 64.516 75.178 Sumber Data : Hasil Penelitian Diolah Dari hasil analisis pada tabel 9 mempunyai tingkat utilitas/kesibukan yang berada dalam kisaran 34% sampai 37% dari waktu kerjanya. Sedangkan utilitas yang rendah terjadi pada jam 13.00 sampai jam 15.00 dengan nilai 31% sampai 26%. Pada waktu sibuk yaitu jam 08.00 sampai jam 12.00 berdasarkan rata – rata kedatangan perhari menunjukan bahwa probabilitas tidak ada nasabah dalam sistem (Po) adalah kecil, 0,35 atau 35% sampai dengan 0,33 atau 33% sedangkan pada jam 13.00 sampai jam 15.00 probabilitasnya 40% sampai 44%. sebanyak 1,07 sampai dengan 3,51 menit dalam sistem (lama konsumen dalam antrian ditambah lama konsumen sedang dilayani). Jumlah konsumen dalam antrian (Lq) adalah 0,02 sampai dengan 0,07 atau sebanyak 1 orang, dan waktu yang dihabiskan oleh seorang konsumen untuk menunggu dalam antrian (Wq) yang tercepat adalah 0,05 menit. Sedangkan waktu terlama konsumen menunggu antrian adalah 0,18 menit. 2. Evaluasi Sistem Antrian Berdasarkan hasil analisis tersebut guna memudahkan dalam mengadakan pemahaman dan pembahasan, maka perhitungan hasil analisis dimasukan kedalam tabel yang sama kemudian dibandingkan. Adapun hasil analisis sistem antrian dengan menggunakan 2 (dua) server dan 3 (tiga) server pada bagian teller bisa dilihat pada tabel berikut ini : Jumlah konsumen dalam sistem (Ls) pada jam 08.00 sampai jam 15.00 adalah sebanyak 1 (satu) orang waktu yang dihabiskan oleh seorang konsumen dalam sistem (Ws) tercepat adalah 1.07 menit. Sedangkan terlama adalah 3,51 menit. Hal ini menunjukan bahwa seorang konsumen menhabiskan waktu Tabel 10. Perbandingan Total Biaya Setelah Penambahan Satu Teller Priode waktu 2 Server 3 Server Selisih Biaya Keterangan Jumlah nasabah dalam sistem Total biaya Jumlah nasabah dalam system Total biaya 08.00- 1,41 62.198 1,09 79.970 16.970 Tidak efektif Tabel 10. Perbandingan Total Biaya Setelah Penambahan Satu Teller JEMI Vol.22/No.1/JUNI/2022 26 09.00 09.00- 10.00 1,50 63.456 1,13 79.791 16.335 Tidak efektif 10.00- 11.00 1,58 64.475 1,17 80.273 15.798 Tidak efektif 11.00- 12.00 1,45 62.727 1,11 79.434 16.707 Tidak efektif 12.00- 13.00 1,46 62.913 1,11 79.526 16.613 Tidak efektif 13.00- 14.00 1,16 58.764 0,95 77.292 18.528 Tidak efektif 14.00- 15.00 0,92 55.418 0,79 75.178 19.760 Tidak efektif Sumber : Diolah Peneliti yang beroprasi. Maka hipotesis yang telah dikemukakan sebelumnya yang menyatakan “Jika sistem transaksi menggunakan struktur antrian Multi Channel - Single Phase, maka efektitivitas pada sistem transaksi akan sangat sulit berjalan dengan baik, dan nantinya dapat menyebabkan antrian yang panjang atau tidak efektif. Dapat diterima dalam arti signifikan dan terbukti kebenarannya. PENUTUP yang beroprasi. Maka hipotesis yang telah dikemukakan sebelumnya yang menyatakan “Jika sistem transaksi menggunakan struktur antrian Multi Channel - Single Phase, maka efektitivitas pada sistem transaksi akan sangat sulit berjalan dengan baik, dan nantinya dapat menyebabkan antrian yang panjang atau tidak efektif. Dapat diterima dalam arti signifikan dan terbukti kebenarannya. PENUTUP Berdasarkan pada pembahasan sebelumnya bahwa PT. pos Indonesia cabang Tenggarong memiliki standar waktu pelayanan pada bagian pelayanan yaitu selama 4 menit waktu untuk melayani nasabah yang bertransaksi. Dari hasil analisis yang telah didapat pada tabel 9 dengan 2 server pelayanan, waktu yang dihabiskan oleh seorang nasabah dalam sistem (Ws) tercepat adalah 1,39 menit dan terlama 4,62 menit. Sedangkan dengan 3 server pelayanan, waktu yang dihabiskan oleh seorang nasabah dalam sistem (Ws) tercepat adalah 1,07 menit dan terlama adalah 3,51 menit. Kesimpulan Jiak sistem transaksi tidak menggunakan struktur antrian Multichannel – Single Phase maka efektivitas pada sistem antrian akan sulit berjalan dengan baik dan nantinya dapat menyebabkan antrian yang panjang atau tidak efektif. Berdasarkan analisis hipotesis yang dikemukakan dapat diterima dan terbukti kebenarannya. 2. y Penerapan sistem pelayanan terhadap konsumen yang menggunakan nomor antrian perlu diperhatikan khususnya nasabah yang ingin mengirim barang pada pihak PT. Pos Indonesia. Hal inilah yang perlu dipertimbangkan kembali oleh PT. Pos Indonesia untuk kebijakan selanjutnya terhadap pelayanan server dan sebagai, sistem pelayanan yang akan dilakukan secara bergantian yaitu setiap 3 sampai 4 orang dengan jenis pelayanan untuk mengirimkan barang selajutnya giliran melayani konsumen dengan dengan menggunakan nomor antrian 3 sampai 4 orang. Hal tersebut bertujuan untuk menimalisir konsumen yang sudah lama mengantri maupun yang baru saja mengambil nomor antrian keluar dari sistem karena nomor antrian yang terakhir dipanggil dengan yang masih menuggu panggilan nomor antrian masih sangat jauh. Kesimpulan Berdasarkan hasil analisis data dan pembahasan, maka kesimpulan yang didapat peneliti kemukakan adalah : Pada tabel 9 diatas terlihat bahwa selisih biaya pada setiap jam operasional menjadi lebih besar, dengan adanya penambahan 1 teller pada PT. Pos Indonesia Cabang Tenggarong dinyatakan tidak efektif karena kantor pos harus menambah baiaya pelayanan, sedangkan jumlah orang mengantri dalam sistem dan perbedaan waktu pelayanan hanya selisih 30 detik sampai 1 menit. Dari uraian diatas dapat diketahui bahwa PT. Pos Indonesia di Tenggarong lebih efektif jika menggunakan struktur antrian Multi channel – Single Phase atau antrian jalur berganda atau satu tahap pelayanan dengan 2 server pelayanan atau 2 teller 1. Sistem antrian yang diterapkan pada Kantor Pos Cabang Tenggarong Multichanel – single Phase atau M/M/S yaitu sistem antrian terdapat beberapa loket pembayaran yang bertugas melayani konsumen namun fase yang dilalui hanya satu tahap saja. 2. Pada Kantor Pos Cabang Tenggarong terdapat fasilitas loket pembayaran yang berfungsi melayani transaksi pembayaran dari konsumen pada waktu tertentu. Pada jam istirahat hanya dua loket saja yang melayani transaksi. JEMI Vol.22/No.1/JUNI/2022 27 3. Hasil analisis sistem kerja antrian pada teller Kantor Pos Cabang Tenggarong diperoleh jumlah rata – rata konsumen yang berada dalam sistem antrian teller sebanyak 1 atau sampai 2 orang ditambah dengan yang sedang mendapatkan pelayanan. Rata – rata waktu yang diperlukan konsumen untuk mendapatkan pelayanan adalah 0,61 menit sampai 3,37 menit menggunakan dua server. atau acak hanya pada hari tertentu namun dapat diprediksi bila kedatangan konsumen akan meningkat pada kondisi minggu pertama dan kedua awal bulan khusunya hari senin, selasa, dan jumat maka pihak PT. Pos Indonesia di Tenggarong perlu mengantisipasi akan jumlah kedatangan pada hari – hari tersebut dengan membuka tiga server yang beroprasi dari jam sibuk yaitu 08.00 – 12.00. 4. Berdasarkan hipotesis dikemukakan yaitu “Jika sitem transaksi menggunakan struktur antrian Multichanel – Single Phase maka efektivitas pada sistem transaksi dapat tercapai. Jiak sistem transaksi tidak menggunakan struktur antrian Multichannel – Single Phase maka efektivitas pada sistem antrian akan sulit berjalan dengan baik dan nantinya dapat menyebabkan antrian yang panjang atau tidak efektif. Berdasarkan analisis hipotesis yang dikemukakan dapat diterima dan terbukti kebenarannya. 4. Berdasarkan hipotesis dikemukakan yaitu “Jika sitem transaksi menggunakan struktur antrian Multichanel – Single Phase maka efektivitas pada sistem transaksi dapat tercapai. Saran-saran Berdasarkan kesimpulan diatas penulis dapat memberikan saran- saran sebagai berikut : 1. Penggunaan dua server pelayanan dinilai sudah cukup apabila dilihat dari hasil analisis total rata – rata kedatangan sebanyak 209 orang perharinya. Akan tetapi mengingat kedatangan kosumen bersifat random JEMI Vol.22/No.1/JUNI/2022 28 DAFTAR PUSTAKA Heizer, Jay. Dan Barry Render, Manajemen Operasi: Manajemen Keberlangsungan dan Rantai Pasokan Edisi 11. Jakarta:Salemba Empat. Badruddin.(2013). Dasar-Dasar Manajemen, Bandung, Penerbit Alfabeta. Gitosudarmo, Indriyo. (2002). Manajemen Operasi Edisis Kedua. Yogyakarta: Badan Penerbit Fakultas Ekonomi– UGM, Yogyakarta. Kusumawardai, Angraini Susanti. (2014) Analisis Sistem Antrian Pelayanan Di PT. Pos Indonesia Kantor Pos Indonesia Semarang II. Jurnal Gaussian Volume 3 Nomor 4. Semarang Universitas Dipenogoro. Ginting, Petrus Lanjong. (2013). Analisis Sistem Antrian Dan Optimalisasi Layanan Teller (Studi Kasus pada Bank X di Kota Semarang). Skripsi Mahasiswa Fakultas Ekonomika Dan Bisnis Universitas Dipenogoro Semarang. Prasetya, Hery. Dan Fitri Lukiastuti. (2011), Manajemen Operasi. Yogyakarta: Cennter For Pulishing Service (CAPS). Hedayanti. (2014). Penerapan Sistem Antrian Sebagai Upaya Mengoptimalkan Pelayanan Terhadap Pasien Pada Loket Pendaftaran Di Puskesmas Rapak mahang Tenggarong. Skripsi mahasiswa Fakultas Ekonomi dan Bisnis Universitas Kutai Kartanegara. Prasetya, Hery. Dan Fitri Lukiastuti (2011). Manajemen Oprasi. Yogyakarta Center For Pulishing service (CAPS). Taufik, Rustam (2012). Analisis Penerapan Sistem Antrian Model M/M/S Pada PT. Bank Negara Indonesia Persero tbk. Kantor Cabang Pembantu Universitas Hassanudin Makassar. Skripsi mahasiswa Fakultas Ekonomi dan Bisnis Universitas Hassanudin . Handoko. T. Hani (2011). Dasar – dasar Manajemen Produksi Dan Operasi. Badan Penerbit Fakultas Ekonomi – UGM, Yogyakarta. Hardiyani, Rini. (2013). Analisis Penerapan Teori Antrian Pada Sistem Pembayaran Supermarket Di Golden Market Jember.Skripsi Mahasiswi Fakultas Ekonomi, Universitas Jember. H I ti (2011) M j Handoko. T. Hani (2011). Dasar – dasar Manajemen Produksi Dan Operasi. Badan Penerbit Fakultas Ekonomi – UGM, Yogyakarta. Hardiyani, Rini. (2013). Analisis Penerapan Teori Antrian Pada Sistem Pembayaran Supermarket Di Golden Market Jember.Skripsi Mahasiswi Fakultas Ekonomi, Universitas Jember. Hasan, Irmayati. (2011) Manajemen Operasional. Malang: UIN Maliki Press JEMI Vol.22/No.1/JUNI/2022 29
https://openalex.org/W3154016041	https://www.frontiersin.org/articles/10.3389/fphys.2021.640295/pdf	English	null	Ganglionated Plexi Ablation Suppresses Chronic Obstructive Sleep Apnea-Related Atrial Fibrillation by Inhibiting Cardiac Autonomic Hyperactivation	Frontiers in physiology	2,021	cc-by	12,792	ORIGINAL RESEARCH published: 09 April 2021 doi: 10.3389/fphys.2021.640295 Ling Zhang 1†, Yankai Guo 1,2,3†, Jiasuoer Xiaokereti 1,2†, Guiqiu Cao 3, Hongliang Li 4, Huaxin Sun 1,2, Kai Li 1,2, Xianhui Zhou 1,2* and Baopeng Tang 1,2* Ling Zhang 1†, Yankai Guo 1,2,3†, Jiasuoer Xiaokereti 1,2†, Guiqiu Cao 3, Hongliang Li 4, Huaxin Sun 1,2, Kai Li 1,2, Xianhui Zhou 1,2* and Baopeng Tang 1,2* 1 Xinjiang Key Laboratory of Cardiac Electrophysiology and Cardiac Remodeling, The First Afﬁliated Hospital of Xinjiang Medical University, Urumqi, China, 2 Cardiac Pacing and Electrophysiology Department, The First Afﬁliated Hospital of Xinjiang Medical University, Urumqi, China, 3 Department of Cardiology, The Fifth Afﬁliated Hospital of Xinjiang Medical University, Urumqi, China, 4 Section of Endocrinology and Diabetes, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States Keywords: atrial ﬁbrillation, obstructive sleep apnea, sympathovagal hyperactivity, ganglionated plexi (GP), ablation Edited by: Jose F. Rodriguez Matas, Politecnico di Milano, Italy Background: Previous studies have reported that right pulmonary artery ganglionated plexi (GP) ablation could suppress the onset of atrial ﬁbrillation (AF) associated with obstructive sleep apnea (OSA) within 1 h. Reviewed by: Richard Gary Trohman, Rush University, United States Matthew W. Kay, George Washington University, United States Objective: This study aimed to investigate the effect of superior left GP (SLGP) ablation on AF in a chronic OSA canine model. Methods and Results: Fifteen beagles were randomly divided into three groups: control group (CTRL), OSA group (OSA), and OSA + GP ablation group (OSA + GP). All animals were intubated under general anesthesia, and ventilation-apnea events were subsequently repeated 4 h/day and 6 days/week for 12 weeks to establish a chronic OSA model. SLGP were ablated at the end of 8 weeks. SLGP ablation could attenuate the atrial effective refractory period (ERP) reduction and decrease ERP dispersion, the window of vulnerability, and AF inducibility. In addition, chronic OSA leads to left atrial (LA) enlargement, decreased left ventricular (LV) ejection fraction, glycogen deposition, increased necrosis, and myocardial ﬁbrosis. SLGP ablation reduced the LA size and ameliorated LV dysfunction, while myocardial ﬁbrosis could not be reversed. Additionally, SLGP ablation mainly reduced sympathovagal hyperactivity and post-apnea blood pressure and heart rate increases and decreased the expression of neural growth factor (NGF), tyrosine hydroxylase (TH), and choline acetyltransferase (CHAT) in the LA and SLGP. After SLGP ablation, the nucleotide-binding oligomerization domain (NOD)-like receptor signaling pathway, cholesterol metabolism pathway, and ferroptosis pathway were notably downregulated compared with OSA. Correspondence: Xianhui Zhou zhouxhuiyf@163.com Baopeng Tang tangbaopeng1111@163.com †These authors share ﬁrst authorship Specialty section: This article was submitted to Cardiac Electrophysiology, a section of the journal Frontiers in Physiology Received: 11 December 2020 Accepted: 02 March 2021 Published: 09 April 2021 Edited by: Edited by: Jose F. Rodriguez Matas, Politecnico di Milano, Italy Citation: Zhang L, Guo Y, Xiaokereti J, Cao G, Li H, Sun H, Li K, Zhou X and Tang B (2021) Ganglionated Plexi Ablation Suppresses Chronic Obstructive Sleep Apnea-Related Atrial Fibrillation by Inhibiting Cardiac Autonomic Hyperactivation. Front. Physiol. 12:640295. doi: 10.3389/fphys.2021.640295 Conclusions: SLGP ablation suppressed AF in a chronic OSA model by sympathovagal hyperactivity inhibition. However, there were no signiﬁcant changes in myocardial ﬁbrosis. April 2021 \| Volume 12 \| Article 640295 Frontiers in Physiology \| www.frontiersin.org 1 GPA Inhibit COSA-Related AF Zhang et al. Blood Gases Blood gases were determined at baseline and at the 4th, 8th, and 12th week before 1 min of apnea. At the same time, after 1 min of apnea in the 12th week of OSA simulation among the three groups, blood gases were also determined. Oxygen partial pressure (PaO2), carbon dioxide pressure (PaCO2), and pH were detected within 1 min once the sample was drawn with an i-STAT300 Analyzer (Abbott Laboratories, USA). However, the long-term eﬀects of GP ablation on chronic OSA-induced AF and the underlying mechanisms of AF suppression have not yet been clearly elucidated. We hypothesized that GP ablation could suppress chronic OSA- related AF by inhibiting cardiac autonomic hyperactivation. In our current study, we constructed a chronic OSA model in canines and explored the long-term eﬀects and underlying mechanisms of SLGP ablation on the development and progression of AF. METHODS g y p The atrial eﬀective refractory period (ERP), ERP dispersion (dERP), AF inducibility, AF duration, sinus node recovery time (SNRT), and window of vulnerability (WOV) were determined at baseline and at the 4th, 8th, and 12th week. Programmed stimulation at the high right atrium (HRA) was administered with eight consecutive stimuli (S1–S1 cycle length 330 ms) followed by premature stimuli (S1–S2) at a 4-fold threshold. The S1 to S2 interval gradually decreased from 180 ms initially in decrements of 10–5 ms until capture no longer occurred. Burst stimulation was performed with consecutive bursts of rapid atrial pacing (S1-S1 cycle length 50 ms, 8 V, lasting 10 s). AF was deﬁned as irregular atrial beats (≥500 bpm, lasting ≥5 s) with irregular atrioventricular (AV) conduction (Gao et al., 2015; Yu All animal protocols were approved by the Ethics Committee of Animal Experiments of the First Aﬃliated Hospital of Xinjiang Medical University (permit number: IACUC201902-K04), which strictly complied with the requirements in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH Publication No. 85-23, revised 1996). All animals were provided by Nanjing Yadong Experimental Animal Research Center [permit number: SCXK (SU): 2013-0001]. Programmed Stimulation At baseline and at the 4th, 8th, and 12th week, the femoral artery and vein were cannulated. Standard limb lead electrocardiograms, intracardiac electrophysiological recordings, and arterial blood pressure (BP) were continuously monitored (LEAD 7000, Jinjiang Inc., Chengdu, China) throughout the experiment. The SLGP was ablated at the end of the 8th week through left thoracotomy at the fourth intercostal space. Establishment of the OSA Model The OSA model was established by blocking the endotracheal cannula at the end of exhalation, as described previously (Zhao et al., 2014; Gao et al., 2015). From the 1st week to the 5th week, the spontaneous breathing time decreased weekly from 9 to 5 min and was maintained for 5 min until the end of the experiment. The apnea time always lasted 1 min. This breath–sleep apnea cycle event was repeated for 4 h/day, 6 days/week, for a total of 12 weeks (Figure 1A). p The role of ganglionated plexi (GP), known as the intrinsic cardiac autonomic nervous system, containing abundant nerve axons, interconnected neurons, and autonomic ganglia clusters, embedded into the epicardial fat pads, has been increasingly recognized (Stavrakis and Po, 2017). Several studies have reported that hyperactivity of the ganglionated plexi (GP) promotes the initiation and maintenance of AF, and both animal (Hou et al., 2007; Lu et al., 2008; Ghias et al., 2009; Yu et al., 2017c) and clinical studies (Scanavacca et al., 2006; Katritsis et al., 2008; Po et al., 2009) have shown that AF can be inhibited by GP ablation. Ghias et al. (2009) reported that right pulmonary artery GP ablation could decrease 1-h apnea-induced AF by suppressing anterior right ganglionated plexus (ARGP) activity. Yu et al. (2017c) also revealed that hyperactivity of superior left GP (SLGP) promoted the initiation and maintenance of AF, and AF inducibility could be suppressed by low-level transcutaneous electrical stimulation (LLTS) in an acute intermittent hypoxia model in dogs. All of these studies demonstrated that GP inhibition/ablation could suppress acute OSA-induced AF by inhibiting the hyperactivity of the autonomic nervous system (ANS). Experiment Protocol Fifteen beagles were randomly divided into three groups: OSA (OSA for 12 weeks with sham GP ablation, n = 5); OSA + GP (OSA for 12 weeks, GP ablation was performed at the end of the 8th week through left thoracotomy at the fourth intercostal space, n = 5); and CTRL (sham OSA without GP ablation, n = 5). For the CTRL, we gave intubation and anesthesia 4 h a day without blocking the endotracheal cannula and GP ablation. Figure 1A illustrates the protocol. INTRODUCTION Virbac SA, France) and xylazine (5 mg/kg, Huamu Animal Health Care Products Co., Ltd., China) was used to induce anesthesia. After the eyelash reﬂex disappeared, the trachea was intubated. During the experiment, 3% sodium pentobarbital solution was continuously pumped intravenously at 2 ml/h to maintain anesthesia. Obstructive sleep apnea (OSA) is one of the most common forms of sleep breathing disorders and may aﬀect ∼24% of men and 9% of women between 30 and 60 years of age (Calkins et al., 2017). A previous study clearly identiﬁed that patients with OSA have a 4- fold risk of developing atrial ﬁbrillation (AF) (Mehra et al., 2006), a higher risk of AF recurrence after cardioversion and catheter ablation (Kanagala et al., 2003; Tang et al., 2009; Fein et al., 2013), and a weak response to anti-arrhythmic drugs (Matiello et al., 2010). Therefore, it is an urgent problem to further explore an eﬀective treatment for patients with OSA-associated AF. Neural Activity Recording y g Neural activities of the left vagal nerve (LVN) and left stellate ganglion (LSG) were recorded among the three groups at the 12th week, as described previously (Zhou et al., 2016; Zhang et al., 2018). All the animals were anesthetized during the neural activity recordings. Brieﬂy, the LVN and LSG were exposed via a left vertical incision in the supraclavicular fossa and bluntly dissected free from surrounding tissues with a glass dissecting needle. Neural activities were recorded through headstage electrodes into the LVN and SLGP, and nerve signals were analyzed with the Analysis Module of Lab Chart 8.0/proV7 Heart Rate Variability Analysis y y Five minutes of ECG recordings were obtained when the dog was awake and quiet to perform heart rate variability (HRV) analysis to reveal changes in ANS activity. A data acquisition system (LabChart Pro, AD Instruments Ltd) was used to analyze its frequency domain indicators: low-frequency power (LF, 0.04–0.15 Hz, assumed to have a dominant sympathetic component), high-frequency power (HF, 0.15–0.40 Hz, reﬂecting cardiac parasympathetic nerve activity), and the low-frequency- to-high-frequency power ratio (LF/HF ratio, quantifying the changing relationship between sympathetic and parasympathetic nerve activities). Three consecutive measurements were averaged for each variable. SLGP Location and Ablation Thoracotomy was performed at the left fourth intercostal space to expose the pericardium, and then the SLGP was found to be located adjacent to the LSPV–LA junction between the LAA and left pulmonary artery (Figures 2Aa,b). High-frequency stimulation (20 Hz, 0.1 ms pulse width, 0.6–4.5 V) (Grass S88, Astro-Med Inc., West Warwick, RI, USA) was administered until the R–R interval increased by 50% or a 2:1 atrioventricular block emerged, indicating that the position of the SLGP was correct (Po et al., 2009). Ablation was performed using an irrigated large- tip (3.5 mm) electrode catheter (Biosense-Webster Inc. Diamond Bar, CA, USA). During ablation energy delivery, marked slowing of the sinus rate and/or AV conduction was observed (Cooper et al., 1980; Schauerte et al., 2000). When applying 12 V to stimulate the SLGP with an electrode catheter after energy delivery, the absence of slowing of the sinus rate and/or AV conduction indicated that the ablation was complete (Lu et al., 2010) (Figures 2Ac–e). Animal Preparation Fifteen adult male beagles weighing 15–18 kg were investigated in this study. Intramuscular injection of Zoletil (0.1 mg/kg, April 2021 \| Volume 12 \| Article 640295 Frontiers in Physiology \| www.frontiersin.org 2 GPA Inhibit COSA-Related AF Zhang et al. FIGURE 1 \| (A) Study protocol performed on the dogs. (B) Changes in blood gas among the three groups. (a) After 1 min of apnea in the 12th week of simulating OSA; (b) During the 12-week period of OSA simulation before apnea. P < 0.05, *P < 0.01, n = 5. OSA, obstructive sleep apnea; GP, ganglionated plexus; CTRL, control; BS, baseline; EP, electrophysiology; ECHO, echocardiography; Pre-, pre-apnea; Post-, post-apnea; W, week. in blood gas among the three groups. (a) After 1 min of apnea in the 12th week of simulating P < 0.05, *P < 0.01, n = 5. OSA, obstructive sleep apnea; GP, ganglionated plexus; CTRL, hy; Pre-, pre-apnea; Post-, post-apnea; W, week. FIGURE 1 \| (A) Study protocol performed on the dogs. (B) Changes in blood gas among the three groups. (a) After 1 min of apnea in the 12th week of simulating OSA; (b) During the 12-week period of OSA simulation before apnea. P < 0.05, *P < 0.01, n = 5. OSA, obstructive sleep apnea; GP, ganglionated plexus; CTRL, control; BS, baseline; EP, electrophysiology; ECHO, echocardiography; Pre-, pre-apnea; Post-, post-apnea; W, week. et al., 2017b). The WOV, deﬁned as the diﬀerence between the maximum and minimum S1–S2 intervals that induced AF, was adopted as an indicator of AF inducibility (Gao et al., 2015; Yu et al., 2017b). The dERP was deﬁned as the diﬀerence between the longest and the shortest value of the ERP at six recording sites, including HRA, left atrial appendage (LAA), right superior pulmonary vein (RSPV), right inferior pulmonary vein (RIPV), left superior pulmonary vein (LSPV), and left inferior pulmonary vein (LIPV). (2D) and M-mode imaging were performed at baseline and at the 4th, 8th, and 12th week. Right atrial end-diastolic diameter (RAd), left atrial end-diastolic diameter (LAd), left ventricular end-diastolic diameter (LVEDD), and left ventricular ejection fraction (LVEF) were determined. Measurements were averaged over three to ﬁve consecutive cardiac cycles, and mean values were used for statistical analysis. Echocardiography Echocardiographic examination was conducted with Doppler echocardiography (Sonos 5500, Philips Ultrasound, USA) at a 3.5-MHz ultrasound probe, and transthoracic 2-dimensional April 2021 \| Volume 12 \| Article 640295 Frontiers in Physiology \| www.frontiersin.org 3 Zhang et al. GPA Inhibit COSA-Related AF FIGURE 2 \| Changes in SLGP before and after ablation. (A) Location of the SLGP. The place marked with an arrow is the position of the SLGP (a,b); before and after SLGP stimulation, different types of arrhythmias were observed (c–e). (B) Silver staining of the SLGP. (C) Representative images of HE staining for SLGP: (a) dozens of neurons in SLGP were observed in subepicardial fat tissue; (b) several neurons were observed between cardiac muscle; (c) The nerve ﬁbers and neurons in a nerve bundle; (d) the nerve ﬁber bundle; (e) disorder of subepicardial collagen tissue structure after SLGP ablation; (f) ganglion cells developed vacuolar degeneration after SLGP ablation. SVC, superior vena cava; IVC, inferior vena cava; RSPV, right superior pulmonary vein; RIPV, right inferior pulmonary vein; LSPV, left superior pulmonary vein; LIPV, left inferior pulmonary vein; SLGP, superior left ganglionated plexus; LOM, ligament of Marshall; HRA, high right atrium; HE, hematoxylin and eosin; other abbreviations as in Figure 1. FIGURE 2 \| Changes in SLGP before and after ablation. (A) Location of the SLGP. The place marked with an arrow is the position of the SLGP (a,b); before and after SLGP stimulation, different types of arrhythmias were observed (c–e). (B) Silver staining of the SLGP. (C) Representative images of HE staining for SLGP: (a) dozens of neurons in SLGP were observed in subepicardial fat tissue; (b) several neurons were observed between cardiac muscle; (c) The nerve ﬁbers and neurons in a nerve bundle; (d) the nerve ﬁber bundle; (e) disorder of subepicardial collagen tissue structure after SLGP ablation; (f) ganglion cells developed vacuolar degeneration after SLGP ablation. SVC, superior vena cava; IVC, inferior vena cava; RSPV, right superior pulmonary vein; RIPV, right inferior pulmonary vein; LSPV, left superior pulmonary vein; LIPV, left inferior pulmonary vein; SLGP, superior left ganglionated plexus; LOM, ligament of Marshall; HRA, high right atrium; HE, hematoxylin and eosin; other abbreviations as in Figure 1. Immunohistochemistry y Tissues sections of the LVN, LSG, SLGP, and LA that were paraﬃn-embedded were used to perform immunohistochemistry (IHC). The avidin–biotin complex method was performed to detect the expression levels of the following proteins: TH (tyrosine hydroxylase, LS C354110, 1:100, LifeSpan BioSciences, Proteomics Analysis Frozen tissue was ground in liquid nitrogen into a cell powder, and lysis buﬀer was added to extract the protein, followed by trypsin digestion and TMT/iTRAQ labeling. Then, an EASY-nLC 1000 UPLC system was used to analyze the expression of each peptide. The resulting tandem mass spectrometry (MS/MS) data were processed using the MaxQuant search engine (v.1.5.2.8) and Paragon protein database. Finally, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to identify enriched pathways. Echocardiography Seattle, WA, USA); CHAT (choline acetyltransferase, LS C79271, 1:100, LifeSpan BioSciences, Seattle, WA, USA); NGF (neural growth factor, LS C388946, 1:100 dilution, LifeSpan BioSciences, Seattle, WA, USA); connexin-40 (Cx40, LS B959, 1:100, LifeSpan BioSciences, Seattle, WA, USA); and connexin-43 (Cx43, LS B9771, 1:100, LifeSpan BioSciences, Seattle, WA, USA). Slices were incubated with diﬀerent antibodies overnight at 4◦C separately and subsequently incubated with peroxidase- conjugated rat anti-rabbit IgG (LS-C60921, LifeSpan BioSciences, Seattle, WA, USA) at 37◦C for 20 min. Finally, a microscope (Leica, Wetzlar, Germany) was used to evaluate the samples. The images were analyzed with Image-Pro Plus 6.0 software (Media Cybernetics, USA). software (Bio Amp; ADInstruments) of PowerLab (Bio Amp; ADInstruments). An ampliﬁer (DP-304; Warner Instruments) was used to amplify nerve signals. Histological Study The SLGP [located at the roof of the left atrium, 1–2 cm medial to the left superior PV (Stavrakis and Po, 2017)], LVN, LSG, and myocardial tissues (SLGP, LA, LAA, RAA, LV, and RV) were collected and ﬁxed in paraformaldehyde after euthanasia. All tissues were embedded in paraﬃn and cut into 4-µm-thick sections. Then, hematoxylin and eosin (HE) staining, Masson’s trichrome staining, periodic acid-Schiﬀ(PAS) staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and silver staining were performed according to the manufacturer’s instructions (Western Biomedical Technology, Hubei, China). A charge-coupled device (CCD) camera attached to an inverted microscope (Zeiss, Germany) was used to capture the image of the stained cells. Image analysis software (Image- Pro Plus: IPP 7.0, Media Cybernetics LP) was used to estimate the area of interstitial ﬁbrosis, glycogen volume fraction of the tissues, and apoptotic rate of cardiomyocytes. Effects of SLGP Ablation on Structural As shown in Figures 2B,C, the structure of the SLGP was shown with silver and HE staining, indicating that the nerve bundle was located in the fat tissue and connective tissue between the cardiac muscle (Figures 2Ba,b). Once the GP was ablated, disorder of the subepicardial collagen tissue structure and the development of ganglion cell vacuolar degeneration could be observed in the SLGP with HE staining (Figures 2Ca–f). Remodeling in Chronic OSA-Induced AF As shown in Figure 6A, after 8 weeks of simulating OSA, the LAd and LVEDD in the OSA and OSA + GP groups were signiﬁcantly increased compared to the CTRL group (P < 0.01), while the LVEF was signiﬁcantly decreased (P < 0.01). Interestingly, 4 weeks after SLGP ablation, decreased LAd and LVEDD and increased LVEF were observed (all P < 0.01). In addition, no signiﬁcant change in RAd was observed among the three groups. Remodeling in Chronic OSA-Induced AF As shown in Figure 3A, the ERP of the HRA (Figure 3Aa) was recorded from the beginning to the end of the study. No diﬀerence in the ERP value was observed among the three groups at baseline (P > 0.05). With the progression of OSA, ERP was gradually and signiﬁcantly shortened at the 4th and 8th week in the OSA group and the OSA + GP group compared to the CTRL group (P < 0.01). Four weeks after SLGP ablation (week 12), ERP in the OSA + GP group gradually returned to the baseline level (P < 0.01). In addition, similar diﬀerences in ERP were also detected at all paced sites among the three groups at week 12. Compared with the CTRL group, the ERP value in the OSA group was obviously decreased, while the ablation of SLGP inhibited this eﬀect (Figures 3Ab–f). To evaluate cardiac injury in chronic OSA, HE staining was performed (Figure 6B). Compared with the CTRL group, considerable fatty inﬁltration of the LAA, extensive ﬁbrosis of the RAA, and mild fatty inﬁltration and ﬁbrosis of the left ventricle (LV) and RV were notably observed in the OSA group. SLGP ablation did not attenuate this structural remodeling within 4 weeks. Because obvious changes were manifested in the LAA, Masson staining, PAS staining, and TUNEL staining were further conducted in LA tissues (Figure 6C). Cardiomyocyte ﬁbrosis, glycogen deposition, and apoptosis were present in the OSA group, and SLGP ablation failed to reverse this negative eﬀect. Blood Gases As shown in Figure 1Ba, after 1 min of apnea in the 12th week of OSA simulation, PaO2 and pH were signiﬁcantly decreased, while PaCO2 was markedly increased (P < 0.05). Moreover, artery blood gases in pre-apnea were also detected at baseline and at the 4th, 8th, and 12th week, and no statistical signiﬁcance in terms of pH, PaCO2, and PaO2 was shown among the three groups at baseline (P > 0.05). As the experiment progressed, PaO2 and pH decreased gradually, while PaCO2 increased notably (P < 0.05) (Figure 1Bb). Additionally, SNRT was also analyzed among the three groups (Figure 4B). From baseline to the 4th week, the value of SNRT was markedly increased in the OSA and OSA + GP groups, and it gradually returned to baseline. Furthermore, the expression levels of Cx43 and Cx40 were also determined with IHC (Figure 5). Compared with the CTRL group, the expression levels of Cx43 and Cx40 were decreased in the OSA group, while SLGP ablation increased their expression in the OSA + GP group. In short, during the early and later period of OSA, hypoxemia, hypercapnia, and acidosis manifested, while SLGP ablation did not alter this hypoxemic environment. In summary, the chronic OSA model is prone to increased AF inducibility, and the eﬀect could be inhibited by SLGP ablation. Statistical Analysis Data are presented as the mean ± SD. Three group comparisons were performed via two-way ANOVA followed by Tukey’s tests. April 2021 \| Volume 12 \| Article 640295 Frontiers in Physiology \| www.frontiersin.org 4 GPA Inhibit COSA-Related AF Zhang et al. signiﬁcantly decreased after 4 weeks of SLGP ablation (P < 0.01) (Figure 3Bb). Analysis of variance with post-hoc Tukey’s test was used to compare continuous variables among diﬀerent time points of apnea. A paired t-test was used for comparisons of BP, HR, and blood gases values before and after apnea was induced. A two- tailed Fisher’s exact test was used to test the enrichment of the diﬀerentially expressed proteins against all identiﬁed proteins. SPSS 19.0 (IBM Corp.) was used for statistical analysis. GraphPad Prism 6.0 was used to draw all the graphics. Statistical signiﬁcance was set at P < 0.05. Furthermore, with the progression of OSA, the AF duration and AF inducibility following S1–S2 stimulation in the OSA group gradually increased, and AF inducibility in the OSA group was as high as 100% at week 12, whereas AF could not be induced with S1–S2 stimulation in the OSA + GP group once the SLGP was ablated at week 8 (Figures 3Bc,d). In addition, AF duration and AF inducibility following S1–S1 burst stimulation in the OSA group were obviously increased, and the change tendency was similar to S1–S2 stimulation (Figures 3Be,f). The AF inducibility was 100% at week 4 until the end of week 12. After SLGP ablation, the AF inducibility via burst pacing in the OSA + GP group was <30%. A representative AF episode is shown in Figure 4A. Frontiers in Physiology \| www.frontiersin.org Effects of SLGP Ablation on Structural In summary, the above results indicated that OSA caused cardiac dysfunction and cardiac remodeling. SLGP ablation treatment ameliorated cardiac dysfunction, but failed to reverse structural remodeling eﬀects within 4 weeks. As shown in Figure 3Ba, the dERP in the OSA group was signiﬁcantly increased compared to that in the CTRL group (P < 0.01) and was obviously decreased in the OSA + GP group. The WOV among the three groups was not signiﬁcantly diﬀerent at baseline (P > 0.05). As OSA was prolonged, the WOV increased notably in the OSA group and the OSA + GP group compared to the CTRL group at the 4th week and 8th week, and it was Effects of SLGP Ablation on the ANS in Effects of SLGP Ablation on the ANS in Chronic OSA-Induced AF As shown in Figure 7A, with the progression of OSA, HR in the OSA and OSA + GP groups was signiﬁcantly increased, reaching April 2021 \| Volume 12 \| Article 640295 Frontiers in Physiology \| www.frontiersin.org 5 GPA Inhibit COSA-Related AF Zhang et al. GURE 3 \| Effect of SLGP ablation on electrical remodeling. (A) Change in the mean ERP at different paced sites among the three groups (a–f). (B) Change in dERP and WOV (b) among the three groups. The mean value of AF duration and AF inducibility (atrial tachyarrhythmia ≥5 s) after 10 S1–S2 pacing (c, d) and burst 1–S1) pacing (e, f) events for each dog. P < 0.05 vs. CTRL, P < 0.01 vs. CTRL, #P < 0.05 vs. OSA, ##P < 0.01 vs. OSA, n = 5. AF, atrial ﬁbrillation; dERP, ective refractory period dispersion; WOV, window of vulnerability; other abbreviations as in Figures 1, 2. GURE 4 \| (A) Typical examples of AF episodes induced by OSA after S1–S2 and burst stimulation among the three groups; (B) Changes in SNRT among the three oups. P < 0.01 vs. CTRL, n = 5. AF, atrial ﬁbrillation; SNRT, sinus node recovery time; other abbreviations as in Figure 1. maximum at the 8th week, and there was no statistically niﬁcant diﬀerence between the OSA and OSA + GP groups. eek 12 HR in the OSA gro p decreased and the scale of the At the same time, changes in systolic blood pressure (SBP) in the OSA and OSA + GP groups at baseline (BS), the 6th h, and the 12th eek ere also detected After 1 min of apnea SBP in the FIGURE 3 \| Effect of SLGP ablation on electrical remodeling. (A) Change in the mean ERP at different paced sites among the three groups (a–f). (B) Change in dERP (a) and WOV (b) among the three groups. The mean value of AF duration and AF inducibility (atrial tachyarrhythmia ≥5 s) after 10 S1–S2 pacing (c, d) and burst (S1–S1) pacing (e, f) events for each dog. P < 0.05 vs. CTRL, P < 0.01 vs. CTRL, #P < 0.05 vs. OSA, ##P < 0.01 vs. OSA, n = 5. AF, atrial ﬁbrillation; dERP, effective refractory period dispersion; WOV, window of vulnerability; other abbreviations as in Figures 1, 2. FIGURE 3 \| Effect of SLGP ablation on electrical remodeling. Effects of SLGP Ablation on the ANS in (A) Change in the mean ERP at different paced sites among the three groups (a–f). (B) Change in dERP (a) and WOV (b) among the three groups. The mean value of AF duration and AF inducibility (atrial tachyarrhythmia ≥5 s) after 10 S1–S2 pacing (c, d) and burst (S1–S1) pacing (e, f) events for each dog. P < 0.05 vs. CTRL, P < 0.01 vs. CTRL, #P < 0.05 vs. OSA, ##P < 0.01 vs. OSA, n = 5. AF, atrial ﬁbrillation; dERP, effective refractory period dispersion; WOV, window of vulnerability; other abbreviations as in Figures 1, 2. FIGURE 4 \| (A) Typical examples of AF episodes induced by OSA after S1–S2 and burst stimulation among the three groups; (B) Changes in SNRT among the three groups. P < 0.01 vs. CTRL, n = 5. AF, atrial ﬁbrillation; SNRT, sinus node recovery time; other abbreviations as in Figure 1. FIGURE 4 \| (A) Typical examples of AF episodes induced by OSA after S1–S2 and burst stimulation among the three groups; (B) Changes in SNRT among the three groups. *P < 0.01 vs. CTRL, n = 5. AF, atrial ﬁbrillation; SNRT, sinus node recovery time; other abbreviations as in Figure 1. At the same time, changes in systolic blood pressure (SBP) in the OSA and OSA + GP groups at baseline (BS), the 6th h, and the 12th week were also detected. After 1 min of apnea, SBP in the OSA group dramatically increased, while the increased value in a maximum at the 8th week, and there was no statistically signiﬁcant diﬀerence between the OSA and OSA + GP groups. At week 12, HR in the OSA group decreased, and the scale of the decrease was larger in the OSA + GP group (P < 0.05). a maximum at the 8th week, and there was no statistically signiﬁcant diﬀerence between the OSA and OSA + GP groups. At week 12, HR in the OSA group decreased, and the scale of the decrease was larger in the OSA + GP group (P < 0.05). a maximum at the 8th week, and there was no statistically signiﬁcant diﬀerence between the OSA and OSA + GP groups. At week 12, HR in the OSA group decreased, and the scale of the decrease was larger in the OSA + GP group (P < 0.05). Effects of SLGP Ablation on the ANS in April 2021 \| Volume 12 \| Article 640295 Frontiers in Physiology \| www.frontiersin.org 6 GPA Inhibit COSA-Related AF Zhang et al. IGURE 5 \| Effect of SLGP ablation on electrical conduction heterogeneity. (A) Representative immunohistochemical staining of Cx43 and Cx40 in LA among the hree groups. (B) Quantitative analysis of Cx43- and Cx40-positive cells among the three groups. P < 0.05, n = 5. Cx43, connexin 43; Cx40, connexin 40; other bbreviations as in Figure 1. IGURE 6 \| Effects of SLGP ablation on structural remodeling. (A) Changes of echocardiographic parameters among the three groups; (B) HE staining of typical myocardial histopathological changes. (C) Myocardial histopathological changes in LA were observed with different staining (a–c): quantitative analysis of Masson taining (b), PAS staining (d), and TUNEL staining (f) among the three groups. P < 0.05 vs. CTRL, P < 0.01 vs. CTRL, ##P < 0.01 vs. OSA. RAd, right atrial nd-diastolic diameter; LAd, left atrial end-diastolic diameter; LVEDD, left ventricular end-diastolic diameter; LVEF, left ventricular ejection fraction. RAA, right atrial ppendage; LV, left ventricular; RV, right ventricular; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; PAS, periodic acid-Schiff; other bbreviations as in Figure 1. k 12 l th th t t BS d 6 h (P 0 01) (Fi 7B) A h i Fi 7D I LF HF d th LF/HF ti FIGURE 5 \| Effect of SLGP ablation on electrical conduction heterogeneity. (A) Representative immunohistochemical staining of Cx43 and Cx40 in LA among the three groups. (B) Quantitative analysis of Cx43- and Cx40-positive cells among the three groups. P < 0.05, n = 5. Cx43, connexin 43; Cx40, connexin 40; other abbreviations as in Figure 1. FIGURE 5 \| Effect of SLGP ablation on electrical conduction heterogeneity. (A) Representative immunohistochemical staining of Cx43 and Cx40 in LA among the three groups. (B) Quantitative analysis of Cx43- and Cx40-positive cells among the three groups. P < 0.05, n = 5. Cx43, connexin 43; Cx40, connexin 40; other abbreviations as in Figure 1. FIGURE 6 \| Effects of SLGP ablation on structural remodeling. (A) Changes of echocardiographic parameters among the three groups; (B) HE staining of typical myocardial histopathological changes. (C) Myocardial histopathological changes in LA were observed with different staining (a–c): quantitative analysis of Masson staining (b), PAS staining (d), and TUNEL staining (f) among the three groups. P < 0.05 vs. CTRL, *P < 0.01 vs. Effects of SLGP Ablation on the ANS in CTRL, ##P < 0.01 vs. OSA. RAd, right atrial end-diastolic diameter; LAd, left atrial end-diastolic diameter; LVEDD, left ventricular end-diastolic diameter; LVEF, left ventricular ejection fraction. RAA, right atrial appendage; LV, left ventricular; RV, right ventricular; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; PAS, periodic acid-Schiff; other abbreviations as in Figure 1. FIGURE 6 \| Effects of SLGP ablation on structural remodeling. (A) Changes of echocardiographic parameters among the three groups; (B) HE staining of typical myocardial histopathological changes. (C) Myocardial histopathological changes in LA were observed with different staining (a–c): quantitative analysis of Masson staining (b), PAS staining (d), and TUNEL staining (f) among the three groups. P < 0.05 vs. CTRL, *P < 0.01 vs. CTRL, ##P < 0.01 vs. OSA. RAd, right atrial end-diastolic diameter; LAd, left atrial end-diastolic diameter; LVEDD, left ventricular end-diastolic diameter; LVEF, left ventricular ejection fraction. RAA, right atrial appendage; LV, left ventricular; RV, right ventricular; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; PAS, periodic acid-Schiff; other abbreviations as in Figure 1. week 12 was lower than that at BS and 6 h (P <0.01) (Figure 7B). In the OSA + GP group, the post-SBP at BS and 6 h was also increased, but no signiﬁcant diﬀerence was observed in the 12th week (Figure 7C). As shown in Figures 7D–I, LF, HF, and the LF/HF ratio were not signiﬁcantly changed at baseline. With the progression of OSA, LF, HF, and the LF/HF ratio were signiﬁcantly increased in the OSA group, and the scale of change in LF was much larger week 12 was lower than that at BS and 6 h (P <0.01) (Figure 7B). In the OSA + GP group, the post-SBP at BS and 6 h was also increased, but no signiﬁcant diﬀerence was observed in the 12th week (Figure 7C). As shown in Figures 7D–I, LF, HF, and the LF/HF ratio were not signiﬁcantly changed at baseline. With the progression of OSA, LF, HF, and the LF/HF ratio were signiﬁcantly increased in the OSA group, and the scale of change in LF was much larger April 2021 \| Volume 12 \| Article 640295 Frontiers in Physiology \| www.frontiersin.org 7 GPA Inhibit COSA-Related AF Zhang et al. FIGURE 7 \| Effect of SLGP ablation on the HRV. (A) Changes in HR among the three groups, P < 0.05 vs. CTRL, P < 0.01 vs. CTRL, #P < 0.05 vs. Effects of SLGP Ablation on the ANS in OSA. Changes in SBP in the OSA group (B) and the OSA + GP group (C) at baseline, 6 h, and the 12th week, P < 0.01 vs. Pre-. Change in LF (D), HF (E), and LF/HF (F) among the three groups at baseline and the 4th, 8th, and 12th week, P < 0.05, P < 0.01. Change in LF (G), HF (H), and LF/HF (I) in each group at baseline and the 4th, 8th, and 12th week; P < 0.05, *P < 0.01. HR, heart rate; SBP, systolic blood pressure; LF, low frequency; HF, high frequency; LF/HF, the ratio between LF and HF; other abbreviations as in Figure 1. FIGURE 7 \| Effect of SLGP ablation on the HRV. (A) Changes in HR among the three groups, P < 0.05 vs. CTRL, P < 0.01 vs. CTRL, #P < 0.05 vs. OSA. Changes in SBP in the OSA group (B) and the OSA + GP group (C) at baseline, 6 h, and the 12th week, P < 0.01 vs. Pre-. Change in LF (D), HF (E), and LF/HF (F) among the three groups at baseline and the 4th, 8th, and 12th week, P < 0.05, P < 0.01. Change in LF (G), HF (H), and LF/HF (I) in each group at baseline and the 4th, 8th, and 12th week; P < 0.05, P < 0.01. HR, heart rate; SBP, systolic blood pressure; LF, low frequency; HF, high frequency; LF/HF, the ratio between LF and HF; other abbreviations as in Figure 1. group were decreased, and the scale of the decrease was larger in the OSA + GP group. than that in HF. In addition, after GP ablation was administered at the end of the 8th week, LF, HF, and the LF/HF ratio were decreased, and HF decreased more. Compared with the 8th week, LF decreased 41.1% at the 12th week in the OSA group, and 67.3% in the OSA + GP group, so compared with the OSA group, LF decreased 1.64-fold in the OSA + GP group; similarly, compared with the OSA group, HF and LF/HF ratio in the OSA + GP group decreased 2.98 and 1.57-fold, respectively. As shown in Figure 9, neural activity of the LVN and LSG was signiﬁcantly increased after simulating OSA at the 12th week in the OSA group compared to the CTRL group. Frontiers in Physiology \| www.frontiersin.org Effects of SLGP Ablation on the ANS in FIGURE 8 \| Dynamic consecutive changes in LF (A), HF (B), and LF/HF (C) among the three groups at baseline and the 4th, 8th, and 12th week, P < 0.01 vs. CTRL, ##P < 0.01 vs. OSA; abbreviations as in Figures 1, 7. FIGURE 9 \| Effect of GP ablation on the neural activity. Representative examples of neural recordings from the LSG and LVN in different groups at the 12th week. LVN, left vagal nerve; LSG, left stellate ganglion; other abbreviations as in Figure 1. FIGURE 9 \| Effect of GP ablation on the neural activity. Representative examples of neural recordings from the LSG and LVN in different groups at the 12th week. LVN, left vagal nerve; LSG, left stellate ganglion; other abbreviations as in Figure 1. FIGURE 9 \| Effect of GP ablation on the neural activity. Representative examples of neural recordings from the LSG and LVN in different groups at the 12th week. LVN, left vagal nerve; LSG, left stellate ganglion; other abbreviations as in Figure 1. Effects of SLGP Ablation on the ANS in However, the hyperactivity of the LVN and LSG was notably inhibited in the OSA + GP group compared to the OSA group. Furthermore, neural indicators that could reﬂect the activity of the ANS were also detected by IHC. As shown in Figures 10, 11, the expression levels of NGF, TH, and CHAT were enhanced in the LA, SLGP, LVN, and LSG in the OSA group, and all of them were decreased in the OSA + GP group. Additionally, we observed consecutive changes in HRV every 4 days throughout the experiment (Figures 8A–C). At the beginning of 12 days, there was no signiﬁcant change in LF, HF, or the LF/HF ratio. From 12 to 56 days, higher LF, HF, and LF/HF ratios were observed in the OSA group and the OSA + GP group than in the CTRL group. From 56 to 84 days, the values of LF, HF, and the LF/HF ratio in the OSA group and the OSA + GP In summary, in the dog model of chronic OSA-induced AF, the activity of the ANS manifested a dynamic change, and the hyperactivity of the ANS could be inhibited by SLGP ablation. Frontiers in Physiology \| www.frontiersin.org April 2021 \| Volume 12 \| Article 640295 8 GPA Inhibit COSA-Related AF Zhang et al. FIGURE 8 \| Dynamic consecutive changes in LF (A), HF (B), and LF/HF (C) among the three groups at baseline and the 4th, 8th, and 12th week, P < 0.01 vs. CTRL, ##P < 0.01 vs. OSA; abbreviations as in Figures 1, 7. FIGURE 8 \| Dynamic consecutive changes in LF (A), HF (B), and LF/HF (C) among the three groups at baseline and the 4th, 8th, and 12th week, P < 0.01 vs. CTRL, ##P < 0.01 vs. OSA; abbreviations as in Figures 1, 7. FIGURE 9 \| Effect of GP ablation on the neural activity. Representative examples of neural recordings from the LSG and LVN in different groups at the 12th week. LVN, left vagal nerve; LSG, left stellate ganglion; other abbreviations as in Figure 1. ne and the 4th, 8th, and 12th week, P < 0.01 vs. FIGURE 8 \| Dynamic consecutive changes in LF (A), HF (B), and LF/HF (C) among the three groups at baseline and the 4th, 8th, and 12th week, P < 0.01 vs. CTRL, ##P < 0.01 vs. OSA; abbreviations as in Figures 1, 7. Proteomics Analysis The results of proteomics analysis are shown in Figure 12. Statistically signiﬁcant diﬀerences were manifested between the OSA and CTRL groups in the interleukin (IL)-17 signaling pathway and indicate its role in autoimmune inﬂammatory disorders (Figure 12A). Moreover, after SLGP was ablated, the OSA + GP group (when compared with the OSA group) showed that the nucleotide-binding oligomerization domain (NOD)- like receptor signaling pathway was notably downregulated. The NOD-like receptors are responsible for detecting speciﬁc pathogen-associated molecules or host-derived damage signals in the cytosol and initiating the innate immune response. Also, cholesterol metabolism pathway and ferroptosis pathway were notably downregulated (Figure 12B). The speciﬁc genes are listed in the Supplementary Data. Major Findings j g To the best of our knowledge, our study is the ﬁrst to report that SLGP ablation inhibits electrical and neural remodeling in chronic OSA-associated AF in canines. We mainly found that (1) from baseline to the 12th week, ERP was gradually shortened and the WOV, AF inducibility, and AF duration were signiﬁcantly increased, while SLGP ablation attenuated this eﬀect and (2) chronic OSA led to left atrial (LA) enlargement, left ventricular (LV) ejection fraction decrease, glycogen deposition, increased necrosis, and myocardial ﬁbrosis. SLGP ablation reduced the LA size and ameliorated LV dysfunction, while myocardial ﬁbrosis could not be reversed. Additionally, SLGP ablation mainly reduced sympathovagal hyperactivity and the post-apnea blood April 2021 \| Volume 12 \| Article 640295 Frontiers in Physiology \| www.frontiersin.org 9 GPA Inhibit COSA-Related AF Zhang et al. FIGURE 10 \| Effect of SLGP ablation on the expression level of TH and CHAT among the three groups. Representative immunohistochemical staining (A) and quantitative analysis of TH (B) and CHAT (C) in LVN and LSG; P < 0.01 vs. CTRL, ##P < 0.01 vs. OSA; TH, tyrosine hydroxylase; CHAT, choline acetyltransferase; abbreviations as in Figures 1, 9. FIGURE 10 \| Effect of SLGP ablation on the expression level of TH and CHAT among the three groups. Representative immunohistochemical staining (A) and quantitative analysis of TH (B) and CHAT (C) in LVN and LSG; P < 0.01 vs. CTRL, ##P < 0.01 vs. OSA; TH, tyrosine hydroxylase; CHAT, choline acetyltransferase; abbreviations as in Figures 1, 9. silence and conduction delay, which are prone to induce AF, could be observed in patients with OSA (Latina et al., 2013). Several studies reported that ERP was signiﬁcantly decreased while the WOV was increased in an acute OSA-induced AF model (Yu et al., 2017a,b). A chronic OSA-induced AF model manifested the same result (Zhao et al., 2014). Furthermore, the AF duration and inducibility were also studied at baseline and the 12th week, whereas the changes during this process were not elucidated in detail (Zhao et al., 2014). silence and conduction delay, which are prone to induce AF, could be observed in patients with OSA (Latina et al., 2013). Several studies reported that ERP was signiﬁcantly decreased while the WOV was increased in an acute OSA-induced AF model (Yu et al., 2017a,b). A chronic OSA-induced AF model manifested the same result (Zhao et al., 2014). Change in Blood Gases in Chronic OSA Hypoxia, hypercapnia, and acidosis manifested in the present study, similar to OSA human features, indicating that our chronic OSA model had been successfully established, consistent with a previous study, which reported changes in blood gases at baseline and week 12 (Yang et al., 2019). Additionally, we also found that hypoxia, hypercapnia, and acidosis presented as early as the 4th week, and this abnormal state became serious at the 8th and 12th week regardless of whether the SLGP was ablated. Furthermore, our study also revealed that AF inducibility was signiﬁcantly decreased under the same hypoxic environment, suggesting that the anti-arrhythmia eﬀect of GP ablation is not mediated by modulating changes in blood gases. Our study ﬁrst reported consecutive changes in electrophysiological parameters at baseline and at the 4th, 8th, and 12th week. Compared to the baseline, ERP decreased signiﬁcantly, while the WOV, AF inducibility, and AF duration increased notably at the 4th week. This change tendency deteriorated more at the 8th and 12th week, while SLGP ablation partly or completely reversed these changes within 1 month. Additionally, we also ﬁrst reported the change tendency of SNRT throughout the experiment and found that SNRT in the OSA group was signiﬁcantly increased at the 4th week and gradually recovered toward baseline at the 8th and 12th week. No statistically signiﬁcant diﬀerence could be observed in the OSA group and the OSA + GP group at the 12th week. Major Findings Furthermore, the AF duration and inducibility were also studied at baseline and the 12th week, whereas the changes during this process were not elucidated in detail (Zhao et al., 2014). pressure and heart rate increases and decreased the expression of NGF, TH, and CHAT in LA. Our data suggested that SLGP ablation suppresses chronic obstructive sleep apnea-related atrial ﬁbrillation by inhibiting cardiac autonomic hyperactivity, not by reversing myocardial structural remodeling. SLGP Ablation Reversed Atrial Electrical Remodeling in OSA-Induced AF Previous studies reported that AF from any cause leads to atrial tissue conduction velocity change, thereby increasing the stability of the re-entrant circuits and promoting the progression of AF (van der Velden et al., 2000; Ai et al., 2010; Yang et al., 2019). The conduction velocity was mainly determined by the communication between cells, especially related to Cx40 and g It has been widely accepted that electrical remodeling contributes to the initiation and maintenance of AF associated with OSA (Oliveira et al., 2009; Latina et al., 2013). Clinical studies have demonstrated that extensive areas of low voltage or electrical April 2021 \| Volume 12 \| Article 640295 Frontiers in Physiology \| www.frontiersin.org 10 GPA Inhibit COSA-Related AF Zhang et al. FIGURE 11 \| Effect of SLGP ablation on the expression level of NGF, TH, and CHAT among the three groups. Representative immunohistochemical staining (A) and quantitative analysis of NGF, TH, and CHAT in LA (B) and SLGP (C); P < 0.01 vs. CTRL, ##P < 0.01 vs. OSA. NGF, neural growth factor; LA, left atrial; other abbreviations as in Figures 1, 10. FIGURE 11 \| Effect of SLGP ablation on the expression level of NGF, TH, and CHAT among the three groups. Representative immunohistochemical staining (A) and quantitative analysis of NGF, TH, and CHAT in LA (B) and SLGP (C); P < 0.01 vs. CTRL, ##P < 0.01 vs. OSA. NGF, neural growth factor; LA, left atrial; other abbreviations as in Figures 1, 10. FIGURE 12 \| The X-axis represents the –log10 (p-value) showing the overrepresented pathways according to the identiﬁed differentially expressed genes for (A) OSA vs. CTRL (up expression) and (B) OSA + GP vs. OSA (down expression). The supplement that lists the speciﬁc genes for each pathway is also shown (n = 3 animals per intervention); abbreviations as in Figure 1. FIGURE 12 \| The X-axis represents the –log10 (p-value) showing the overrepresented pathways according to the identiﬁed differentially expressed genes for (A) OSA vs. CTRL (up expression) and (B) OSA + GP vs. OSA (down expression). The supplement that lists the speciﬁc genes for each pathway is also shown (n = 3 animals per intervention); abbreviations as in Figure 1. Cx43 (Kanagaratnam et al., 2002; Dhein et al., 2014). Yang et al. SLGP Ablation Failed to Reverse Myocardial Tissue Changes in OSA-Induced AF Myocardial tissue changes in chronic repetitive apneic events play an important role in OSA-induced AF, mainly including atrial enlargement, ﬁbrosis, hypertrophy, myolysis, and other degenerative changes (De Jong et al., 2011). Atrial enlargement and ﬁbrosis are thought to be the two core functions of atrial remodeling (Nattel et al., 2008). Both Zhao et al. (2014) and Sun et al. (2017) reported that atrial apoptosis and ﬁbrosis could be detected in a dog model of chronic OSA-induced AF. In our present study, we also revealed the existence of ﬁbrosis and adipose tissue in the left atrial tissues, while GP ablation failed to reverse those changes in AF associated with OSA within 4 weeks. Myocardial tissue changes in chronic repetitive apneic events play an important role in OSA-induced AF, mainly including atrial enlargement, ﬁbrosis, hypertrophy, myolysis, and other degenerative changes (De Jong et al., 2011). Atrial enlargement and ﬁbrosis are thought to be the two core functions of atrial remodeling (Nattel et al., 2008). Both Zhao et al. (2014) and Sun et al. (2017) reported that atrial apoptosis and ﬁbrosis could be detected in a dog model of chronic OSA-induced AF. In our present study, we also revealed the existence of ﬁbrosis and adipose tissue in the left atrial tissues, while GP ablation failed to reverse those changes in AF associated with OSA within 4 weeks. In addition, we also ﬁrst comprehensively reported the changes in myocardial tissue in other chambers. Compared with the CTRL group, considerable fatty inﬁltration of the LAA, extensive ﬁbrosis of the RAA, and mild fatty inﬁltration and ﬁbrosis of the LV and RV were present in the OSA + GP group. At the same time, glycogen deposition and cardiomyocyte apoptosis were also observed in myocardial tissues. Previous studies have also shown that chronic intermittent hypoxia (CIH) causes endoplasmic reticulum stress, inﬂammatory inﬁltration, increased cardiomyocyte apoptosis, and destruction of mitochondrial structure (Kuang et al., 2004; Zhou et al., 2014). Taken together, these results clearly showed that all of these Additionally, we detected that the expression levels of markers of neuronal activity in atrial tissues and the SLGP, not only markers of sympathetic neuron activation (NGF and TH) but also vagus nerve activation markers (CHAT), were notably enhanced. SLGP Ablation Reversed the Echocardiographic Changes in OSA-Induced AF Previous studies have illustrated that OSA has a strong correlation with LA size, mainly because repetitive swings in intrathoracic pressure are transmitted to the thin-walled atria and lead to atrial dilation and ﬁbrosis, ultimately promoting the occurrence of AF (Orban et al., 2008; Linz et al., 2011a,b; Valenza et al., 2014). Clinical studies have shown that LA enlargement and left ventricular diastolic dysfunction could promote OSA, leading to a predisposition to AF (Latina et al., 2013). Zhao et al. (2014) and Sun et al. (2017) reported that RAd and LAd were enlarged and that LVEF decreased at 3 months in an AF-associated OSA canine model; this change was also observed in our study. Furthermore, we also found that these echocardiographic parameters changed as early as the 4th week and deteriorated more at weeks 8 and 12. In our study, we ﬁrst recorded the dynamic changes in HRV in non-anesthetized dogs throughout the experiment, thus eﬀectively avoiding the eﬀects of anesthetics on the inhibition of sympathetic nerve activity (Liu et al., 2019). In the initial stage of chronic OSA, we did not observe excessive activation of sympathetic nerve activity (SNA) and vagus nerve activity (VNA); with the prolongation of OSA time, both SNA and VNA increased and reached a plateau at the end of the 8th week. At this time, SNA and VNA were the highest, and neural activity tends to decrease. Therefore, we chose this time point to ablate the GP. We found that regardless of whether GP ablation was performed, SNA and VNA were decreased in the OSA group and OSA + GP group, and the scale of decrease was larger in the OSA + GP group. We hypothesized that the autonomic hyperinnervation might trigger an autonomic reﬂex to reduce the SNA and VNA (especially the SNA) as a protective mechanism after 2 months. We just showed this phenomenon, and more experiments are needed to explore possible mechanisms. In the ﬁnal stage of OSA, both SNA and VNA in the OSA group decreased, compared with baseline, and still remained active (especially SNA). In addition, the change range of HR and SBP before and after SLGP ablation also supported this opinion (Figures 7A–C). This was partly consistent with Zhao et al. (2014) study, which showed that increasing vagal activation promotes AF inducibility in chronic OSA. SLGP Ablation Reversed the Echocardiographic Changes in OSA-Induced AF We ﬁrst reported the change tendency of echocardiography in a 3-month OSA-induced AF canine model and furthermore ﬁrst investigated whether SLGP ablation could reverse this eﬀect. In addition, our study also showed that eﬀective interventions made at an early stage could result in a better prognosis. Change in ANS Activity in the Process of AF Induced by OSA In short, SLGP ablation could alleviate the deterioration of AF-related electrophysiological parameters and subsequently decrease the occurrence of AF. Some human and animal studies have indicated that sympathovagal imbalance also participates in the onset of OSA-related AF (Linz et al., 2012; Galal, 2017). HRV is known and widely used to evaluate cardiac autonomic activity (Usui and Nishida, 2017). Galal (2017) reported that LF, HF, and the LF/HF ratio were increased in patients with OSA. Yu et al. (2017c) showed that both LF and HF were increased in a 1-h intermittent hypoxia-induced AF dog model, revealing that sympathovagal imbalance contributed to the initiation of AF. However, these parameters in animal studies were acquired under anesthesia, and changes in ANS when awake did not reﬂect, let alone describe, the dynamic trend of ANS activity. SLGP Ablation Reversed Atrial Electrical Remodeling in OSA-Induced AF (2019) demonstrated that the decreased expression level of Cx43 played an important role in the initiation and development of a chronic intermittent hypoxia-induced AF rat model, which was paralleled by another study in which lower expression of Cx43 was also detected in aged rabbit atrial tissue with AF (Yan et al., 2018). However, Zhao et al. (2014) and Sun et al. (2017) reported that Cx40, Cx43, and the Cx40/Cx43 ratio were not signiﬁcantly changed in a dog model of chronic OSA-induced AF. In our present study, we found that both Cx40 and Cx43 were evidently April 2021 \| Volume 12 \| Article 640295 Frontiers in Physiology \| www.frontiersin.org 11 Zhang et al. GPA Inhibit COSA-Related AF changes in myocardial structure constitute the arrhythmogenic substrate for OSA-induced AF. decreased in the OSA group, and this change could be eﬀectively reversed by SLGP ablation, suggesting that a low expression level of connexin might also be involved in the development of OSA-induced AF. Frontiers in Physiology \| www.frontiersin.org SLGP Ablation Failed to Reverse Myocardial Tissue Changes in OSA-Induced AF Previous studies (Zhao et al., 2014; Sun et al., 2017) have shown that the expression levels of TH and CHAT-positive ﬁbers were signiﬁcantly increased in a dog model of chronic OSA-induced AF, indicating that the ANS plays a vital role in OSA-induced neural remodeling. Other studies (Wang et al., 2016; Yu et al., 2016, 2017a,b) have also shown that high expression of NGF promotes cardiac autonomic remodeling and cardiac arrhythmia inducibility, and inhibiting NGF expression could have the eﬀect of treating AF. Our results were consistent with those studies, and we also found that the expression of these neuronal factors was evidently decreased after the SLGP was ablated. g In addition, we also ﬁrst comprehensively reported the changes in myocardial tissue in other chambers. Compared with the CTRL group, considerable fatty inﬁltration of the LAA, extensive ﬁbrosis of the RAA, and mild fatty inﬁltration and ﬁbrosis of the LV and RV were present in the OSA + GP group. At the same time, glycogen deposition and cardiomyocyte apoptosis were also observed in myocardial tissues. Previous studies have also shown that chronic intermittent hypoxia (CIH) causes endoplasmic reticulum stress, inﬂammatory inﬁltration, increased cardiomyocyte apoptosis, and destruction of mitochondrial structure (Kuang et al., 2004; Zhou et al., 2014). Taken together, these results clearly showed that all of these April 2021 \| Volume 12 \| Article 640295 12 GPA Inhibit COSA-Related AF Zhang et al. and Yu et al. (2017c) showed that GP ablation could eﬀectively suppress the onset of AF in acute simulated OSA-induced AF. However, one more important limitation of those studies was that the chronic eﬀect of GP ablation was not evaluated. Taken together, these results indicated that SLGP ablation suppressed hyperactivity of the ANS and subsequently inhibited AF inducibility. The imbalanced state of the ANS has been gradually recognized in AF associated with OSA (Carnagarin et al., 2019; Huang et al., 2020). Treatment measures that modulate the ANS, including metoprolol (Li et al., 2015; Sun et al., 2017; Dai et al., 2019) and LLTS (Yu et al., 2017b), could inhibit the inducibility of AF. Recently, atrial GP ablation was also recognized as an important measurement to regulate the ANS. Ghias et al. (2009) In our study, we conﬁrmed that dozens of neurons were contained in the SLGP, and the myelinated nerve ﬁbers and neurons were obviously destroyed once the SLGP was ablated. SLGP Ablation Failed to Reverse Myocardial Tissue Changes in OSA-Induced AF Additionally, the dynamic change in HRV and neural markers also showed hyperactivity of the ANS in chronic OSA-induced AF, and the SLGP rebalanced the ANS to a degree. Therefore, we FIGURE 13 \| The schematic diagram illustrates the underlying mechanism by which SLGP ablation attenuates chronic OSA-associated AF. 3 \| The schematic diagram illustrates the underlying mechanism by which SLGP ablation attenuates chronic OSA-associated AF. Frontiers in Physiology \| www.frontiersin.org April 2021 \| Volume 12 \| Article 640295 13 GPA Inhibit COSA-Related AF Zhang et al. have reason to infer that SLGP ablation suppressed autonomic remodeling and subsequently inhibited the occurrence of AF. FIGURE 14 \| The schematic diagram illustrates the changes in the intrinsic and extrinsic nervous system activity of OSA-related chronic AF. Previous studies reported that mediators of the inﬂammatory response could alter atrial electrophysiology and structural substrates, thereby leading to increased vulnerability to AF (Hu et al., 2015), and anti-inﬂammatory measures might be therapeutic for AF patients (Aschar-Sobbi et al., 2015; Yao et al., 2018). In our current study, proteomics analysis also showed inﬂammatory signaling pathway participation in the progression of OSA-induced AF, and SLGP ablation downregulated inﬂammatory expression, which was consistent with ameliorated electrophysiological parameters and echocardiography parameters and lower AF inducibility. However, the evident ﬁbrosis in the myocardial tissue was not ameliorated after the GP ablation. To date, just one manuscript by Zhao et al. (2011) has described the relationship between GP ablation and inﬂammation in AF. They reported that concentrations of TNF-α and IL-6 in the atrium increased signiﬁcantly 8 weeks post GP ablation, which was a predictor for recurrence of AF after GP ablation. This was contradictory to our research ﬁndings. We just demonstrated inﬂammatory signaling pathway participation in the progression of OSA-induced AF, and SLGP ablation downregulated inﬂammatory expression. Additional investigation will be required to resolve this discrepancy. FIGURE 14 \| The schematic diagram illustrates the changes in the intrinsic and extrinsic nervous system activity of OSA-related chronic AF. Taken together, all of this evidence suggested that electrical remodeling was independent of atrial structural remodeling, and SLGP ablation could improve the occurrence of electrical remodeling by regulating the abnormal activity of the ANS and inﬂammation levels, which could suppress the appearance of AF. SLGP Ablation Failed to Reverse Myocardial Tissue Changes in OSA-Induced AF aspect was that we observed electrical remodeling and structural remodeling in the early stage of our experiment, so early intervention should be considered in the early progression of OSA to acquire a better prognosis. However, we have only conducted animal studies, so more clinical studies are needed for conﬁrmation. CONCLUSION Canines with chronic OSA demonstrate signiﬁcant atrial electrical and structural remodeling characterized by shortening ERP, increased AF inducibility, increased conduction heterogeneity, and sinus node dysfunction. In addition, chamber enlargement, glycogen deposition, increased cardiomyocyte necrosis, and ﬁbrous hyperplasia accompanied by adipose tissue inﬁltration were noted in the LA. Meanwhile, chronic OSA dramatically increased activities of the intrinsic (SLGP) and extrinsic cardiac ANS (LVN and LSG). All of the factors suggest Canines with chronic OSA demonstrate signiﬁcant atrial electrical and structural remodeling characterized by shortening ERP, increased AF inducibility, increased conduction heterogeneity, and sinus node dysfunction. In addition, chamber enlargement, glycogen deposition, increased cardiomyocyte necrosis, and ﬁbrous hyperplasia accompanied by adipose tissue inﬁltration were noted in the LA. Meanwhile, chronic OSA dramatically increased activities of the intrinsic (SLGP) and extrinsic cardiac ANS (LVN and LSG). All of the factors suggest Study Limitations g A schematic diagram illustrates the underlying mechanism by which SLGP ablation attenuates chronic OSA-associated AF (Figures 13, 14). On the one hand, OSA increases aﬀerent signaling to the central nervous system, which leads to overactivity of the intrinsic and extrinsic cardiac autonomic nervous system, thereby increasing the nerve activity of the SLGP, LSG, and LVN, which increases AERP dispersion and AF inducibility and sympathovagal innervation in the LA. On the other hand, chronic repeated OSA leads to hypoxia, negative intrathoracic pressure surges, decreased Cx43 levels, LA enlargement, glycogen deposition, increased necrosis, and ﬁbrosis in cardiomyocytes. All of these changes promote increased conduction heterogeneity and decreased conduction velocity, thereby contributing to the occurrence and maintenance of OSA-induced AF. However, SLGP ablation reduces input to the central nervous system and downregulates the activity of the autonomic nervous system, thereby inhibiting OSA-induced AF. Several limitations must be noted in this study. (1) Due to our technical limitations, we failed to implant a recorder to monitor neural activity in the body in the long term, while we dynamically monitored the change in HRV to reﬂect the activity of the ANS. (2) Previous studies have conﬁrmed that the bilateral stellate ganglia and vagus nerve have diﬀerent roles in atrial remodeling. We only focused on the activity of the left stellate ganglion and cervical vagus nerve, which might not fully reﬂect the extrinsic autonomic nervous system. (3) The third limitation is that the inﬂammatory signaling pathway was involved in the progression of OSA as determined through proteomics analysis, while the exact pathway has not yet been analyzed and needs further veriﬁcation. REFERENCES Galal, I. H. (2017). Nocturnal heart rate variability in obstructive sleep apnea syndrome: eﬀect of automatic positive airway pressure. Egyptian J. Bronchol. 11:372. doi: 10.4103/ejb.ejb_36_17 Ai, X., Zhao, W., and Pogwizd, S. M. (2010). Connexin43 knockdown or overexpression modulates cell coupling in control and failing rabbit left ventricular myocytes. Cardiovasc. Res. 85, 751–762. doi: 10.1093/cvr/ cvp353 Gao, M., Zhang, L., Scherlag, B. J., Huang, B., Stavrakis, S., Hou, Y. M., et al. (2015). Low-level vagosympathetic trunk stimulation inhibits atrial ﬁbrillation in a rabbit model of obstructive sleep apnea. Heart Rhythm. 12, 818–824. doi: 10.1016/j.hrthm.2014.12.024 Aschar-Sobbi, R., Izaddoustdar, F., Korogyi, A. S., Wang, Q., Farman, G. 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Med. 31, 127–132. doi: 10.1016/j.tcm.2020. 01.006 Dai, H., Yuan, Y., Yin, S., Zhang, Y., Han, Y., Sun, L., et al. (2019). ACKNOWLEDGMENTS The animal study was reviewed and approved by the Committee of the Ethics of Animal Experiments of the First Aﬃliated Hospital of Xinjiang Medical University. We would like to thank Mei Ma for his surgery and anesthesia support. We thank AJE (American Journal Experts) for its linguistic assistance during the preparation of this manuscript. DATA AVAILABILITY STATEMENT This work was supported by the National Natural Science Foundation of China (Project No: 81860067 and 81560064) and the Department of Education, Xinjiang Uygur Autonomous Region (CN) (2018Q046). The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Clinical Implications The long-term treatment eﬀect of SLGP ablation could eﬀectively suppress AF inducibility in chronic OSA in our current study. Previous clinical studies also showed that GP ablation + PVI signiﬁcantly increased the success rate of AF ablation (Katritsis et al., 2011, 2013; Kampaktsis et al., 2017). Therefore, we hypothesized that SLGP ablation could be considered one of the strategies for chronic OSA-induced AF. Another important April 2021 \| Volume 12 \| Article 640295 Frontiers in Physiology \| www.frontiersin.org 14 Zhang et al. GPA Inhibit COSA-Related AF statistical analysis and interpretation. All authors contributed to the writing, critical reading, and approval of the manuscript. that the hyperactivity of intrinsic and extrinsic cardiac ANS plays a crucial role in the development of chronic OSA-induced AF. Therefore, SLGP ablation might serve as a potential therapy to treat AF in OSA patients. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2021.640295/full#supplementary-material LZ and BT contributed to the funding acquisition, conception, and design of the study. LZ, YG, JX, HS, and KL contributed to the animal experiments. GC, HL, and XZ contributed to the REFERENCES Combined blockade of early and late activated atrial potassium currents suppresses atrial ﬁbrillation in a pig model of obstructive apnea. Heart Rhythm. 8, 1933–1939. doi: 10.1016/j.hrthm.2011.07.018 van der Velden, H. M., Ausma, J., Rook, M. B., Hellemons, A. J., van Veen, T. A., Allessie, M. A., et al. (2000). Gap junctional remodeling in relation to stabilization of atrial ﬁbrillation in the goat. Cardiovasc. Res. 46, 476–486. doi: 10.1016/S0008-6363(00)00026-2 j Linz, D., Schotten, U., Neuberger, H. R., Böhm, M., and Wirth, K. (2011b). 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Inﬂammation abnormalities and inducibility of atrial ﬁbrillation after Frontiers in Physiology \| www.frontiersin.org April 2021 \| Volume 12 \| Article 640295 16 Zhang et al. GPA Inhibit COSA-Related AF epicardial ganglionated plexi ablation. Arch. Cardiovasc. Dis. 104, 227–233. doi: 10.1016/j.acvd.2011.01.007 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. Zhou, Q., Zhou, X., Wang, H., Yin, T., Li, Y., Zhang, L., et al. (2016). Renal sympathetic denervation suppresses atrial ﬁbrillation induced by acute atrial ischemia/infarction through inhibition of cardiac sympathetic activity. Int. J. Cardiol. 203, 187–195. doi: 10.1016/j.ijcard.2015.10.120 Copyright © 2021 Zhang, Guo, Xiaokereti, Cao, Li, Sun, Li, Zhou and Tang. 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. Zhou, S., Yin, X., Zheng, Y., Miao, X., Feng, W., Cai, J., et al. (2014). Metallothionein prevents intermittent hypoxia-induced cardiac endoplasmic reticulum stress and cell death likely via activation of Akt signaling pathway in mice. Toxicol. Lett. 227, 113–123. doi: 10.1016/j.toxlet.2014. 03.011 April 2021 \| Volume 12 \| Article 640295 Frontiers in Physiology \| www.frontiersin.org 17
https://openalex.org/W2166742793	https://www.matec-conferences.org/articles/matecconf/pdf/2014/07/matecconf_csndd2014_08005.pdf	English	null	Galloping of wind-excited tower under external excitation and parametric damping	MATEC web of conferences	2,014	cc-by	4,048	1 Introduction unsteady wind is present, IPD decreases the amplitude of galloping in all cases of loading, but has no inﬂuence on the galloping onset. Notice that the IPD can be introduced via a damper device in the interﬂoors damping as reported in [13]. Its use as a control strategy was motivated by its simple implementation and beneﬁcial eﬀect in reducing vi- bration in many engineering applications. The control of large amplitude oscillations in tall ﬂexible building constitutes an important issue in designing stable tall structures. Considerable eﬀorts have been made to re- duce the amplitude of oscillations induced by steady and unsteady wind. In general, taller ﬂexible towers are aero- dynamically sensitive to the natural wind environment and their failure may be expected if the nonlinear dynamic ef- fects on the structure are neglected. It is well known that above a certain threshold of the wind speed, tall buildings develop galloping [1–5] causing the structure to oscillate with large amplitudes. In this context, considerable eﬀorts have been done to quench such wind-induced oscillations including, for instance, mass tuned dampers, tuned liquid dampers or external excitation. A review of some control methods and their full-scale implementation to civil infras- tructure applications is given in [6]. In this paper, we extend the results given in [10,12] by focusing on the combined eﬀect of external FHE and fast IPD on periodic galloping. The main purpose is to exam- ine how FHE and IPD can inﬂuence the galloping of the wind-excited tower when both expiations are introduced simultaneously. The paper is organized as follows: In Section 2, the equation of motion including the eﬀect of both external excitation and parametric damping is given. The method of DPM [14,15] is performed and the MSM is applied in section 3 to derive the modulation equations of the slow dynamic near the primary resonance. In Section 4, we an- alyze the additional eﬀect of FHE and IPD on the periodic galloping in the cases where the unsteady wind activates diﬀerent excitations. Section 5 concludes the work. The eﬀect of unsteady wind on the galloping onset of towers has been considered by several authors. In [4] a sin- gle degree of freedom (sdof) model has been considered and the multiple scales method (MSM) [7] has been used to analyze the inﬂuence of the unsteady wind on the critical wind speed above which galloping occurs. Galloping of wind-excited tower under external excitation and parametric damping L. Mokni, I. Kirrou, M. Belhaq Laboratory of Mechanics, University Hassan II-Casablanca, Morocco Laboratory of Mechanics, University Hassan II-Casablanca, Morocco Abstract. This paper investigates the inﬂuence of combined fast external excitation and fast parametric damp- ing on the amplitude and the onset of galloping of a tower submitted to steady and unsteady wind ﬂow. A lumped single degree of freedom model is considered and the cases where the turbulent wind activates either external ex- citation, parametric one or both are studied. The methods of direct partition of motion and the multiple scales are used to drive a slow dynamic near primary resonance. The inﬂuence of the combined excitations on the galloping is examined showing the beneﬁcial eﬀect on the amplitude and the onset of galloping when the excitations are introduced together. 1 Introduction It was shown that the unsteady wind decreases signiﬁcantly the gallop- ing onset only near the primary resonance. In [8,9], the eﬀect of parametric, external and self-induced excitation on periodic galloping of a single tower and of two towers linked by a nonlinear viscous device was examined. DOI: 10.1051/ Owned by the authors, published by EDP Sciences, 2014 , / 0 0 (2014) 201 conf Web of Conferences 4 0 0 1 1 MATEC matec 6 6 ⃝ C 0 05 8 8 5 DOI: 10.1051/ Owned by the authors, published by EDP Sciences, 2014 , / 0 0 (2014) 201 conf Web of Conferences 4 0 0 1 1 MATEC matec 6 6 ⃝ C 0 05 8 8 5 DOI: 10.1051/ Owned by the authors, published by EDP Sciences, 2014 , / 0 0 (2014) 201 conf Web of Conferences 4 0 0 1 1 MATEC matec 6 6 ⃝ C 0 05 8 8 5 This is an Open Access article distributed under the terms of the Creative Commons Attribution License 3.0, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Article available at http://www.matec-conferences.org or http://dx.doi.org/10.1051/matecconf/20141608005 DOI: 10.1051/ Owned by the authors, published by EDP Sciences, 2014 , / 0 0 (2014) 201 conf Web of Conferences 4 0 0 1 1 MATEC matec 6 6 ⃝ C 0 05 8 8 5 DOI: 10.1051/ Owned by the authors, published by EDP Sciences, 2014 , / 0 0 (2014) 201 conf Web of Conferences 4 0 0 1 1 MATEC matec 6 6 ⃝ C 0 05 8 8 5 2 Equations of motion and slow dynamic A single mode approach of the tower motion is considered and a sdof lumped mass system is introduced [4,8]. It is assumed that the tower is subjected to steady and unsteady wind ﬂow and to a combined eﬀect of FHE and IPD. In this case, the dimensionless sdof equation of motion can be written in the form [8] Recently, the eﬀect of fast harmonic excitation (FHE) on periodic and quasiperiodic galloping onset of a tower exposed to steady and unsteady wind was studied near pri- mary resonance [10]. The introduction of a FHE as a con- trol strategy was motivated by an experimental work made for vibrating testing purpose of a full size tower [11]. The mechanical vibration exciter system used in such an ex- periment is placed on the top of the structure and debits a harmonic excitation to the structure. More recently, the inﬂuence of internal parametric damping (IPD) on peri- odic galloping onset of a tower under steady and unsteady wind was examined [12]. It was concluded that when the ¨x + x + ca(1 −¯U) −b1u(t) ˙x + Yν2 cos(νt)˙x + b2 ˙x2 + b31 ¯U + b32 ¯U2 u(t) ˙x3 = η1 ¯Uu(t) + η2 ¯U2 + Y cos(νt) (1) (1) where the dot denotes diﬀerentiation with respect to the non-dimensional time t. Equation (1) contains, in addition to the elastic, viscous and inertial linear terms, quadratic MATEC Web of Conferences where ti = εit (i = 0, 1). In terms of the variables ti, the time derivatives become d dt = d0 + εd1 + O(ε2) and d2 dt2 = d2 0 + 2εd0d1 + O(ε2), where d j i = ∂j ∂jti . Substituting Eq. (4) into Eq. (3), equating coeﬃcients of the same power of ϵ, we obtain the two ﬁrst orders of approximation and cubic components in the velocity generated by the aero- dynamic forces. The steady component of the wind veloc- ity is represented by ¯U and the turbulent wind ﬂow is ap- proximated by a periodic force, u(t), which is assumed to include the two ﬁrst harmonics, u(t) = u1 sin Ωt+u2 sin 2Ωt, where u1, u2 and Ω are, respectively, the amplitudes and the fundamental frequency of the response. 2 Equations of motion and slow dynamic We shall analyze the case of external excitation, u(t) = u1 sin Ωt, paramet- ric one, u(t) = u2 sin 2Ωt, and the case where external and parametric excitations are present simultaneously. The co- eﬃcients of Eq. (1) are given in Appendix I and Y, ν are, re- spectively, the dimensionless amplitude and the frequency of the FHE and IPD. In order to simplify the analysis, one considers the particular case where the FHE and the IPD have the same amplitude and the same frequency. d2 0z0 + z0 = G (5) (5) d2 0z1 + z1 = −2d0d1z0 + (caV + b1u(t0) + H0 −H1(b31 + b32u(t0)))(d0z0) −(B −B0(b31 + b32u(t0)))(d0z0)2 −H2(b31 + b32u(t0))(d0z0)3 + η1u(t0) + η2(6) d2 0z1 + z1 = −2d0d1z0 + (caV + b1u(t0) + H0 −H1(b31 + b32u(t0)))(d0z0) −(B −B0(b31 + b32u(t0)))(d0z0)2 −H2(b31 + b32u(t0))(d0z0)3 + η1u(t0) + η2(6) A solution of Eq. (5) is given by A solution of Eq. (5) is given by (7) z0 = A(t1) exp(it0) + ¯A(t1) exp(−it0) + G Equation (1) includes a slow dynamic due to the steady and unsteady wind and a fast dynamic induced by the FHE and the IPD. To separate these dynamics, we perform the method of DPM on Eq. (1) by deﬁning a fast time T0 = νt and a slow time T1 = t, and splitting up x(t) into a slow part z(T1) and a fast part φ(T0, T1) as where i is the imaginary unit and A is an unknown complex amplitude. Equation (6) can be solved for the complex am- plitude A by introducing its polar form as A = 1 2aeiφ. Sub- stituting the expression of A into Eq. (6) and eliminating the secular terms, the modulation equations of the ampli- tude a and the phase φ can be extracted as ⎧⎪⎪⎪⎪⎪⎪⎪⎪⎨⎪⎪⎪⎪⎪⎪⎪⎪⎩ ˙a = [S 1 −S 3 sin(2φ)]a −S 5 cos(φ)a2 +[−S 2 + 2S 4 sin(2φ)]a3 −β cos(φ) a˙φ = [σ −S 3 cos(2φ)]a + 3S 5 sin(φ)a2 +[S 4 cos(2φ)]a3 + β sin(φ) (8) x(t) = z(T1) + μφ(T0, T1) (2) (2) (8) where z describes the slow main motions at time-scale of oscillations, μφ stands for an overlay of the fast motions and μ indicates that μφ is small compared to z. Since ν is considered as a large parameter, we choose μ ≡ν−1 for convenience. 4.1 Case of non-turbulent wind where H0 = 4b2Y2, H1 = 6( Y ν )2, H2 = 1 + 6Y2ν2, B = b2(1 + 2Y2ν2), B0 = 12Y2 and G = −2b2( Y ν )2. Note that the case without FHE has been studiend in [10], the case without IPD was considered in [12], while the case without FHE and IPD (Y = 0) was talked in [8]. Fixed points of Eq. (8), representing periodic oscillations of the system, are giving by setting ˙a = ˙φ = 0. In the absence of turbulence (u1 = u2 = 0), only the ﬁrst equation of system (8) is used. Besides the trivial solution, a = 0, the amplitude of the self-excitation is also obtained such as ⎧⎪⎪⎪⎪⎪⎨⎪⎪⎪⎪⎪⎩ a = 0 a = 4(caV + H0 −H1b31) 3b31H2 (9) (9) 2 Equations of motion and slow dynamic The fast part μφ and its derivatives are as- sumed to be 2π−periodic functions of fast time T0 with zero mean value with respect to this time, so that < x(t) >= z(T1) where <>≡ 1 2π 2π 0 () dT0 deﬁnes time-averaging op- erator over one period of the fast excitation with the slow time T1 ﬁxed. Averaging procedure gives the following equation governing the slow dynamic of motion where S 1 = 1 2(caV + H0 −H1b31), S 2 = 3 8b31H2, S 3 = 1 4(b1 −H1b32)u2, S 4 = 1 8b32H2u2, S 5 = 1 8b32B0u1 and β = η1u1 2 . It can be seen that the FHE and the IPD inﬂuence the dynamic of the tower via the coeﬃcients Hi(i = 0, 1, 2) and B0. where S 1 = 1 2(caV + H0 −H1b31), S 2 = 3 8b31H2, S 3 = 1 4(b1 −H1b32)u2, S 4 = 1 8b32H2u2, S 5 = 1 8b32B0u1 and β = η u where S 1 = 1 2(caV + H0 −H1b31), S 2 = 3 8b31H2, S 3 = 1 4(b1 −H1b32)u2, S 4 = 1 8b32H2u2, S 5 = 1 8b32B0u1 and β = where S 1 = 1 2(caV + H0 −H1b31), S 2 = 3 8b31H2, S 3 = 1 4(b1 −H1b32)u2, S 4 = 1 8b32H2u2, S 5 = 1 8b32B0u1 and β = 4 8 8 η1u1 2 . It can be seen that the FHE and the IPD inﬂuence the dynamic of the tower via the coeﬃcients Hi(i = 0, 1, 2) and B0. 4 Applications and results ¨z + z + ca(1 −¯U) −b1u(t) −H0 + b31 ¯U +b32 ¯U2 u(t) H1 ˙z + B −B0 b31 ¯U + b32 ¯U2 u(t) ˙z2 + b31 ¯U +b32 ¯U2 u(t) H2˙z3 = η1 ¯Uu(t) + η2 ¯U2 + G (3) In this section, we analyze the eﬀect of the amplitude Y of the excitations on the vibration of the tower for diﬀerent types of turbulent wind ﬂow. The parameter values used in the present study are taken from [8]. 4.2 Case where turbulent wind activates external excitation Figure 4 shows the amplitude versus the velocity V, as given by (11), in the absence of external excitation (u1 = 0, u2 0). The solid lines correspond to the stable branches, dots correspond to the unstable ones. The plots show that as Y increases the amplitude of the response decreases sig- niﬁcantly and a shift to the right in the frequency response is obtained. Figure 5 shows the decrease of the galloping amplitude versus σ as Y is increased. In this case (u1 0, u2 = 0), the modulation equations (8) lead to the following response equation (S 1a −S 2a3)2 (β + S 5a2)2 + (−σa)2 (β + 3S 5a2)2 = 1 (10) (10) In Figs. 2, 3, we show the eﬀect of the amplitude Y on the frequency response versus V and σ, respectively, for diﬀerent values of Y. −0.4 −0.2 0 0.2 0.4 0.6 0.8 1 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 V a Y = 0 Y = 0.15 Fig. 4. Amplitude versus V when σ = 0, u1 = 0, u2 = 0.1 and ν = 8. Solid line: stable, dashed line: unstable, circles: numerical simulation. Figure 2 indicates that as the amplitude Y increases, the amplitude of tower oscillation decreases and the frequency response shifts right. In Fig. 3 is shown the decreasing in the amplitude when Y is increased. One can observe from this ﬁgure that for Y = 0, three solutions exist within the interval (σ ∈[−0.0001, 0.0001]). As Y is increased, only one solution exists. The solid lines correspond to the stable branches, while the dots correspond to the unstable ones. Validation of the analytical approximation is made using numerical simulation (circles). Fig. 4. Amplitude versus V when σ = 0, u1 = 0, u2 = 0.1 and ν = 8. Solid line: stable, dashed line: unstable, circles: numerical simulation. −0.4 −0.2 0 0.2 0.4 0.6 0.8 1 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 V a Y = 0.15 Y = 0 Fig. 2. Amplitude versus velocity of the wind when σ = 0, u2 = 0, u1 = 0.1 and ν = 10. Solid line: stable; dashed: unstable. 3 The multiple scales analysis To obtain the modulation equations of the slow dynamic (3) near primary resonance, the MSM is performed by in- troducing a bookkeeping parameter ε, scaling as z = ε 1 2 z, b1 = εb1, H0 = εH0, H1 = εH1, B = ε 1 2 B, B0 = ε 1 2 B0, η1 = ε 3 2 η1, η2 = ε 3 2 η2 and assuming that ¯U = 1 + εV [8]. With the resonance condition Ω = 1 + εσ where V stands for the mean wind velocity and σ is a detuning parameter, a two-scale expansion of the solution is sought in the form To obtain the modulation equations of the slow dynamic (3) near primary resonance, the MSM is performed by in- troducing a bookkeeping parameter ε, scaling as z = ε 1 2 z, b1 = εb1, H0 = εH0, H1 = εH1, B = ε 1 2 B, B0 = ε 1 2 B0, η1 = ε 3 2 η1, η2 = ε 3 2 η2 and assuming that ¯U = 1 + εV [8]. Figure 1 shows the galloping amplitude a versus the wind velocity V in the absence of the unsteady wind (u1 = 0, u2 = 0), as given by Eq. (9), for σ = 0 and for diﬀer- ent values of the amplitude Y of the FHE and the IPD. The trivial solution exists everywhere and changes its stability at the bifurcation point. It can be seen from this ﬁgure that increasing the amplitude Y, the amplitude of the tower re- sponse decreases signiﬁcantly and the location of the gal- loping onset shifts toward higher values of the steady wind velocity. With the resonance condition Ω = 1 + εσ where V stands for the mean wind velocity and σ is a detuning parameter, a two-scale expansion of the solution is sought in the form z(t) = z0(t0, t1) + εz1(t0, t1) + O(ε2) (4) z(t) = z0(t0, t1) + εz1(t0, t1) + O(ε2) 08005-p.2 Fig. 1. Equilibrium branches in the absence of turbulent wind, for ν = 8. Solid line: stable, dashed line: unstable. CSNDD CSNDD 2014 −1 −0.8 −0.6 −0.4 −0.2 0 0.2 0.4 0.6 0.8 1 x 10 −3 0 0.005 0.01 0.015 0.02 0.025 σ a Y = 0 Y = 0.1 Fig. 3. 4.2 Case where turbulent wind activates external excitation −0.4 −0.2 0 0.2 0.4 0.6 0.8 1 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 V a Y = 0.15 Y = 0 −1 −0.8 −0.6 −0.4 −0.2 0 0.2 0.4 0.6 0.8 1 x 10 −3 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 σ a Y = 0 Y = 0.05 Y = 0.08 Fig. 5. Eﬀect of Y on galloping; u2 = 0.1, V = 0.167, ν = 8. Solid line: stable, dashed line: unstable, circles: numerical simulation. Fig. 2. Amplitude versus velocity of the wind when σ = 0, u2 = 0, u1 = 0.1 and ν = 10. Solid line: stable; dashed: unstable. Fig. 5. Eﬀect of Y on galloping; u2 = 0.1, V = 0.167, ν = 8. Solid line: stable, dashed line: unstable, circles: numerical simulation. 3 The multiple scales analysis Amplitude versus σ when V = 0.117, u1 = 0.033, u2 = 0 and ν = 10. Solid line: stable, dashed line: unstable, circles: numerical simulation. −1 −0.8 −0.6 −0.4 −0.2 0 0.2 0.4 0.6 0.8 1 x 10 −3 0 0.005 0.01 0.015 0.02 0.025 σ a Y = 0 Y = 0.1 Fig. 1. Equilibrium branches in the absence of turbulent wind, for ν = 8. Solid line: stable, dashed line: unstable. Fig. 3. Amplitude versus σ when V = 0.117, u1 = 0.033, u2 = 0 and ν = 10. Solid line: stable, dashed line: unstable, circles: numerical simulation. References the stable branches, dots correspond to the unstable ones and circles are obtained by numerical integration. The plots indicate the eﬀect the amplitude Y on the frequency re- sponse indicating that increasing Y eliminates the bistabil- ity in the amplitude response, thereby the coexistence of two diﬀerent amplitudes of oscillation. 1. G.V. Parkinson, J.D. Smith, The square prism as an aeroelastic non-linear oscillator. Quarterly Journal of Mechanics and Applied Mathematics 17, (1964) 225-239 echanics and Applied Mathematics 17, (1964) 225-239 2. M. Novak, Aeroelastic galloping of prismatic bodies. ASCE, Journal of Engineering Mechanics Division 96, (1969) 115-142 −1.5 −1 −0.5 0 0.5 1 1.5 x 10−3 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 σ a Y = 0 Y = 0.12 Fig. 6. Amplitude versus σ when V = 0.11, u1 = 0.033, u2 = 0.033 and ν = 8. Solid line: stable, dashed line: unstable, circles: numerical simulation. −1.5 −1 −0.5 0 0.5 1 1.5 x 10−3 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 σ a Y = 0 Y = 0.12 3. A.H. Nayfeh, M. Abdel-Rohman, Galloping of squared cantilever beams by the method of multiple scales. J. Sound Vib. 143, (1990) 87-93 4. M. Abdel-Rohman, Eﬀect of unsteady wind ﬂow on galloping of tall prismatic structures, Nonlinear Dyn. 26, (2001) 231-252 5. R. Clark, A. Modern, Course in Aeroelasticity, fourth ed (Kluwer Academic Publishers, Dordrecht, The Nether- lands, 2004) 6. B.F. Spencer Jr, S. Nagarajaiah, State of the art of struc- tural control. Journal of Structural Engineering, 129, (2003) 845-865 Fig. 6. Amplitude versus σ when V = 0.11, u1 = 0.033, u2 = 0.033 and ν = 8. Solid line: stable, dashed line: unstable, circles: numerical simulation. 7. A. H. Nayfeh, B. Balachandran, Applied Nonlinear Dy- namics (John Wiley, New York, 1995) 8. A. Luongo, D. Zulli, Parametric, external and self- excitation of a tower under turbulent wind ﬂow, J. Sound Vib. 330, (2011) 3057-3069 9. D. Zulli, A. Luongo, Bifurcation and stability of a two- tower system under wind-induced parametric, external and self-excitation, J. Sound Vib. 331, (2012) 365-383 4.4 Case where turbulent wind activates external and parametric excitations In this case (u1 = 0, u2 0), the corresponding amplitude response equation is written as In the case where the external and parametric excitations are both present (u1 0, u2 0), the amplitude frequency response is shows in Fig. 6. The solid lines correspond to (−S 1a + S 2a3)2 (−S 3a + 2S 4a3)2 + (−σa)2 (−S 3a + S 4a3)2 = 1 (11) (11) 08005-p.3 MATEC Web of Conferences 5 Conclusion 10. M.Belhaq, I. Kirrou, L. Mokni, Periodic and quasiperiodic galloping of a wind-excited tower under external excitation, Nonlinear Dyn. 74, (2013) 849-867 In this work, we have investigated the eﬀect of FHE and IPD on the amplitude and the onset of periodic galloping of a tower when submitted to steady and turbulent wind ﬂow. A lumped mass sdof model was considered and at- tention was focused on the case where the turbulent wind activates either external excitation, parametric one or both. The method of DPM and the MSM are used to drive a slow dynamic near primary resonance. 11. W.O. Keightley, G.W. Housner, D.E. Hudson, Vibra- tion tests of the Encino dam intake tower, (California In- stitute of Technology, Report No. 2163, Pasadena, Cali- fornia 1961) 12. L. Mokni, I. Kirrou , M. Belhaq, Galloping of a wind- excited tower under internal parametric damping, J. Vib. Acoust. (2014), doi: 10.1115/1.4026505 In the case of steady wind, the combined eﬀect of FHE and IPD retards substantially the galloping onset and de- creases signiﬁcantly its amplitude. 13. Munteanu, L., Chiroiu, V., Sireteanu, T.: On the re- sponse of small buildings to vibrations. Nonlinear Dyn. 73, (2013) 1527-1543 , ( ) 14. I. I. Blekhman, Vibrational Mechanics, Nonlinear Dy- namic Eﬀects, General Approach, Applications (Singa- pore: World Scientiﬁc 2000) In the case of turbulent wind, the FHE and IPD also decreases the amplitude of the galloping and increases the galloping onset of the tower in all cases of turbulent wind. p 15. J.J. Thomsen, Vibrations and Stability: Advanced Theory, Analysis, and Tools (Springer- Verlag, Berlin- Heidelberg 2003) Appendix The expressions of the coeﬃcients of Eq. (1) are 16. F. Lakrad, M. Belhaq, Suppression of pull-in instabil- ity in MEMS using a high-frequency actuation. Commun Nonlinear Sci Numer Simulat. 15, (2010) 3640-3646 ω = π √ 3EI hℓ√m, ca = ρA1bhℓ¯Uc 2π √ 3EIm, b1 = ca, b2 = −4ρA2b1ℓ 3πm b31 = −3πρA3bℓ √ 3EI 8h ¯Uc √ m3 , b32 = −b31, η1 = 4ρA0bh2ℓ¯U2 c 3π3EI η2 = η1 2 , U(t) = ¯U + u(t), nlinear Sci Numer Simulat. 15, (2010) 3640-3646 17. I. Kirrou, L. Mokni, M. Belhaq, On the quasiperiodic galloping of a wind-excited tower, J. Sound Vib. 332 (2013) 4059-4066. 2 where ℓis the height of the tower, b is the cross-section wide, EI the total stiﬀness of the single story, m is the mass longitudinal density, h is the inter story height, and ρ is the air mass density. y Ai, i = 0, ...3 are the aerodynamic coeﬃcients for the squared cross-section. y Ai, i = 0, ...3 are the aerodynamic coeﬃcients for the squared cross-section. The dimensional critical velocity is given by √ The dimensional critical velocity is given by ¯Uc = 4πξ √ 3EIm ρbA1hℓ. ρ 1 Here ξ is the modal damping ratio, depending on both the external and internal dampings: ρ Here ξ is the modal damping ratio, depending on both the external and internal dampings: ξ = ηh2 24EI ω + c 2mω 08005-p.4 08005-p.4
https://openalex.org/W4384557994	https://www.researchsquare.com/article/rs-3128174/latest.pdf	English	null	The Impact of Urban Population on Housing Cost: The Case of Australia	Research Square (Research Square)	2,023	cc-by	9,592	DOI: https://doi.org/10.21203/rs.3.rs-3128174/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: (Not answered) Version of Record: A version of this preprint was published at npj Urban Sustainability on December 20th, 2023. See the published version at https://doi.org/10.1038/s42949-023-00136-7. Abstract 6 Rapid population expansions in urban areas have signiﬁcant implications for housing costs, 7 creating challenges for housing aﬀordability. However, estimating the causal eﬀect of popu- 8 lation on housing costs is challenging due to various confounding issues, such as unobserved 9 location-speciﬁc attributes, measurement error, and the potential bi-directional relationship 10 between population and housing costs. To address them, we adopt a city-level analysis and 11 introduce a novel instrumental variable (IV) that enables us to employ ﬁxed eﬀects IV es- 12 timation. Our ﬁndings indicate that housing costs tend to increase at a faster rate than 13 population growth. As individuals and households with lower incomes tend to allocate a 14 larger proportion of their earnings to housing expenses, an upward trajectory of housing 15 costs may dramatically widen the inequality in income after housing expenditure. 16 The Impact of Urban Population on Housing Cost: The Case 1 of Australia 2 Nicholas Simb Nicholas Simb Chris Leishmana Weidong Lianga a School of Business, University of South Australia b School of Business, Singapore University of Social Sciences 1 Introduction 17 Population expansions contribute to urban agglomeration and productivity growth, but they 18 also come with the potential drawback of increasing social cost, such as the cost of housing. 19 Balancing the beneﬁts and costs of urban agglomeration is a crucial objective for policymakers 20 in population planning. While the economic advantages of larger cities, including population 21 size and density, have been extensively studied (Ciccone and Hall, 1996; Ciccone, 2002), there 22 is a scarcity of quantiﬁed evidence regarding the speciﬁc costs associated with population 23 growth, particularly in relation to housing. For policymakers, having such evidence on the 24 costs and beneﬁts of population growth is important for managing the expansion of city 25 population. 26 As such, estimating the eﬀects of city population on urban costs has garnered signiﬁcant 27 interest over the years. However, obtaining such estimates is far from straightforward - besides 28 the usual concerns like omitted variable bias, the presence of measurement error could lead to 29 2 2 attenuation bias. Moreover, the high cost of living in urban areas may discourage population 30 growth (Cannari, Nucci and Sestito, 2000), leading to a reverse eﬀect. To address these 31 concerns, we propose a new approach to estimate the elasticity of housing cost with respect 32 to city population using Australian panel data. Our work employs ﬁxed eﬀects model to 33 eliminate the confounding eﬀects of unobserved heterogeneity, known as ﬁxed eﬀects, such as 34 unobserved location attributes that might jointly inﬂuence population growth and housing 35 cost. To address the confounding issues of reverse causality and measurement error, we 36 propose a new instrumental variable (IV) for city population to identify its eﬀect on housing 37 cost. 38 For our IV to be applicable within a panel ﬁxed eﬀects framework, it must contain both 39 cross-sectional and time variations. To this end, we construct a Bartik-style IV by interacting 40 data on city climate and visa issuance. A Bartik-style IV is one that is created by taking 41 the interaction between a time-varying variable common across all cross-sectional units and 42 a time-invariant variable that varies cross-sectionally. This interaction term will create an IV 43 that exhibits variations across both time and space, ensuring that its eﬀect on the endogenous 44 variable (i.e. city population) will not be washed out by the inclusion of ﬁxed eﬀects into the 45 regression model. Results 74 In this section, we present the estimation results from the OLS, reduced form, and 2SLS 75 regressions. Details on the data and statistical models are provided under Methods. Further 76 results from our robustness checks are provided under Supplementary Information. 77 Introduction 17 46 In our case, our time-varying variable is related to overseas immigration, which directly 47 contributes to population growth. Immigration has been a signiﬁcant driver of Australia’s 48 population expansion since 1995, and visa issuance, decided by the Australian federal gov- 49 ernment in response to the country’s labor market and economic conditions (Productivity 50 Commission, 2016), determines the annual number of new overseas migrants entering Aus- 51 tralia. For our work, it is important to emphasize that Australian cities do not have the 52 authority to inﬂuence visa issuance levels, which implies that visa issuance from a city plan- 53 ner’s perspective is taken as given. Our cross-sectional variable is city climate, which in- 54 ﬂuences people’s location choices and, consequently, city population levels (see, e.g., Roos, 55 2005; Jordan, 2007). Like visa issuance, it is reasonable to assume that climate is exogenous 56 3 3 (Dell, Jones and Olken, 2014). Thus, our IV for city population, which is constructed by 57 interacting climate and visa issuance, should be plausibly exogenous. 58 We implement two-stage least squares (2SLS) regression using our climate-visas IV as an 59 instrument for city population. In the ﬁrst stage, we ﬁnd a positive relationship between the 60 number of visas issued and population, particularly in cities with favorable climates. This 61 result aligns with the hypothesis that attractive climates draw in more migrants, leading to 62 more population growth (Combes, Duranton and Gobillon, 2019). Our second-stage estimates 63 indicate that a 1% increase in city population is associated with an average increase in home 64 prices ranging from 1.16% to 1.59%, and an average increase in rental prices ranging from 65 1.84% to 1.97%. These elasticities suggest that housing costs tend to increase at a faster rate 66 than population growth. 67 Overall, our study highlights the concerning trend that housing costs, especially rental 68 costs, tend to rise more quickly than population growth. As individuals and households with 69 lower incomes tend to allocate a larger proportion of their earnings to housing expenses, 70 an increase in housing cost induced by population growth can signiﬁcantly exacerbate the 71 inequality of income net of housing expenditure. Therefore, proactive measures are needed 72 to address the challenges posed by population growth on housing aﬀordability. 73 OLS estimates 78 Table 1 presents the OLS estimates of the elasticities of home and rental prices with respect 79 to current (populationist) and lagged (populationist−1) city population based on Eq. (1). The 80 4 results indicate that the elasticities of home prices are 0.461 and 0.412, respectively, while 81 the elasticities of rental prices are 0.384 and 0.297, respectively. All the OLS estimates show 82 that city population is statistically signiﬁcant for housing cost at the 1% level. Interestingly, 83 although the log of housing supply and employment rate show the expected signs, they are 84 statistically insigniﬁcant, suggesting that population growth plays a more signiﬁcant role in 85 driving home and rental prices in Australia than housing supply and employment. 86 Table 1: OLS Estimates of City Population on Housing Cost (1) (2) (3) (4) Dependent Variable log(home priceis,t) log(rental priceis,t) log(populationis,t) 0.461* (0.115) 0.384* (0.108) log(populationis,t −1) 0.412* (0.113) 0.297* (0.096) log(housing supplyis,t) -0.028 (0.025) -0.031 (0.025) -0.685 (0.029) -0.059 (0.026) employment rateis,t 0.001 (0.004) 0.001 (0.004) 0.002 (0.003) 0.002 (0.003) Adj. R2 0.72 0.72 0.79 0.78 City FE Yes Yes Yes Yes State Year FE Yes Yes Yes Yes Observations 4988 4821 4415 4248 Note: Robust standard errors clustered in the city level are reported in the parentheses. * p < 0.05, p < 0.01, * p < 0.001 Two-stage least squares estimates 100 Two-stage least squares estimates 100 Reduced form estimates 87 Table 2 presents the reduced form estimates of the eﬀect of our instrument (favourable climateis× 88 log(visast−1) or favourable climateis × log(visast−2)) on home and rental prices based on Eq. 89 (3). In Columns (1) and (2), the estimated eﬀects of our instrument on home prices are 0.578 90 and 0.772, respectively. This indicates that, on average, a 1% increase in visa issuance in 91 the previous year (t −1) or previous two years (t −2) would lead to an additional 0.578% or 92 5 0.772% increase in home prices in cities with a favourable climate compared to those without. 93 In Columns (3) and (4), the estimated eﬀects of our instrument on rental prices are 0.611 94 and 0.678, respectively. This indicates that, on average, a 1% increase in visa issuance in the 95 previous year or two years would lead to an additional increase in rental prices by 0.611% 96 to 0.678% in cities with a favourable climate compared to those without. These estimates 97 are statistically signiﬁcant at the 1% level, which suggests that population growth through 98 immigration is a signiﬁcant driver of housing cost especially in cities with favorable climates. 99 Table 2: Reduced Form Estimates of Instruments on Housing Cost (1) (2) (3) (4) Dependent Variable log(home priceis,t) log(rental priceis,t) favourable climateis × log(visast−1) 0.578* (0.015) 0.611* (0.020) favourable climateis × log(visast−2) 0.772* (0.017) 0.678* (0.020) log(housing supplyis,t) -0.052 (0.025) -0.033 (0.022) -0.028* (0.017) -0.029* (0.017) employment rateis,t 0.002 (0.004) 0.001 (0.004) 0.003 (0.003) 0.002 (0.003) Adj. R2 0.72 0.72 0.78 0.78 City FE Yes Yes Yes Yes State Year FE Yes Yes Yes Yes Observations 4988 4821 4415 4248 Note: Robust standard errors clustered in the city level are reported in the parentheses. * p < 0.05, p < 0.01, * p < 0.001 Table 2: Reduced Form Estimates of Instruments on Housing Cost Two-stage least squares estimates 100 Two-stage least squares estimates 100 Table 3 presents the two-stage least squares (2SLS) estimates of Eq. (1) with city population 101 instrumented as expressed by Eq. (2). The ﬁrst-stage estimation results show that the 102 coeﬃcients on our IV are positive and statistically signiﬁcant at the 1% level. Speciﬁcally, 103 an increase of 1% in visa issuance by the federal government in year t −1 is associated 104 6 with an additional increase of 0.035% to 0.050% in population in cities with a favourable 105 climate compared to those without it (see Columns (1) and (3) of the lower panel in Table 106 3). Similarly, an increase of 1% in visa issuance in year t −2 results in an additional increase 107 of 0.036 to 0.049% in population in cities with a favourable climate. Furthermore, the 108 Kleibergen-Paap ﬁrst-stage F-statistics are all greater than the critical value at the 10% 109 level, indicating that our IV is a powerful instrument for city population. 110 with an additional increase of 0.035% to 0.050% in population in cities with a favourable 105 climate compared to those without it (see Columns (1) and (3) of the lower panel in Table 106 3). Similarly, an increase of 1% in visa issuance in year t −2 results in an additional increase 107 of 0.036 to 0.049% in population in cities with a favourable climate. Furthermore, the 108 Kleibergen-Paap ﬁrst-stage F-statistics are all greater than the critical value at the 10% 109 level, indicating that our IV is a powerful instrument for city population. 110 The second-stage estimation results in Table 3 conﬁrm that city population expansions 111 would drive up both home and rental prices. For example, a 1% increase in city population 112 would, on average, lead to a 1.164% to 1.589% increase in home prices (see Columns (1) and 113 (2) of Table 3). Similarly, a 1% increase in city population would result in a 1.843% to 1.972% 114 increase in rental prices on average (see Columns (3) and (4) of Table 3). Comparing the 115 OLS estimates of the eﬀects of city population reported in Table 1 with the 2SLS estimates 116 reported here, we observe that the latter are about three times larger than the former. 1Refer to Table A1 on page 1586 in Combes, Duranton and Gobillon (2019) for their two-stage least squares estimates. It should be emphasized that Combes, Duranton and Gobillon (2019) focused on at land prices. However, the appreciation of existing homes can be attributed mainly to the increase in land prices, which suggests that our analysis is comparable to Combes, Duranton and Gobillon (2019). Two-stage least squares estimates 100 This 117 discrepancy highlights the downward bias in the OLS estimates if confounding eﬀects such as 118 reverse causality and measurement error are not accounted for. The 2SLS results therefore 119 underscore the importance of addressing these issues when estimating the impact of city 120 populations on housing costs as the impact size could be vastly understated. 121 Our 2SLS results are consistent with the conclusion drawn by Combes, Duranton and 122 Gobillon (2019), namely, that city population expansions inevitably lead to increases in 123 housing costs. However, our second-stage estimates of city population on housing costs are 124 considerably larger than those reported by Combes, Duranton and Gobillon (2019).1 This 125 disparity may be attributed to the application of an instrumental variable approach within a 126 panel data setting, which enables us to address the issues of reverse causality, measurement 127 errors, and unobserved heterogeneity. By contrast, Combes, Duranton and Gobillon (2019) 128 conducted their analysis using a pooled cross-sectional regression without accounting for city 129 7 ﬁxed eﬀects, which could confound the impact of city population on housing costs. 130 Table 3: 2SLS Estimates of City Population on Housing Cost (1) (2) (3) (4) Dependent Variable (second stage) log(home priceis,t) log(rental priceis,t) log(populationis,t) 1.164* (0.297) 1.589* (0.334) 1.843* (0.555) 1.972* (0.559) log(housing supplyis,t) -0.009 (0.031) -0.620* (0.038) -0.217 (0.102) -0.234 (0.111) employment rateis,t -0.006 (0.005) -0.010** (0.005) -0.009 (0.006) -0.011* (0.007) Dependent Variable (ﬁrst-stage) log(populationis,t) log(populationis,t) favourable climateis × log(visast−1) 0.050* (0.015) 0.035* (0.004) favourable climateis × log(visast−2) 0.049* (0.005) 0.036* (0.004) Kleibergen-Paap Wald F-Statistic 97 106 73 77 Stock and Yogo Critical Value (10%) 16.38 16.38 16.38 16.38 City FE Yes Yes Yes Yes State Year FE Yes Yes Yes Yes Observations 4988 4821 4415 4248 Note: Robust standard errors clustered in the city level are reported in the parentheses. * p < 0.05, p < 0.01, * p < 0 001 Table 3: 2SLS Estimates of City Population on Housing Cost Discussion 131 The study explores an implication of rising urban population in terms of housing costs in 132 Australia. We ﬁnd strong evidence that city populations have a signiﬁcant impact on housing 133 costs. Furthermore, our elasticity estimates suggest that housing costs, particularly rental 134 costs, tend to increase at a faster rate than population growth. 135 As such, our paper underscores the potential for population growth to exacerbate the 136 8 inequality of income after housing expenditure, which can be driven by rising housing costs. 137 Several studies have highlighted the link between population growth and housing cost. Us- 138 ing data from Organisation for Economic Co-operation and Development (OECD) counties, 139 Gevorgyan (2019) found that if population growth increases by one percentage point, house 140 price growth increases by 1.4 percentage points. Using data from Amsterdam and Paris, 141 Francke and Korevaar (2022) found that a one percentage point increase in the current birth 142 rate increases house prices about 25-30 years later by 4 to 5%. Our paper contributes to this 143 literature by showing that housing costs may escalate at a faster rate than population growth 144 in the Australian city-level context. As individuals and households with lower incomes tend 145 to allocate a larger proportion of their earnings to housing expenses (Dustmann, Fitzenberger 146 and Zimmermann, 2021), an upward trajectory of housing costs may dramatically widen the 147 inequality in income net of housing expenses. 148 inequality of income after housing expenditure, which can be driven by rising housing costs. 137 Several studies have highlighted the link between population growth and housing cost. Us- 138 ing data from Organisation for Economic Co-operation and Development (OECD) counties, 139 Gevorgyan (2019) found that if population growth increases by one percentage point, house 140 price growth increases by 1.4 percentage points. Using data from Amsterdam and Paris, 141 Francke and Korevaar (2022) found that a one percentage point increase in the current birth 142 rate increases house prices about 25-30 years later by 4 to 5%. Our paper contributes to this 143 literature by showing that housing costs may escalate at a faster rate than population growth 144 in the Australian city-level context. Discussion 131 As individuals and households with lower incomes tend 145 to allocate a larger proportion of their earnings to housing expenses (Dustmann, Fitzenberger 146 and Zimmermann, 2021), an upward trajectory of housing costs may dramatically widen the 147 inequality in income net of housing expenses. 148 Methodologically, our paper contributes to the existing literature in the following ways. 149 Firstly, to the best of our knowledge, our study is among the ﬁrst to estimate the eﬀect of 150 city population on housing costs by implementing an instrumental variable panel data ap- 151 proach. Previous studies by Thomas (1980), Richardson (1987), and Henderson (2002) have 152 examined the association between urban costs and population expansion. These studies have 153 found a positive relationship between population size and the cost of living (Thomas, 1980), 154 and have documented that infrastructure spending, commuting time, and rental prices may 155 increase due to urbanization (Richardson, 1987; Henderson, 2002). However, as they do not 156 consider an identiﬁcation strategy, their point estimates could be biased and inconsistent. 157 Combes, Duranton and Gobillon (2019), on the other hand, employed an instrumental vari- 158 able approach as an identiﬁcation strategy to estimate the elasticity of housing costs (i.e. 159 house and land prices) with respect to city population using French data. However, their 160 study was based on a pooled cross-sectional setting. Without employing panel data, their 161 estimation approach was unable to account for city ﬁxed eﬀects, which could jointly deter- 162 mine both population and housing cost. In our study, we employ an instrumental variable 163 9 approach in a panel data setting so that we may address both the issue of reverse causality 164 and unobserved heterogeneity. 165 Secondly, our paper introduces a new method for estimating the relationship between 166 urban population and housing cost. Previous studies have instrumented city population using 167 historical population levels (Ciccone and Hall, 1996; Combes, Duranton and Gobillon, 2008; 168 Duranton, 2016; Combes, Duranton and Gobillon, 2019). However, historical population 169 levels may be unsuitable as instruments as they are endogenous to housing cost (Sharpe, 2019; 170 Broxterman and Larson, 2020). Discussion 131 Other studies have explored using geological characteristics 171 such as fertile soil to explain population size (Combes et al., 2010; Combes, Duranton and 172 Gobillon, 2011), but using such data to construct an instrument for population size may be 173 diﬃcult to justify for a country like Australia whose economy is not primarily driven by the 174 agricultural sector. Finally, there are studies that linked city population to city amenities 175 such as the number of hotel rooms (Carlino and Saiz, 2008; Combes, Duranton and Gobillon, 176 2019), but the theoretical justiﬁcation for this relationship may be challenging and such 177 ﬁne-level data may not be available. Our approach has the advantage of constructing an 178 instrument using publicly accessible data on national-level visa issuance and city climate, 179 which makes the construction of such an instrument potentially more feasible for studies 180 based on other countries. 181 2The employment rate data are derived from the ABS Census conducted in 2001, 2006, and 2011, and are not available on a yearly basis. Therefore, we employ extrapolation methods to estimate the employment rates for the missing years between Censuses. 4For additional information regarding these data, please refer to https://www.homeaffairs.gov.au/ research-and-statistics/statistics/visa-statistics/live/migration-program. g p g 5Please note that our study considers only seven climate zones instead of eight, as previously mentioned in Footnote 10. g y 3These categories account for over 70% of the total visas issued. However, visitor visas, corresponding to short-term stays in Australia, are excluded from our analysis. 4 Data 183 Our dataset comprises a panel of 513 Australian cities, speciﬁcally deﬁned as Local Govern- 184 ment Areas (LGA), covering the period from 2003 to 2016. The housing cost and supply 185 data are obtained from the Australian Urban Research Infrastructure Network (AURIN) and 186 are reported on a monthly basis. To capture housing costs in cities, we utilize the transacted 187 average home and rental prices for each city. Additionally, the total number of houses listed 188 10 in the market is used as a proxy for city housing supply. In order to align with the frequency 189 of our data (population, visa issuance, and employment), we aggregate the monthly data into 190 yearly frequencies. 191 Data on city populations and employment rates are sourced from the Australian Bureau 192 of Statistics (ABS).2 For visa issuance, we consider visas issued to permanent and temporary 193 skilled migrants (residents), international students, and long-stay businessmen.3 The visa 194 issuance data are obtained from the Department of Home Aﬀairs.4 The summary statistics 195 on the variables are presented in Table 4. 196 Table 4: Summary Statistics j p p yp p p 7In this context, “overseas migrants” refers to the sum of all Australian temporary and permanent resi- dents/migrants. Temporary migrants include individuals with temporary visas for purposes such as studying and working. Our study excludes temporary visitors who come to Australia as tourists. 6For more information, please visit the climate zone map in ABCB and the maps on the Bureau of Me- teorology’s website at http://www.bom.gov.au/jsp/ncc/climate_averages/climate-classifications/ index.jsp?maptype=tmp_zones#maps. Table 4: Summary Statistics Variable obs. mean s.d. min. max. log(population) 7,140 9.43 1.72 4.53 14.00 log(visas) 7,182 5.06 0.19 4.68 5.25 log(rental price) 4,582 5.55 0.36 4.17 7.55 log(house price) 5,621 12.49 0.70 8.72 15.07 log(house supply) 6,656 8.10 1.88 5.62 12.62 employment rate 5,515 0.56 0.07 0.36 0.84 For the climate of cities, we utilize the city climate zone data provided by the Bureau 197 of Meteorology (BoM). The BoM categorized all Australian cities into seven climate zones 198 based on historical climate conditions, speciﬁcally precipitation, temperature, and humidity 199 levels between 1961 and 1990. The climate zone classiﬁcation is depicted in Figure 7 above.5 200 For instance, if a city’s average annual temperature, average annual 9 am humidity, and 201 For the climate of cities, we utilize the city climate zone data provided by the Bureau 197 of Meteorology (BoM). The BoM categorized all Australian cities into seven climate zones 198 based on historical climate conditions, speciﬁcally precipitation, temperature, and humidity 199 levels between 1961 and 1990. The climate zone classiﬁcation is depicted in Figure 7 above.5 200 For instance, if a city’s average annual temperature, average annual 9 am humidity, and 201 2The employment rate data are derived from the ABS Census conducted in 2001, 2006, and 2011, and are not available on a yearly basis. Therefore, we employ extrapolation methods to estimate the employment rates for the missing years between Censuses. 3 4For additional information regarding these data, please refer to https://www.homeaffairs.gov.au/ research-and-statistics/statistics/visa-statistics/live/migration-program. 11 average annual rainfall levels fell within the ranges of 18 to 24 degrees Celsius, 50 to 70 202 percent, and 1,000 to 2,000 millimeters, respectively, between 1961 and 1990, it is classiﬁed 203 under “Zone 2: Warm humid summer and mild winter”. On the other hand, cities with 204 average annual temperatures between 9 and 18 degrees Celsius, average annual 9 am humidity 205 levels of 70 to 80 percent, and average annual rainfall of 600 to 1500 millimeters within the 206 same period are assigned to “Zone 7: Cool temperate”. 207 To conserve space, we will not present the detailed construction of the remaining zones 208 based on average humidity, temperature, and precipitation levels between 1961 and 1990. 209 Interested readers can refer to the climate zone map provided by the Australian Building 210 Codes Board (ABCB) and the maps available on the Bureau of Meteorology’s website.6 211 Population growth and visa issuance 212 Australia has experienced signiﬁcant population growth over the years. Figure 1 illustrates 213 this trend, showing an increase in population from 3.7 million in 1901 to 25 million in 2019, 214 with a projected growth to 40 million by 2051. Among OECD countries, Australia had 215 the third fastest growing population (OECD, 2019). Notably, the population expansion in 216 Australia has been particularly rapid in the last two decades, with an annual increase of over 217 325,000 people. 218 This population growth can be attributed to two main factors: new births and overseas 219 migration. Figure 2 sheds some light on the contribution of each factor. Before 2006, the 220 quarterly average of new births remained around 60,000, but modestly increased to 78,000 221 in 2017. By contrast, the quarterly average of new overseas migrants arriving in Australia 222 had more than tripled, rising from below 50,000 in 1982 to 138,000 in 2017.7 223 6For more information, please visit the climate zone map in ABCB and the maps on the Bureau of Me- teorology’s website at http://www.bom.gov.au/jsp/ncc/climate_averages/climate-classifications/ index.jsp?maptype=tmp_zones#maps. j p p yp p p 7In this context, “overseas migrants” refers to the sum of all Australian temporary and permanent resi- dents/migrants. Temporary migrants include individuals with temporary visas for purposes such as studying and working. Our study excludes temporary visitors who come to Australia as tourists. 12 Figure 1: Australian Population Level (1901-2050) Figure 1: Australian Population Level (1901-2050) Notes: The population data are estimated data and extracted from the ABS. Population data after 2019 are projected by the ABS. Figure 1: Australian Population Level (1901-2050) Notes: The population data are estimated data and extracted from the ABS. Population data after 2019 are projected by the ABS. Notes: The population data are estimated data and extracted from the ABS. Population data after 2019 are projected by the ABS. 13 Figure 2: Australian Population Expansion: New Births vs Overseas Migrants (Time Series) Source: Australian Bureau of Statistics. Figure 2: Australian Population Expansion: Figure 2: Australian Population Expansion: g p p New Births vs Overseas Migrants (Time Series) New Births vs Overseas Migrants (Time Series) New Births vs Overseas Migrants (Time Series) Source: Australian Bureau of Statistics. 8To calculate the percentage of new births (or overseas migration) in Australian population expansion each year, we divide the annual number of new births (or new overseas migrants) by the annual total new population (i.e., the sum of new births and overseas migrants). For example, if there were 10 new births and 20 new overseas migrants in 2002, the total new population would be 30. The percentage of new births in the new population would be approximately 33% in 2002. 9As depicted in Figure 4, the federal government temporarily reduced the number of visas issued in 2009 and 2010 in response to the economic downturn caused by the Global Financial Crisis. Population growth and visa issuance 212 Examining the proportion of population growth attributed to new births and overseas 224 migration, Figure 3 reveals that between 1982 and 1995, approximately 54% of population 225 expansion came from new births while 46% was due to new overseas migrant intakes.8 How- 226 ever, in 2017, new overseas migrants accounted for 65% of the population growth while 227 births contributed to the remaining 35%. Thus, new overseas migrant intakes have become 228 the primary driving force behind Australia’s population expansion in recent years. 229 14 Figure 3: Australian Population Expansion: Figure 3: Australian Population Expansion: New Births vs Overseas Migrants (Proportions) Source: Australian Bureau of Statistics. g p p New Births vs Overseas Migrants (Proportions) Source: Australian Bureau of Statistics. g p p New Births vs Overseas Migrants (Proportions) g p p New Births vs Overseas Migrants (Proportions) Source: Australian Bureau of Statistics. Unsurprisingly, the growth in population driven by overseas migration is closely linked to 230 the number of visas issued by the Australian federal government. The government carefully 231 manages the inﬂux of overseas migrants into Australia, taking into account the prevailing la- 232 bor market conditions in the country (Productivity Commission, 2016). This approach allows 233 the government to respond to labor supply shortages in speciﬁc sectors such as healthcare, in- 234 formation technology, engineering, and construction trades (Productivity Commission, 2016). 235 For example, starting from 1995, the federal government increased the issuance of visas to at- 236 tract immigrants from these occupational groups (Spinks, 2010).9 Consequently, the number 237 15 of visas issued had increased signiﬁcantly since 1995, as shown in Figure 4. of visas issued had increased signiﬁcantly since 1995, as shown in Figure 4. of visas issued had increased signiﬁcantly since 1995, as shown in Figure 4. 238 Figure 4: Populations vs Visa Issuance in Australia Data Sources: ABS and the Department of Home Aﬀairs Figure 4: Populations vs Visa Issuance in Australia Data Sources: ABS and the Department of Home Aﬀairs To examine the relationship between visa issuance and Australia’s population, we analyze 239 the log forms of both variables from 1996 to 2016, as shown in Figure 4. The upward trends of 240 log(population) and log(visa issuance) indicate a positive relationship between visa issuance 241 and Australia’s population at the national level. Population growth and visa issuance 212 Since national populations are composed 242 of city populations, we can also infer a positive association between visa issuance and city- 243 level populations. To further explore this relationship, we select four Australian capital 244 cities and plot their populations against the number of visas issued, as depicted in Figure 245 5. The similarity in the trends of the log number of visas issued and the log populations of 246 these selected cities supports the argument that population growth in these cities is driven 247 16 rily by migration, which in turn depends on the number of visas issued. primarily by migration, which in turn depends on the number of visas issued. Figure 5: City Populations and Visa Issuance Data Sources: ABS and Department of Home Aﬀairs Figure 5: City Populations and Visa Issuance ions and Visa Issuance Figure 5: City Populat Data Sources: ABS and Department of Home Aﬀairs Climate and population spatial variation in Australia 249 Climate and population spatial variation in Australia 249 Climate and population spatial variation in Australia 249 The population distribution in Australia exhibits signiﬁcant spatial variation. As shown in 250 Figure 6, the populations of certain cities, such as MacDonnell and Diamantina, located in 251 the middle of Australia, average less than 10,000 between 2001 and 2020. By contrast, the 252 coastal city of Brisbane has consistently housed over 1 million people during the same period. 253 The considerable disparity in city populations across Australia can be attributed, in 254 part, to variations in climate conditions (Cheshire and Magrini, 2006; Albouy and Stuart, 255 2014). When selecting their residential locations, households take into account local climate 256 17 conditions and are more inclined to settle in areas with a more favorable climate for living 257 (Jordan, 2007). For instance, individuals often prefer suburban, sunny, and coastal areas 258 characterized by mild, warm, or cool climates (see, e.g., Cragg and Kahn, 1997; Jordan, 259 2007; Albouy, Leibovici and Warman, 2013; Albouy and Lue, 2015). On the other hand, 260 they are generally reluctant to reside in remote areas characterized by hot-dry summers or 261 extremely cold winters (Maddison and Bigano, 2003; Sinha and Cropper, 2013; Albouy et al., 262 2016). 263 conditions and are more inclined to settle in areas with a more favorable climate for living 257 (Jordan, 2007). For instance, individuals often prefer suburban, sunny, and coastal areas 258 characterized by mild, warm, or cool climates (see, e.g., Cragg and Kahn, 1997; Jordan, 259 2007; Albouy, Leibovici and Warman, 2013; Albouy and Lue, 2015). On the other hand, 260 they are generally reluctant to reside in remote areas characterized by hot-dry summers or 261 extremely cold winters (Maddison and Bigano, 2003; Sinha and Cropper, 2013; Albouy et al., 262 2016). 263 To gain further insight into how climate conditions are associated with population spatial 264 variation in Australia, we adopt the climate zone classiﬁcation provided by the Bureau of 265 Meteorology (BoM) in Australia. As shown in Figure 7, the Australian cities are categorized 266 into seven climate zones.10 Examining the population levels of cities within each climate 267 zone, Table 5 provides the average city population between 2001 and 2018, as well as the 268 average number of overseas migrants in each city from 2016 to 2019, for each climate zone. 10These climate zone data are developed by BoM to assist the Australian Building Codes Board (ABCB) in regulating the building and construction industry. The data can be accessed at https://www.abcb.gov.au/Resources/Tools-Calculators/Climate-Zone-Map-Australia-Wide. BoM developed eight zones for ABCB, but for this study, we consider the cities with some alpine-climate areas as having a cool temperate climate, resulting in seven climate zones being analyzed. Climate and population spatial variation in Australia 249 269 The data reveal that cities characterized by a warm-summer, mild, or cool climate have 270 signiﬁcantly higher populations compared to those with hot or hot-dry summer climates. 271 Hence, the disparity in climate conditions may explain the spatial distribution of Australian 272 city populations, with cities possessing a more livable climate (i.e., warm, mild, or cool) 273 attracting larger populations and overseas migrants. 274 18 Figure 6: Average City Population (2001-2020) Notes: City Population data are from ABS Notes: City Population data are from ABS Notes: City Population data are from ABS 19 19 Figure 7: Climate Zones in the Australia Figure 7: Climate Zones in the Australia Figure 7: Climate Zones in the Australia Table 5: Climate and Population and Migrant Spatial Variation Climate Zone Average City Population (2001-2018) Average City Overseas Migrants (2016-2019) Zone 2: Warm humid summer and mild winter 128,223 116,980 Zone 6: Mild temperate 64,053 84,472 Zone 5: Warm temperate 60,329 30,126 Zone 7: Cool temperate 25,243 10,127 Zone 1: Hot humid summer and warm winter 15,022 3,364 Zone 4: Hot dry summer and cool winter 7,428 4,590 Zone 3: Hot dry summer and mild winter 7,140 841 Table 5: Climate and Population and Migrant Spatial Variation Average City Population Average City Overseas Migrants Average City Population Average City Overseas Migrants 20 The model 275 Our primary model examines the relationship between the logarithm of housing costs (i.e., 276 home and rental prices), denoted as log(pricek is,t), and the logarithm of population, denoted 277 as log(populationis,t), for city i, state s, and year t. The equation is speciﬁed as follows: 278 log(pricek is,t) = c + α log(populationis,t) + γ′xis,t + µi + µst + ǫis,t (1) log(pricek is,t) = c + α log(populationis,t) + γ′xis,t + µi + µst + ǫis,t (1) (1) where the superscript k indexes home or rental price. The vector xis,t consists of a set of 279 control variables that include the log of housing supply and employment rates. The terms 280 µi and and µst represent the city ﬁxed eﬀects and state-year ﬁxed eﬀects, respectively. The 281 variable ǫis,t denotes the idiosyncratic error term clustered at the city level. 282 The main focus of this study is to estimate the parameter α in Eq. (1). This represents 283 the elasticity of housing cost with respect to city population. To achieve this objective, we 284 incorporate various ﬁxed eﬀects and control variables into Eq. (1). The inclusion of city 285 ﬁxed eﬀects, µi, enable us to control for time-invariant city-speciﬁc characteristics such as 286 location and land size and other unobserved location-related attributes. The inclusion of 287 state-year ﬁxed eﬀects, µst, enable us to control for the inﬂuence of macroeconomic variables 288 and macroeconomic shocks (e.g., interest rates and Global Financial Crisis), as well as for 289 factors that vary across states and years such as annual economic and labor market conditions 290 within states. We also incorporate city housing supply and employment rate in Eq. (1) 291 to control for local housing and labor market conditions, which are likely correlated with 292 population sizes and housing costs (Zabel, 2012). 293 Despite the inclusion of these ﬁxed eﬀects and control variables, we may still encounter 294 challenges that would hinder the identiﬁcation of α, the eﬀect of city population on housing 295 costs. The ﬁrst challenge arises from measurement errors in log(populationis,t). Since the 296 city population data used in our study are estimated, measurement errors could be prevalent. 297 21 Consequently, if these measurement errors were classical, the estimate of α would be biased 298 toward zero. 299 The second challenge is related to reverse causality. The model 275 On the one hand, the expansion 300 of city population can drive up local housing costs (see, for example, Gonzalez and Ortega 301 (2013); Accetturo et al. (2014); Combes, Duranton and Gobillon (2019). On the other hand, 302 high housing costs may discourage people from moving to certain cities (Cannari, Nucci and 303 Sestito, 2000). Therefore, given this bi-directional relationship, it is important to disentangle 304 the eﬀect of city population on housing cost from the reverse confounding eﬀect. 305 Lastly, other determinants of city housing costs that are correlated with population may 306 be captured in the error term ǫis,t. For example, the safety of a city can inﬂuence both 307 population levels and house prices (Klimova and Lee, 2014). If the inﬂuence of unobserved 308 characteristics on housing cost and city population is not eliminated, the OLS estimates 309 could still be susceptible to omitted variable bias. 310 Estimation strategy 311 To address the aforementioned issues, we propose an instrumental variable (IV) approach 312 within a panel data framework to estimate the relationship between city populations and 313 housing costs. Our IV strategy involves interacting two variables that exhibit exogenous 314 variations across cities and over time. 315 The ﬁrst variable is city climate, which is considered to be exogenous to economic out- 316 comes such as housing cost (Roos, 2005; Dell, Jones and Olken, 2014). Climate can inﬂuence 317 residential choices, and cities with more favorable climates, characterized by mild, warm, 318 or cool conditions, tend to have larger populations compared to cities with less favorable 319 climates, such as those with hot-dry summers or extremely cold winters (Cragg and Kahn, 320 1997; Jordan, 2007; Albouy and Lue, 2015).11 In the context of Australia, cities with more 321 livable climates tend to have larger populations compared to cities with hot-dry summer cli- 322 11Please also refer to Albouy, Leibovici and Warman (2013). 22 mates. Therefore, we classify mild, warm, and cool climates as favorable climates and indicate 323 them with a dummy variable, favorable climateis. This variable serves as the cross-sectional 324 component of our IV. 325 The second variable is the number of visas issued, which we argue is plausibly exogenous 326 with respect to housing cost. As shown earlier, overseas migration is the primary driver of 327 population expansion in Australia since 1995. The Australian federal government operates 328 the Migration Program, and the issuance of visas positively inﬂuences overseas migration 329 to Australia. Thus, the annual number of visas issued can be considered a determinant of 330 Australian city populations (see Figure 5). Importantly, since the number of visas issued 331 is determined by the federal government based on the country’s labor market needs, visa 332 issuance should be exogenous to current housing costs in the cities. Therefore, we utilize 333 log(visast−j), the log of the number of visas issued at time t −j where j = 1 or 2, as the 334 time-varying component of our IV. 335 Our ﬁrst-stage regression model is deﬁned as follows: log(populationis,t) =c + βj × favorable climateis × log(visast−j) + θ′xis,t log(populationis,t) =c + βj × favorable climateis × log(visast−j) + θ′xis,t + µi + µst + wis,t, + µi + µst + wis,t, (2) (2) where our IV is the interaction between favorable climateis and log(visast−j). 12It should be noted that there are visa programs that are associated with investments in real estate. However, this is not the case for Australia. Estimation strategy 311 The main iden- 336 tifying assumption is that the number of visas issued aﬀects housing cost solely through its 337 impact on city populations. This assumption would be violated if: 1) visa issuance directly 338 aﬀects local housing costs, implying that it is not an excluded factor, or 2) housing costs 339 reverse causally inﬂuence visa issuance. The ﬁrst concern is irrelevant since the number of 340 visas issued by itself does not directly impact city housing costs, but rather, potentially in- 341 ﬂuences them through its eﬀect on city population size.12 The second concern is also unlikely 342 as visa issuance is inﬂuenced by the Migration Program designed by the Australian federal 343 23 government to address the labor conditions of the entire country (Productivity Commission, 344 2016). Nevertheless, to ensure the validity of our instrument, we utilize the lagged number 345 of visas issued, which is predetermined with respect to home and rental prices. 346 Our main estimation approach employs two-stage least squares (2SLS) regression, where Eq. (1) is estimated as a second-stage model in conjunction with Eq. (2) as the ﬁrst- stage model. This allows us to address the issues of reverse causality and measurement error associated with city populations. In addition, we also estimate the inﬂuence of our instrument on housing costs via the following reduced form regression: log(pricek is,t) =c + δj × favorable climateis × log(visast−j) + ψ′xis,t (3) + µi + µst + ηis,t. (3) + µi + µst + ηis,t. This speciﬁcation allows us to explore the combined eﬀect of favorable climate and visas 347 issuance on housing costs, while controlling for other factors captured by the vector of control 348 variables ψ′xis,t. 349 Further remarks on the estimation strategy 350 Further remarks on the estimation strategy 350 Further remarks on the estimation strategy 350 Our estimation strategy bears similarities to the shift-share instrument commonly used in 351 urban and housing literature (e.g., Saiz, 2007; Gonzalez and Ortega, 2013; Accetturo et al., 352 2014; Sharpe, 2019). These studies construct instruments by interacting the historical share 353 of migrant population to total population at the local level, which provides cross-sectional 354 variation, with the current national migrant level, which provides time variation. The ratio- 355 nale is that the current location decisions of immigrants are expected to be inﬂuenced by the 356 location decisions of earlier immigrants (say, from the same country of origin). Therefore, 357 this interaction term can be interpreted as an approximation of the yearly immigration level 358 to a local area. 359 However, the validity of such an IV has been subject to debate. For it to be valid, the 360 24 cross-sectional variation, i.e. the historical share of migrant population to total population, 361 must be exogenous. However, Sharpe (2019) and Broxterman and Larson (2020) have argued 362 that the historical migrant population share could be inﬂuenced by housing costs. Moreover, 363 it could also be correlated with initial economic conditions, city characteristics, and housing 364 cost (Sharpe, 2019). Consequently, the exclusion restriction assumption necessary for the 365 validity of such instruments may not hold. 366 By contrast, our new instrument addresses these issues by relying on climate conditions 367 to generate the cross-sectional variation in our instrument rather than the historical share 368 of migrant population. Unlike the latter, a city’s climate is exogenous to economic vari- 369 ables including housing costs (Hsiang, Burke and Miguel, 2013; Dell, Jones and Olken, 2014; 370 Hsiang, 2016). Additionally, the time-varying component in our instrument - national-level 371 visa issuance - is determined by the Australian federal government based on the country’s 372 overall labor market conditions. Therefore, our proposed instrument, which is based on the 373 interaction between city climate and visa issuance, is plausibly exogenous to city housing 374 costs. 375 Data Availability 376 All data related to this article, as well as the codes used to process the data, are available 377 from the authors upon request. 378 25 25 References 379 Accetturo, Antonio, Francesco Manaresi, Sauro Mocetti and Elisabetta Olivieri. 2014. “Don’t 380 stand so close to me: The urban impact of immigration.” Regional Science and Urban 381 Economics 45:45–56. 382 Albouy, David and Bert Lue. 2015. “Driving to opportunity: Local rents, wages, commuting, 383 and sub-metropolitan quality of life.” Journal of Urban Economics 89:74–92. 384 Albouy, David and Bryan Stuart. 2014. Urban population and amenities: The neoclassical 385 model of location. 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The case of Sydney.” Economic record 90:16–40. 439 Maddison, David and Andrea Bigano. 2003. “The amenity value of the Italian climate.” 440 Journal of Environmental Economics and Management 45(2):319–332. 441 OECD. 2019. “The new immigrants global trends in migration towards OECD countries 442 between 2000/01 and 2015/16.” https://www.oecd.org/migration/ . 443 Productivity Commission. 2016. “Migrant intake into Australia.” Inquiry Report (77):313– 444 332. 445 27 Richardson, Harry W. 1987. “The costs of urbanization: A four-country comparison.” Eco- 446 nomic Development and Cultural Change 35(3):561–580. 447 Roos, Michael WM. 2005. “How important is geography for agglomeration?” Journal of 448 Economic Geography 5(5):605–620. 449 Saiz, Albert. 2007. “Immigration and housing rents in American cities.” Journal of Urban 450 Economics 61(2):345–371. 451 Sharpe, Jamie. 2019. “Re-evaluating the impact of immigration on the US rental housing 452 market.” Journal of Urban Economics 111:14–34. 453 Sinha, Paramita and Maureen L Cropper. 2013. 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Corresponding author 481 Correspondence to Nicholas Sim. 482 Authors and Aﬃliations 470 School of Business, University of South Australia, Adelaide, SA 5000, Australia. 471 Chris Leishman and Weidong Liang 472 473 School of Business, Singapore University of Social Sciences, 463 Clementi Road, 474 Singapore 599494, Singapore. 475 Nicholas Sim 476 School of Business, Singapore University of Social Sciences, 463 Clementi Road, 474 Singapore 599494, Singapore. 475 Competing interest 467 The Authors declare no competing ﬁnancial or non-ﬁnancial interests. 468 The Authors declare no competing ﬁnancial or non-ﬁnancial interests. 468 28 28 Author information 469 Author information 469 Authors and Aﬃliations 470 School of Business, University of South Australia, Adelaide, SA 5000, Australia. 471 Chris Leishman and Weidong Liang 472 473 School of Business, Singapore University of Social Sciences, 463 Clementi Road, 474 Singapore 599494, Singapore. 475 Nicholas Sim 476 Contributions 477 CL: Research design and conceptualization, manuscript writing and editing. WL: Data 478 preparation, methods design, data analysis, manuscript writing and editing. NS: Methods 479 design, data analysis, manuscript writing and editing. 480 Corresponding author 481 Supplementary information 483 The supplementary information provides further details on two additional robustness checks 484 that we conducted for the reduced form and 2SLS regressions. 485 The ﬁrst robustness check examines how sensitive these estimates are to the way “favourable” 486 climates are classiﬁed. In the baseline model, we have considered mild, warm and cool cli- 487 mates as favourable climates in the estimations above. Here, we add hot-humid summer cli- 488 mate to the list, construct a new favorable climate indicator ^ climateis to reﬂect this change, 489 mate to the list, construct a new favorable climate indicator climateis to reﬂect this change, 489 29 and interact it with log(visat−j) to construct our IV for our reduced form and 2SLS regres- 490 sion. The second robustness check uses a new measure of visa, log(g visat−j), that includes only 491 visas issued for permanent skill migrants and all migrants with an intention of staying over 492 one year, to construct our IV (i.e. log(g visat−j) excludes visas for temporary and short-term 493 visitors to Australia). Thus, we may think of log(g visat−j) as measuring the expansion of a 494 stable population who live in Australia over a longer term. 495 and interact it with log(visat−j) to construct our IV for our reduced form and 2SLS regres- 490 sion. The second robustness check uses a new measure of visa, log(g visat−j), that includes only 491 visas issued for permanent skill migrants and all migrants with an intention of staying over 492 one year, to construct our IV (i.e. log(g visat−j) excludes visas for temporary and short-term 493 visitors to Australia). Thus, we may think of log(g visat−j) as measuring the expansion of a 494 stable population who live in Australia over a longer term. 495 Robustness checks on reduced form model 496 Tables S1 and S2 present the reduced form estimates when ^ climateis or log(g visat−j) are used to 497 construct our alternative instruments. The results show that, under these alternative IVs, the 498 combined eﬀects of favourable climates and visa issuance on home and rental prices remain 499 positive. This suggests that population growth through immigration drives up housing costs 500 more in cities with better climates compared to those without. Additionally, the magnitudes 501 of the coeﬃcients on the alternative IVs are similar to those presented in Table 2. Therefore, 502 our estimates appear to be robust to diﬀerent deﬁnitions of favourable climates and the use 503 of alternative measures for visa issuance. 504 30 30 30 able S1: Reduced Form Estimates of New Instruments on Housing Cost (Alternative F rable Climate Deﬁnition) (1) (2) (3) (4) Dependent Variable log(home priceis,t) log(rental priceis,t) ^ favourable climateis × log(visast−1) 0.62* (0.016) 0.40 (0.016) ^ favourable climateis × log(visast−2) 0.82* (0.018) 0.45 (0.017) log(housing supplyis,t) -0.052** (0.025) -0.033 (0.022) -0.029* (0.018) -0.029* (0.017) employment rateis,t 0.001 (0.004) 0.001 (0.004) 0.003 (0.003) 0.003 (0.002) Adj. R2 0.72 0.69 0.78 0.77 City FE Yes Yes Yes Yes State Year FE Yes Yes Yes Yes Observations 4988 4821 4415 4248 Note: Robust standard errors clustered in the city level are reported in the parentheses. * p < 0.05, p < 0.01, * p < 0.001 Table S1: Reduced Form Estimates of New Instruments on Housing Cost (Alternative Fa- vorable Climate Deﬁnition) 31 31 Table S2: Reduced Form Estimates of New Instruments on Housing Cost (Excluding Short Term Visitors in Visa Measure) (1) (2) (3) (4) Dependent Variable log(home priceis,t) log(rental priceis,t) favourable climateis × log(] visast−1) 0.57* (0.015) 0.61 (0.020) favourable climateis × log(] visast−2) 0.77* (0.017) 0.68* (0.020) log(housing supplyis,t) -0.052 (0.025) -0.033 (0.022) -0.028* (0.017) -0.029* (0.017) employment rateis,t 0.002 (0.004) 0.001 (0.004) 0.003 (0.003) 0.002 (0.003) Adj. R2 0.72 0.24 0.78 0.78 City FE Yes Yes Yes Yes State Year FE Yes Yes Yes Yes Observations 4988 4821 4415 4248 Note: Robust standard errors clustered in the city level are reported in the parentheses. * p < 0.05, p < 0.01, * p < 0.001 Robustness checks on 2SLS regression 505 Tables S3 and S4 present the 2SLS estimates when ^ climateis or log(g visat−j) are used to 506 construct our alternative instruments. The ﬁrst-stage regression results show that these 507 alternative IVs are positive and statistically signiﬁcant for city population at the 1% level. 508 Additionally, the Kleibergen-Paap F-statistics all exceed the critical value from Stock and 509 Yogo at the 10% level, indicating that these IVs are strong. In the second-stage regression, the 510 new elasticity estimates of housing costs with respect to city population remain statistically 511 signiﬁcant and fall within the two standard deviation bands of the baseline estimates in Table 512 3. Therefore, the baseline 2SLS estimates are robust to diﬀerent approaches of constructing 513 the variables to measure favourable climates and visa issuance. 514 32 32 Table S3: 2SLS Estimates of City Population on Housing Cost Using New Instruments Alternative Favorable Climate Deﬁnition) (1) (2) (3) (4) Dependent Variable (second stage) log(home priceis,t) log(rental priceis,t) log(populationis,t) 1.177* (0.290) 1.603* (0.343) 1.218* (0.459) 1.322* (0.459) log(housing supplyis,t) -0.010 (0.034) -0.063* (0.038) -0.153 (0.074) -0.167 (0.080) employment rateis,t -0.006 (0.005) -0.011 (0.005) -0.004 (0.005) -0.006 (0.005) Dependent Variable (ﬁrst-stage) log(populationis,t) log(populationis,t) ^ favourable climateis × log(visast−1) 0.054* (0.015) 0.035* (0.019) ^ favourable climateis × log(visast−2) 0.052* (0.005) 0.036*** (0.004) Kleibergen-Paap Wald F-Statistic 104 108 74 77 Stock and Yogo Critical Value (10%) 16.38 16.38 16.38 16.38 City FE No No No No State Year FE Yes Yes Yes Yes Observations 4988 4821 4415 4248 Note: The variable ^ climate is an indicator for climates that are mild, warm, cool and hot-humid. Robust standard errors clustered in the city level are reported in the parentheses. Note: The variable ] visas is the number visas issued for permanent skill migrants/residents and all migrants with an intention of staying over one year. Robust standard errors clustered in the city level in parentheses. * p < 0.05, p < 0.01, * p < 0.001 Robustness checks on 2SLS regression 505 * p < 0.05, p < 0.01, * p < 0.001 33 33 Table S4: 2SLS Estimates of City Population on Housing Cost Using New Instruments Excluding Short-Term Visitors in Visa Measure) (1) (2) (3) (4) Dependent Variable (second stage) log(home priceis,t) log(rental priceis,t) log(populationis,t) 0.528* (0.268) 0.662* (0.295) 1.852* (0.544) 1.974* (0.545) house supply -0.024 (0.031) -0.006 (0.029) -0.217 (0.102) -0.234 (0.110) employment rate -0.001 (0.004) -0.003 (0.004) -0.009 (0.006) -0.011* (0.006) Dependent Variable (ﬁrst-stage) log(populationis,t) log(populationis,t) favourable climateis × log(] visast−1) 0.068* (0.007) 0.045* (0.005) favourable climateis × log(] visast−2) 0.068* (0.007) 0.046* (0.005) Kleibergen-Paap Wald F-Statistic 90 89 72 76 Stock and Yogo Critical Value (10%) 16.38 16.38 16.38 16.38 City FE No No No No State Year FE Yes Yes Yes Yes Observations 4988 4821 4415 4248 Note: The variable ] visas is the number visas issued for permanent skill migrants/residents and all migrants with an i t ti f t i R b t t d d l t d i th it l l i th * < 0 05 ** Table S4: 2SLS Estimates of City Population on Housing Cost Using New Instruments (Excluding Short-Term Visitors in Visa Measure) 34 34 NPJreplication.zip Supplementary Files This is a list of supplementary ¦les associated with this preprint. Click to download.
https://openalex.org/W1979699711	https://europepmc.org/articles/pmc2873391?pdf=render	English	null	Increased Rac1 activity and Pak1 overexpression are associated with lymphovascular invasion and lymph node metastasis of upper urinary tract cancer	BMC cancer	2,010	cc-by	7,695	RESEARCH ARTICLE Open Access BioMed Central © 2010 Kamai et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Lymphovascular invasion (LVI) and lymph node metastasis are conventional pathological factors associated with an unfavorable prognosis of urothelial carcinoma of the upper urinary tract (UC-UUT), but little is known about the molecular mechanisms underlying LVI and nodal metastasis in this disease. Rac1 small GTPase (Rac1) is essential for tumor metastasis. Activated GTP-bound Rac1 (Rac1 activity) plays a key role in activating downstream effectors known as Pak (21-activated kinase), which are key regulators of cytoskeletal remolding, cell motility, and cell proliferation, and thus have a role in both carcinogenesis and tumor invasion. Methods: We analyzed Rac1 activity and Pak1 protein expression in matched sets of tumor tissue, non-tumor tissue, and metastatic lymph node tissue obtained from the surgical specimens of 108 Japanese patients with UC-UUT. Results: Rac1 activity and Pak1 protein levels were higher in tumor tissue and metastatic lymph node tissue than in non-tumor tissue (both P < 0.0001). A high level of Rac1 activity and Pak1 protein expression in the primary tumor was related to poor differentiation (P < 0.05), muscle invasion (P < 0.01), LVI (P < 0.0001), and lymph node metastasis (P < 0.0001). Kaplan-Meier survival analysis showed that an increase of Rac1 activity and Pak1 protein was associated with a shorter disease-free survival time (P < 0.01) and shorter overall survival (P < 0.001). Cox proportional hazards analysis revealed that high Rac1 activity, Pak1 protein expression and LVI were independent prognostic factors for shorter overall and disease-free survival times (P < 0.01) on univariate analysis, although only Pak1 and LVI had an influence (P < 0.05) according to multivariate analysis. Conclusions: These findings suggest that Rac1 activity and Pak1 are involved in LVI and lymph node metastasis of UC- UUT, and may be prognostic markers for this disease. Metastasis involves the spread of tumor cells from the primary tumor to a distant site [3], and is the major cause of human cancer death. Various pathological studies have shown that poorly differentiated cancer, muscle invasion, lymph node metastasis, and lymphovascular invasion (LVI) are associated with recurrence and are unfavorable prognostic factors for UC-UUT [2,4,5]. Thus, LVI and lymph node metastasis are used to predict the prognosis. Despite their clinical importance, little is known about the molecular mechanisms of LVI and lymph node metastasis, making it important to examine the factors Kamai et al. BMC Cancer 2010, 10:164 http://www.biomedcentral.com/1471-2407/10/164 Kamai et al. BMC Cancer 2010, 10:164 http://www.biomedcentral.com/1471-2407/10/164 Kamai et al. BMC Cancer 2010, 10:164 http://www.biomedcentral.com/1471-2407/10/164 Research article Increased Rac1 activity and Pak1 overexpression are associated with lymphovascular invasion and lymph node metastasis of upper urinary tract cancer Takao Kamai1, Hiromichi Shirataki2, Kimihiro Nakanishi1, Nobutaka Furuya1, Tsunehito Kambara1, Hideyuki Abe1, Tetsunari Oyama3 and Ken-Ichiro Yoshida1 Background Urothelial carcinoma of the upper urinary tract (UC- UUT) is relatively uncommon, accounting for <10% of all urothelial malignancies, but its incidence is increasing [1]. Many patients who undergo curative resection develop systemic metastases within a few years, so the prognosis of this cancer is poor [2], presumably due to occult micrometastasis at the time of surgery because of the thin walls and rich lymphatic drainage of the ureter. Correspondence: kamait@dokkyomed.ac.jp 1 Department of Urology, Dokkyo Medical University, Mibu, Tochigi, Japan Full list of author information is available at the end of the article * Correspondence: kamait@dokkyomed.ac.jp 1 Department of Urology, Dokkyo Medical University, Mibu, Tochigi, Japan Full list of author information is available at the end of the article Kamai et al. BMC Cancer 2010, 10:164 http://www.biomedcentral.com/1471-2407/10/164 Page 2 of 13 playing a role in LVI and lymph node metastasis of UC- UUT. active GTP-binding form (active GTPase) recognizes tar- get proteins and generates a response. Increased activity of Rac1 and overexpression of Pak1 are associated with the progression of cancer, but most of the evidence has come from cell culture studies. Therefore, the role of active Rac1 GTPase and its downstream effector needs to be studied by using surgically resected samples of human tumors to better assess their contribution to human can- cer. Accordingly, we examined the expression of active GTP-bound Rac1 (Rac1 activity) and its downstream effector Pak1 in the primary tumors and metastatic lymph nodes of patients with UC-UUT by Western blot- ting, and also assessed the relation of these molecules with clinicopathological features. There have been few reports about the simultaneous analysis of Rac1 and Pak1 in human tumor tissues. Such information could be use- ful for developing individualized treatment strategies and could potentially improve the design and application of adjuvant therapy for UC-UUT. Members of the Rho small GTPases family, prototype RhoA, Rac1, and Cdc42, are involved in the regulation of a variety of cellular processes, such as organization of the microfilament network, cell-cell contact, and malignant transformation, and also perform essential and special- ized functions during organization of the actin cytoskele- ton [6]. RhoA regulates the formation of stress fibers and focal adhesions in cells, while Rac1 regulates the forma- tion of lamellipodia and membrane ruffling, and Cdc42 regulates the formation of filopodia [6,7]. Background In addition, a number of investigations have established a significant role of GTPases from the Rho family in several human tumors, including UC-UUT [7,8]. Rac1 is ubiquitously expressed and exists in two conformational states. In response to extracellular signals, interconversion of these two states occurs via guanine nucleotide exchange factors (GEFs), which convert the inactive GDP-bound form of Rac1 to its active GTP-bound form, while GTPase-acti- vating proteins (GAPs) inactivate proteins (GAPs) inacti- vate Rac1. After activation, Rac1 interacts with various specific effectors to coordinate the activation of a multi- tude of signaling cascades that influence diverse physio- logical outcomes. The Pak (p21-activated kinase) serine/ threonine kinases have recently been found to be key reg- ulators of cytoskeletal remolding, cell motility, and cell proliferation, with a role in both carcinogenesis and cellu- lar invasion [9]. It has been reported that Pak1, the best characterized member of this family, shows increased expression and activity in human cancers [9-11]. Multiple signalling pathways converge to promote activation of Pak1 through both small GTPases and several of the tyrosine kinases. In turn, activated Pak1 regulates diverse cellular functions. Pak1 binds to Rac1 in a GTP-depen- dent manner, after which activated Pak1 regulates cellular functions such as cytoskeletal dynamics, cell adhesion, and transcription [9]. Rac1 signals through Pak1 to acti- vate c-Jun N-terminal kinase (JNK) [9], placing Rac1 between the Ras small GTPases (Ras) and mitogen-acti- vated protein kinase (MAPK) in the signaling cascade from growth factor receptors and v-Src to activation of JNK [12,13]. Gao et al. reported that a low molecular weight inhibitor of Rac GTPase targeting the activation of Rac by GEF was able to reverse the tumor cell phenotype associated with deregulation of Rac [14]. In addition, sev- eral low molecular weight inhibitors have been shown to interfere with Pak kinase activity or function [9]. These findings suggest that Rac1 and Pak may be potential molecular targets for the treatment of cancer. Regarding the expression of Rho family GTPases in human cancers most previous reports were based on the Patients and tissues Between 1995 and 2006, surgical specimens of UC-UUT were obtained from 108 consecutive Japanese patients (77 men and 31 women) aged 42 to 89 years (mean age: 71.9 years) with newly diagnosed primary transitional cell carcinoma (TCC) of the renal pelvis and ureter without distant metastasis (cTanyNanyM0). These specimens were reviewed in the present study. All patients routinely underwent imaging investigations (CT and/or MRI) before surgery to acquire information for staging. The follow-up time ranged from 5 to 132 months, with a median follow-up period of 41 months. Patients under- went surgery before receiving any other therapy. g y g y y During nephroureterectomy, the patients also under- went lymphadenectomy when enlarged lymph nodes were confirmed. In all patients, tumor tissue, non-tumor tissue, and lymph node tissue were acquired from the resected specimens after removing excess stromal tissue. Then the tissues were embedded in OCT tissue com- pound (Miles, Elkhart, IN, USA) and stored at - 80°C according to the method described previously [15]. The grade and stage of each tumor were classified according to the TNM system [16]. This study was conducted in accordance with the Helsinki Declaration and institu- tional review board approval in Dokkyo Medical Univer- sity Hospital was obtained. In addition, each patient signed a consent form approved by the Committee on Human Rights in Research of our institution. Systemic chemotherapy with methotrexate, vinblastine, Adriamycin, and cisplatin (M-VAC) was given to the 16 patients who had lymph node metastasis at nephroure- terectomy, as well as those in whom lymph node metasta- Regarding the expression of Rho family GTPases in human cancers, most previous reports were based on the investigation of protein expression levels by Western blotting and immunohistochemistry. However, only the Page 3 of 13 Kamai et al. BMC Cancer 2010, 10:164 http://www.biomedcentral.com/1471-2407/10/164 sis (14 patients) or distant metastasis (5 patients) was detected postoperatively [17]. then incubated with horseradish peroxidase-conjugated secondary antibodies. Antibody-bound protein bands were visualized by chemiluminescence, the blotted mem- brane was scanned for densitometry with a PDI imaging scanner (Agfa Japan, Tokyo), and the data were analyzed with NIH Image software. Expression of active Rac1 and Pak1 was determined relative to that of beta actin in the tumor tissue and corresponding normal tissue speci- mens, after which relative expression was calculated. Rac1 activation assay and Western blotting Tumor tissue and normal tissue specimens were carefully dissected free of stromal tissue. To measure Rac1 activa- tion, we performed a Rho-binding domain (RBD) affinity precipitation assay for Rac1-GTP using a specific Rac1 antibody according to the manufacturer's protocol (Cytoskeleton, BK126, Denver, CO) [18-20]. Tissue extracts were obtained from 50 mg of each type of tissue and the protein concentration was determined by Brad- ford's method (BIO-RAD, Hercules, CA). Then the extracts were equalized with ice cold cell lysis buffer con- taining 25 mM Tris (pH 7.5), 10 mM MgCl2, 0.5 M NaCl, and 1% Triton X-100 to obtain identical protein concen- trations. Next, equivalent amounts of protein were added to 15 μl of Pak (p21-activated kinase)-RBD beads and incubated at 4°C on a rotor for 1 hr. Then the Pak-RBD beads solution was centrifuged at 3,000 g for 1 min at 4°C and supernatant was removed. After washing the pellet three times, the bound proteins were analyzed by West- ern blotting, as described previously [21]. Briefly, 25 μg of GTP-Rac1 bound to Pak-RBD beads was separated by SDS-PAGE (5-20% gradient gel) and electrotransferred to a polyvinylidene difluoride membrane (Sequi-Blot PVDF membranes; BIO-RAD). Rac1 His-tagged protein (100, 50, and 10 ng/μl) was also run on the gel as a standard. If the intensity of the immunoreactive band was outside the range of 10-100 ng/μl, samples were diluted with loading buffer and run on the gel again. Total cell lysate with GDP was employed as a negative control. After membranes were blocked to eliminate nonspecific binding, mem- brane-bound proteins were probed with an anti-Rac1 monoclonal antibody (Cytoskeleton, BK126). Then the membranes were washed and incubated with a horserad- ish peroxidase-conjugated secondary antibody. Anti- body-bound protein bands were visualized by chemiluminescence, the blotted membrane was sub- jected to densitometry by scanning with a ChemiDoc XRS-J imaging scanner (BIO-RAD), and the data were analyzed with NIH Image software. The mean value for three experiments was obtained with each tissue speci- men. Immunohistochemistry To support the data obtained by Western blotting, immu- nohistochemistry was performed with the same antibod- ies utilized for Western blotting on 3 tumors from patients with lymph node metastases (pN+) and 5 tumors from patients without nodal metastasis (pN-), as described previously [20-22]. Patients and tissues For quantification of these proteins, the relative amount of Rac1 or Pak1 in tumor tissue was expressed as a ratio of the optical density of the band obtained from the tumor specimen to that from the corresponding normal tissue (which was set at 1.0) by densitometric analysis as described previously [21-23]. The mean values for tumor tissue and non-tumor tissue were calculated from three experiments [21,22]. Statistical analysis Western blotting data were analyzed by the Mann-Whit- ney U test to compare two groups [21,22], or by the Kruskal-Wallis test for comparisons among three or more groups. Spearman's rank correlation coefficient analysis was employed to determine the relation between Rac1 activity and Pak1 expression. Rac1 activity and Pak1 expression, as well as tumor grade, pT stage, lymph node metastasis, and LVI, were assessed for their relation to disease-free survival and overall survival by univariate and multivariate analyses with the Cox proportional haz- ards model. The Kaplan-Meier method was used to esti- mate survival, and differences of survival were assessed by the log-rank test. Probability values of less than 0.05 were considered significant. Data were analyzed with commercially available software. Association of Rac1 activity and Pak1 expression with tumor characteristics For measurement of Pak1, 50 μg of cytosolic protein was separated by SDS-PAGE (12.5% gel) and elec- trotransferred to a polyvinylidene difluoride membrane (Immobilon-P membrane; Millipore, Bedford, MA). After the membrane was blocked, the bound proteins were probed with specific antibodies (Santa Cruz Biotechnol- ogy; sc-881, Santa Cruz, CA) and a primary antibody for beta actin (Santa Cruz Biotechnology, Santa Cruz, CA). Hela cells were used as a positive control for Rac1 and Pak1 expression. Next, the membranes were washed and Rac1 and Pak1 proteins were detected in tumor tissues, non-tumor tissues, metastatic lymph nodes, and normal lymph nodes (Figures. 1, 2). The level of active (GTP- bound) Rac1 was significantly increased in tumor tissues (mean ± S.D. = 2.72 ± 1.72) and metastatic lymph nodes (1.86 ± 0.34) compared with the level in non-cancerous tissues, which was set at 1.0 [21-23], (P < 0.0001, Figure. 3A). An increase of Rac1 activity in primary tumors was Kamai et al. BMC Cancer 2010, 10:164 Page 4 of 13 Kamai et al. BMC Cancer 2010, 10:164 http://www.biomedcentral.com/1471-2407/10/164 http://www.biomedcentral.com/1471-2407/10/164 Figure 1 Expression of GTP-binding (active form) Rac1 (22 kDa), Pak1 (65 kDa) and beta actin (42 kDa) proteins using Western blotting. M; marker, P; positive control using Hela cells, N; negative control, T1,2; tumor tissue, N1,2; non-tumor tissue. LN1; non-tumor lymph node, mLN1; meta- static lymph node, Each number corresponds to a case number. 2.10, P < 0.0001, Figure. 4C), lymph node metastasis (pN0; 2.43 ± 1.09, pN1-3; 3.84 ± 1.35, P < 0.0001, Figure. 4D), and LVI (LVI(-); 2.03 ± 0.68, LVI(+); 3.34 ± 1.36, P < 0.0001, Figure. 4E). associated with poorly differentiated cancer (grade 1; 2.38 ± 1.38, grade 2; 2.33 ± 1.31, grade 3; 3.30 ± 2.08, P = 0.0471, Figure. 3B), local invasion (<pT1; 2.38 ± 1.35, pT2; 2.32 ± 1.31, pT3; 2.91 ± 1.69, pT4; 4.46 ± 3.14, P = 0.1417, Figure. 3C), lymph node metastasis (pN0; 2.55 ± 1.62, pN1-3; 3.52 ± 1.96, P < 0.05, Figure. 3D), and LVI (LVI(-); 2.04 ± 0.97, LVI(+); 3.40 ± 2.02, P < 0.0001, Figure. 3E). We investigated the correlation between Rac1 activity and Pak1 expression in tumor tissues. When Rac1 was used as an independent variable and Pak1 as a dependent variable, a positive correlation between them was observed (r2 = 0.288, P < 0.0001, Figure. 5A). Association of Rac1 activity and Pak1 expression with tumor characteristics However, no such correlation was observed in specimens of metastatic lymph nodes (r2 = 0.307, P = 0.2602, Figure. 5B). The level of Pak1 protein was significantly higher in tumor tissues (mean ± S.D. = 2.68 ± 1.26) and metastatic lymph nodes (2.77 ± 1.60) than the level in non-cancer- ous tissues, which was also set at 1.0 [21-23], (P < 0.0001, Figure. 4A). Higher expression of Pak1 protein in the pri- mary tumor was associated with poorly differentiated cancer (grade 1; 1.67 ± 0.34, grade 2; 2.38 ± 0.96, grade 3; 3.29 ± 1.45, P < 0.0001, Figure. 4B), local invasion (<pT1; 1.89 ± 0.60, pT2; 2.52 ± 0.97, pT3; 3.09 ± 1.12, pT4; 4.56 ± Prognostic influence of Rac1 activity and Pak1 expression Prognostic influence of Rac1 activity and Pak1 expression The mean level of Rac1 activity was 2.72 (± 1.72). Patients were divided into two groups at this value, i.e., a high activity group (46 patients) and a low activity group (62 Kamai et al. BMC Cancer 2010, 10:164 http://www.biomedcentral.com/1471-2407/10/164 Kamai et al. BMC Cancer 2010, 10:164 Page 5 of 13 http://www.biomedcentral.com/1471-2407/10/164 the N0 M0 patients with low Rac1 activity (59 patients) versus high Rac1 activity (33 patients) showed that high Rac1 activity was associated with a shorter disease-free survival time (P = 0.001, Figure. 6C). Similarly, higher expression of Pak1 was a significant unfavorable factor for disease-free survival (P < 0.0001, Figure. 6D). Although tumor grade, pT stage, LVI, active Rac1, and Pak1 were all significant factors by Cox univariate analy- sis, only LVI and Pak1 were independent variables according to multivariate analysis (Table 1). Figure 2 Immunohistochemistry for Rac1 (upper panel) and Pak1 (lower panel) proteins in Grade 3 carcinoma. Cytosolic compart- ments shows intensely brown staining in most of the cancer cells, dis- playing high Rac1 and Pak1 protein levels, but the nuclei of the cancer cells shows very weak staining. g y With regard to the site of first postoperative recurrence among the 92 N0 M0 patients, the Rac1 activity and Pak1 protein levels in the primary tumor tissues of patients with retroperitoneal lymph node metastasis (PRLN) (n = 14; 3.99 ± 1.82, 3.61 ± 0.94, respectively) and other organ metastases (3 of lung, one of liver, and one of bone; 6.35 ± 2.35 and 4.49 ± 1.90, respectively) were significantly higher than the levels in patients with bladder recurrence (n = 18; 1.95 ± 0.89, P < 0.0001, 2.32 ± 1.02, P < 0.01, respectively, Figures. 3F, 4F). Furthermore, we divided this group into two subgroups to study the relationship between LVI and Rac1 activity or Pak1 expression (Fig- ure. 7). When the PRLN and distant organs were the first site of recurrence, the primary tumors had LVI, while pri- mary tumors without LVI showed no PRLN recurrence or distant metastasis. Among the LVI(+) group, both Rac1 activity and Pak1 expression in the primary tumors were higher in patients with RPLN and distant metastases than in those with bladder recurrence. Prognostic influence of Rac1 activity and Pak1 expression The patients with blad- der recurrence had higher tumor levels of Pak1 expres- sion than those with no evidence of disease (NED), but Rac1 activity did not vary. On the other hand, in the LVI(- ) group, both Rac1 activity and Pak1 expression were not different between the primary tumors of patients with bladder recurrence and those with NED. Interestingly, although Rac1 activity showed no difference between LVI(+) and LVI(-) tumors in patients with bladder recur- rence, Pak1 expression was higher in the former tumors. Figure 2 Immunohistochemistry for Rac1 (upper panel) and Pak1 (lower panel) proteins in Grade 3 carcinoma. Cytosolic compart- ments shows intensely brown staining in most of the cancer cells, dis- playing high Rac1 and Pak1 protein levels, but the nuclei of the cancer cells shows very weak staining. patients), according to the method described previously [21-23]. Similarly, the mean level of Pak1 protein expres- sion in the tumor tissues was 2.68 (± 1.26), so a high expression group (49 patients) and a low expression group (59 patients) were separated by using this as the cut-off value. Kaplan-Meier plots of survival for patients with low versus high levels of Rac1 activity showed that increased Rac1 activity was associated with a shorter overall sur- vival time (P < 0.0001, Figure. 6A). High expression of Pak1 protein was also correlated with shorter overall sur- vival (P < 0.0001, Figure. 6B). Univariate analysis of over- all survival with the Cox proportional hazards model revealed that tumor grade, pT stage, lymph node metas- tasis, LVI, active Rac1, and Pak1 were all significant vari- ables (Table 1). However, multivariate analysis revealed that only LVI and Pak1 had an independent influence on overall survival. M-VAC therapy was performed for 16 patients with lymph node metastasis at the time of nephroureterec- tomy, 14 patients with postoperative lymph node involve- ment, and 5 patients with postoperative metastasis to other organs. All of these 35 patients eventually died of progressive cancer. Their primary tumors were all LVI(+) on pathological examination, as well as showing increased Rac1 activity and high Pak1 expression (data not shown). Discussion With regard to disease-free survival of the patients who were N0 M0 at the time of nephroureterectomy (92 patients), the mean level of Rac1 activity and Pak1 expression in tumor tissue was 2.55 ± 1.62 and 2.43 ± 1.09, respectively. Comparison of Kaplan-Meier plots for Rac1 and Pak1 have recently been shown to be key regu- lators of cancer cell signaling networks, and there are sev- eral lines of evidence linking Rac1 and Pak1 to the acquisition of migratory, invasive, and metastatic pheno- types [7,9]. In order to take into account possible inter- Kamai et al. BMC Cancer 2010, 10:164 Page 6 of 13 http://www.biomedcentral.com/1471-2407/10/164 individual variations of Rac1 activity and Pak1 protein expression in UC-UUT, we performed a comparison among paired samples of tumor tissue, metastatic lymph node tissue, and non-tumor tissue from the same patient. The present study showed that Rac1 activity and Pak1 expression were significantly increased in primary Figure 3 The relative expression levels of GTP-binding active Rac1 protein in tumor to those in corresponding non-tumor portion, which was set to 1.0. A; Expression in tumor, non-tumor, and lymph node tissues with metastasis (mLNs) and without (LN). B; Expression in Grade. C; Expres- sion in pT stage. D; Expression in lymph node metastasis. pN(-) is pN0. pN(+) is pN1-3. E; Expression in lymphovascular invasion (LVI). F; Expression in first recurrence site. There were no difference between bladder and none of recurrence. The data show the 95% confidential interval. Figure 3 The relative expression levels of GTP-binding active Rac1 protein in tumor to those in corresponding non-tumor portion, which was set to 1.0. A; Expression in tumor, non-tumor, and lymph node tissues with metastasis (mLNs) and without (LN). B; Expression in Grade. C; Expres- sion in pT stage. D; Expression in lymph node metastasis. pN(-) is pN0. pN(+) is pN1-3. E; Expression in lymphovascular invasion (LVI). F; Expression in first recurrence site. There were no difference between bladder and none of recurrence. The data show the 95% confidential interval. individual variations of Rac1 activity and Pak1 protein expression in UC-UUT, we performed a comparison among paired samples of tumor tissue, metastatic lymph node tissue, and non-tumor tissue from the same patient. The present study showed that Rac1 activity and Pak1 expression were significantly increased in primary Kamai et al. BMC Cancer 2010, 10:164 Page 7 of 13 Kamai et al. Discussion However, it is unclear whether Rac1 and Pak1 play a simi- lar role in lymph node metastasis and bladder recurrence. It is well known that urothelial cancer often behaves like a field change disease, with multiple occurrences and recurrences due to implantation and migration, making it difficult to determine whether a recurrent tumor has been caused by tumor cell implantation, migration, or multifocal carcinogenesis [26]. A recent molecular study revealed that UC-UUT might be less genetically stable than bladder tumors [27]. Therefore, an increase of Rac1 activity and upregulation of Pak1 expression might play a role in lymph node metastasis of UC-UTT after neph- roureterectomy, rather than contributing to bladder recurrence. The differences of molecular mechanisms between LVI and lymph node metastasis or bladder recurrence need to be investigated further. On the other hand, a previous study of bladder cancer showed that tumors and metastatic lymph nodes compared with non- tumor tissues. Also, an increase of Rac1 activity and Pak1 expression in the primary tumor was correlated with poorly differentiated cancer, local invasion, lymph node metastasis, LVI, and an unfavorable prognosis. To our knowledge, this is the first report about Rac1 and Pak1 in UC-UUT. Our data suggested that Rac1 and its down- stream effector Pak1 may be involved in the progression of this cancer. As well as our findings in patients with UC-UUT, over- expression of Rac1 and Pak1 has been reported in several other human cancers [7,9]. Moreover, an increase of Rac1 and Pak1 activity or overexpression have been observed in breast cancer tissues and metastatic lymph nodes [24,25]. In the present study, increased Rac1 activity and higher Pak1 expression in the primary tumors was related to muscle invasion and lymph node metastasis. There- fore, it is likely that Rac1 and Pak1 have a role in deter- mining the local invasive and metastatic potential of various human cancers. Regarding the site of initial postoperative recurrence in patients who were pTanypN0 M0 at the time of neph- roureterectomy, patients with postoperative lymph node recurrence had a worse prognosis than those with blad- der recurrence, probably because many bladder cancers were detected at the superficial stage by active surveil- lance. In the present study, LVI, Pak1 activity, Rac1, pT stage, and tumor grade were related to postoperative recurrence according to univariate analysis, with both LVI and Pak1 still being significant determinants accord- ing to multivariate analysis. Discussion BMC Cancer 2010, 10:164 http://www.biomedcentral.com/1471-2407/10/164 http://www.biomedcentral.com/1471-2407/10/164 Figure 4 The relative expression levels of Pak1 protein in tumor to those in corresponding non-tumor portion, which was set to 1.0. A; Ex- pression in tumor, non-tumor, and lymph node tissues with metastasis (mLNs) and without (LN). B; Expression in Grade. C; Expression in pT stage. D; Expression in lymph node metastasis. pN(-) is pN0. pN(+) is pN1-3. E; Expression in lymphovascular invasion (LVI). F; Expression in first recurrence site. There were no difference between bladder and none of recurrence. The data show the 95% confidential interval. Figure 4 The relative expression levels of Pak1 protein in tumor to those in corresponding non-tumor portion, which was set to 1.0. A; Ex- pression in tumor, non-tumor, and lymph node tissues with metastasis (mLNs) and without (LN). B; Expression in Grade. C; Expression in pT stage. D; Expression in lymph node metastasis. pN(-) is pN0. pN(+) is pN1-3. E; Expression in lymphovascular invasion (LVI). F; Expression in first recurrence site. There were no difference between bladder and none of recurrence. The data show the 95% confidential interval. Kamai et al. BMC Cancer 2010, 10:164 http://www.biomedcentral.com/1471-2407/10/164 Kamai et al. BMC Cancer 2010, 10:164 Page 8 of 13 http://www.biomedcentral.com/1471-2407/10/164 Figure 5 Spearman rank correlation coefficient relationship between expression levels of Rac1 activity and Pak1. X axis is an independent variable. Y axis is a dependent variable. A; Tumor tissues. B; Metastatic lymph node tissues. Figure 5 Spearman rank correlation coefficient relationship between expression levels of Rac1 activity and Pak1. X axis is an independent variable. Y axis is a dependent variable. A; Tumor tissues. B; Metastatic lymph node tissues. tumors of patients with postoperative lymph node metas- tasis than in those of patients with bladder recurrence from the LVI(+) group, but not the LVI(-) group. Pak1 expression was higher in the tumors of patients with bladder recurrence than in recurrence-free patients from the LVI(+) group, but Rac1 did not differ between them. Moreover, all patients with lymph node involvement at nephroureterectomy had LVI(+) tumors on pathological examination. Therefore, LVI might be an important step along the road to lymph node metastasis. Primary tumors with LVI(+) and lymph node metastasis showed an increase of Rac1 activity and Pak1 expression, while met- astatic lymph node tissues showed higher Rac1 activity and Pak1 expression than normal lymph nodes, indicat- ing that Rac1 and Pak1 are involved in tumor metastasis. Discussion As shown in Figure 7, Rac1 activity and Pak1 expression were higher in the primary Kamai et al. BMC Cancer 2010, 10:164 Page 9 of 13 http://www.biomedcentral.com/1471-2407/10/164 Figure 6 Survival curve in the patients based on the mean values of Rac1 activity and Pak1 in primary tumor tissues, the cases were divided into two groups at this levels - high and low expression. A; Overall survival curve based on Rac1 activity in all patients. B; Overall survival curve based on Pak1 in all patients. C; Disease-free survival curve based on Rac1 activity in N0 M0 patients at nephroureterectomy. D; Disease-free survival curve based on Pak1 in N0 M0 patients at nephroureterectomy. P value was analyzed by log-rank test. Figure 6 Survival curve in the patients based on the mean values of Rac1 activity and Pak1 in primary tumor tissues, the cases were divided into two groups at this levels - high and low expression. A; Overall survival curve based on Rac1 activity in all patients. B; Overall survival curve based on Pak1 in all patients. C; Disease-free survival curve based on Rac1 activity in N0 M0 patients at nephroureterectomy. D; Disease-free survival curve based on Pak1 in N0 M0 patients at nephroureterectomy. P value was analyzed by log-rank test. high Pak1 expression was associated with a higher risk of recurrence, even in patients with low grade/stage tumors [28]. Taken together, therefore, it is likely that Pak1 and Rac1 both play an important role in the invasion, metas- tasis, and recurrence of urothelial cancer. GTPases and several tyrosine kinases) that are often acti- vated in cancer cells [9-11]. Therefore, several oncogenic pathways may act through Pak1 to promote cancer pro- gression, so that Pak1 protein expression had a greater impact on overall and recurrence-free survival than Rac1 activity in the present study. We did not assess Pak1 activ- ity in this study, so its activity in tumor tissues needs to be elucidated in the future. Although Pak1 is well known as a downstream effector of Rac1, there have been few simultaneous analyses of Rac1 and Pak1 expression in human tumor tissues. Our study showed a positive correlation between Rac1 activity and Pak1 expression in tumor tissue, while no such rela- tion was observed in metastatic lymph node tissue. Discussion The data show the 95% confidential interval. Figure 7 Rac1 activity (A) and protein levels of Pak1 (B) regarding with postoperative first recurrence site among N0 M0 cases (92 patients). The data show the 95% confidential interval. Figure 7 Rac1 activity (A) and protein levels of Pak1 (B) regarding with postoperative first recurrence site among N0 M0 cases (92 patients). The data show the 95% confidential interval. ) and protein levels of Pak1 (B) regarding with postoperative first recurrence site among N0 M0 cases (92 patients). onfidential interval. These 35 tumors were all LVI(+) and showed increased Rac1 activity and high Pak1 expression. Furthermore, an increase of Rac1 activity and a higher Pak1 expression were associated with a shorter overall survival time in all patients and shorter postoperative disease-free survival in pTanypN0 M0 patients, indicating that Rac1 activity and Pak1 expression may be useful prognostic indicators for UC-UTT. These findings suggests that patients with LVI(+) tumors that have higher Rac1 activity and Pak1 expression are at more risk of developing postoperative RPLN or distant metastases in comparison to patients without these markers. Therefore, we need to assess the potential of chemotherapy for patients who have pTanypN0 M0 tumors that are LVI(+) with increased Rac1 activity and Pak1 expression to prevent RPLN recurrence These 35 tumors were all LVI(+) and showed increased Rac1 activity and high Pak1 expression. Furthermore, an increase of Rac1 activity and a higher Pak1 expression were associated with a shorter overall survival time in all patients and shorter postoperative disease-free survival in pTanypN0 M0 patients, indicating that Rac1 activity and Pak1 expression may be useful prognostic indicators for UC-UTT. These findings suggests that patients with LVI(+) tumors that have higher Rac1 activity and Pak1 expression are at more risk of developing postoperative RPLN or distant metastases in comparison to patients without these markers. Therefore, we need to assess the potential of chemotherapy for patients who have pTanypN0 M0 tumors that are LVI(+) with increased Rac1 activity and Pak1 expression to prevent RPLN recurrence The renal pelvis and ureter have thin walls and a rich lymphatic drainage [1], so many patients present with local invasion and/or lymph node metastasis, while delay in making the initial diagnosis is correlated with a higher stage at presentation. Discussion In contrast to investigation of Rac1 activity, we only mea- sured Pak1 protein expression, but we could still deter- mine the approximate relation between the two molecules. As shown in Figure 5, there was a positive cor- relation in tumor tissues, but the absolute correlation was fairly weak and no correlation was found in metastatic lymph nodes. Pak1 is the best-characterized downstream effector of Rac1, but it is also an important convergence point for many signaling pathways (including small Cell migration is central to metastasis by malignant tumors [3]. Members of the Rho small GTPases family regulate formation of stress fibers, focal adhesions, and cell migration through reorganization of the actin cytoskeleton [6]. Several lines of evidence have directly linked Rac1 and Pak1 to acquisition of a migratory, inva- sive, and metastatic phenotype and to a variety of pro- cesses that occur in tumors, including cell transformation, survival, invasion, metastasis, and angio- genesis [7,9]. Our findings suggested that Rac1 and Pak1 were associated with LVI and RPLN, as well as distant metastasis of UC-UUT. P valu 0.018 0.633 0.000 0.355 < 0.000 < 0.000 0.001 P valu 0.018 0.633 0.000 0.355 0.000 0.000 0.001 nephroureterectomy Analysis Relative risk 95% confidential interval P value No. of Patients Analysis Relative risk 95% confidential interval P valu Univariate (U) 2.626 1.486 - 4.641 0.0009 Univariate (U) 1.947 1.117-3.392 0.018 32/51/9 ultivariate (M) 1.008 0.488 - 2.083 0.6126 Multivariate (M) 1.047 0.427 - 1.678 0.633 U 3.138 2.065 - 4.768 < 0.0001 U 1.905 1.308 - 2.774 0.000 3/37/26/26 M 1.499 0.837 - 2.526 0.1454 M 1.291 0.479 - 1/302 0.355 U 3.049 2.005 - 4.636 < 0.0001 M 1.48 0.894 - 2.448 0.1271 U 11.024 5.550 - 21.896 < 0.0001 U 10.298 4.737 - 22.388 < 0.00 40/52 M 6.923 2432 - 19.707 < 0.0001 M 9.954 4.093 - 24.204 < 0.00 U 4.363 2.188 - 8.702 < 0.0001 U 2.917 1.494 - 5.694 0.001 33/59 M 1.87 0.863 - 4.094 0.1223 M 1.323 0.627 - 2.791 0.462 U 12.633 4.869 - 32.776 < 0.0001 U 4.814 2.384 - 9.721 < 0.00 35/57 M 3.635 1.230 - 10.741 0.0196 M 3.526 1.564 - 7.946 0.002 < Kamai et al. BMC Cancer 2010, 10:164 Page 11 of 13 http://www.biomedcentral.com/1471-2407/10/164 Figure 7 Rac1 activity (A) and protein levels of Pak1 (B) regarding with postoperative first recurrence site among N0 M0 cases (92 patients). Kamai et al. BMC Cancer 2010, 10:164 http://www.biomedcentral.com/1471-2407/10/164 Kamai et al. BMC Cancer 2010, 10:164 http://www.biomedcentral.com/1471-2407/10/164 increased disease specific survival in patients with transitional cell carcinoma of the upper urinary tract. J Urol 2005, 174:2120-2124. or distant metastasis by performing a randomized study in the future. In premenopausal breast cancer patients, Pak1 overexpression has been closely linked with tamox- ifen resistance of tumors [29]. On the other hand, several low molecular weight inhibitors have been shown to interfere with Pak1 kinase activity or function [9]. There- fore, the Rac1-Pak1 pathway might be a potential thera- peutic target for the prevention of tumor invasion and metastasis by inhibition of this signaling pathway. Accordingly, we should examine the effects of various inhibitors using cell lines or tumor tissue samples. Fur- thermore, as the Rac family has three isoforms and the Pak family has six isoforms, each isoform may play a dif- ferent role. Therefore, it is necessary to investigate each of these isoforms in human cancers, and the information thus obtained may shed new light on treatment strategies for UC-UTT and other tumors. increased disease specific survival in patients with transitional cell carcinoma of the upper urinary tract. J Urol 2005, 174:2120-2124. 5. Ozsahin M, Zouhair A, Villa S, Storme G, Chauvet B, Taussky D, Gouders D, Ries G, Bontemps P, Coucke PA, Mirimanoff RO: Prognostic factors in urothelial renal pelvis and ureter tumors: a multicentre Rare Cancer Network study. Eur J Cancer 1999, 35:738-743. Network study. Eur J Cancer 1999, 35:738-743. 6. Etienne-Manneville S, Hall A: Rho GTPases in cell biology. Nature 2002, 420:629-635. 7. Sahai E, Marshall CJ: Rho-GTPases and cancer. Nature Rev Cancer 2002, 2:133-142. 8. Kamai T, Kawakami S, Koga F, Arai G, Takagi K, Arai K, Tsujii T, Yoshida K-I: RhoA is associated with invasion and lymph node metastasis in upper urinary tract cancer. BJU Int 2003, 91:234-238. 9. Kumar R, Gururaj AE, Barnes CJ: P21-activated kinases in cancer. Nature Rev Cancer 2006, 6:459-471. 10. Mira JP, Berard V, Groffen J, Sanders LC, Knaus UG: Endogeneos, hyperactive Rac3 controls proliferation of breast cancer cells by a p21- activated kinase-dependent pathway. Proc Natl Acad Sci USA 2000, 97:185-189. 11. Balasenthil S, Sahin AA, Barnes CJ, Wang RA, Pestell RG, Vadlamudi RK: p21-activated kinase-1 signalingmediates cyclin D1 expression in mammary epithelial and cancer cells. J Biol Chem 2004, 279:1422-1428. 12. Authors' contributions T.K.* and H.S. initiated the study, participated in its design and coordination, carried out the study, performed the statistical analysis, and drafted the manu- script. K.N., N.F., T.K., H.A. and T.O. carried out the study. K-I.Y. participated in the design of the study and helped to draft the manuscript. All authors read and approved the final manuscript. 18. Nakanishi K, Kamai T, Mizuno T, Arai K, Yamanishi T: Expression of RhoA mRNA and activated RhoA in urothelium and smooth muscle, and effects of a Rho-kinase inhibitor on contraction of the porcine urinary bladder. Neurourol Urodynam 2009, 28:521-528. 19. Habas R, Dawid IB, He X: Coactivation of Rac and Rho by Wnt/Frizzled signaling is required for vertebrate gastrulation. Genes Dev 2003, 17:295-307. Acknowledgements The authors are special grateful to Hitomi Yamazaki for her excellent technique in this study. 20. Alstergren R, Zhu B, Glougauer M, Mak TW, Ellen RP, Sodek J: Polarization and directed migration of murine meutrophils is dependent on cell surface expression of CD44. Cell Immunol 2004, 231:146-157. Competing interests h h d l h p g The authors declare that they have no competing interests. 17. Conner JP, Rpoportm F, Olsson CA, Sawczuk IS, Benson MC: Long-term follow-up patients treated with methotrexate, vinblastine, doxorubicin and cisplatin (M-VAC) for transitional cell carcinoma of urinary bladder: Cause for concern. Urology 1990, 34:353-356. Kamai et al. BMC Cancer 2010, 10:164 http://www.biomedcentral.com/1471-2407/10/164 Zohn IM, Campbell SL, Khosravi-Far R, Rossman KL, Der CJ: Rho family proteins and Ras transformation: the RHOad less traveled gets congested. Oncogene 1998, 17:1415-1438. Author Details 21. Abe H, Kamai T, Shirataki H, Oyama T, Arai K, Yoshida K-I: High expression of Ran GTPase is associated with local invasion and metastasis of human clear cell renal cell carcinoma. Int J Cancer 2008, 122:2391-2397. 1Department of Urology, Dokkyo Medical University, Mibu, Tochigi, Japan, 2Department of Molecular and Cell Biology, Dokkyo Medical University, Mibu, Tochigi, Japan and 3Department of Anatomic and Diagnostic Pathology, Dokkyo Medical University, Mibu, Tochigi, Japan 22. Kamai T, Tsujii T, Arai K, Takagi K, Asami H, Ito Y, Oshima H, Yoshida K-I: Significant association of Rho/ROCK pathway with invasion and metastasis of bladder cancer. Clin Cancer Res 2003, 9:2632-2641. Received: 25 September 2009 Accepted: 28 April 2010 Published: 28 April 2010 This article is available from: http://www biomedcentral com/1471-2407/10/164 © 2010 Kamai 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 reproductio BMC Cancer 2010 10:164 Received: 25 September 2009 Accepted: 28 April 2010 Published: 28 April 2010 This article is available from: http://www biomedcentral com/1471 2407/10/164 © 2010 Kamai 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 reproduct BMC Cancer 2010 10:164 23. Fritz G, Just I, Kaina B: Rho GTPase over-expressed in human tumors. Int J Cancer 1999, 81:682-687. 24. Fiegen D, Haeusler LC, Blumenstein L, Herbrand U, Dvorsky R, Vetter IR, Ahmadian MR: Alternative splicing of Rac1 generates Rac1b, a self- activating GTPase. J Biol Chem 2004, 279:4743-4749. Conclusion h 13. Nobes CD, Hall A: Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 1995, 81:53-62. In the present study, increased Rac1 activity and increased expression of Pak1 (a major downstream effec- tor) were associated with poorly differentiated tumors, local invasion, LVI, lymph node metastasis, and an unfa- vorable prognosis. Our findings suggest that the Rac1- Pak1 pathway may be related to the progression of UC- UUT and that these molecules may be indicators for this disease. 14. Gao Y, Dickerson JB, Guo F, Zheng J, Zheng Y: Rational design and charaterization of a Rac GTPase-specific small molecular inhibitor. Proc Natl Acad Sci USA 2004, 101:7618-7623. 15. Suwa H, Ohshio G, Imamura T, Watanabe G, Arii S, Imamura M, Narumiya S, Hiai H, Fukumoto M: Overexpression of the rhoC gene correlates with progression of ductal adenocarcinoma of the pancreas. Br J Cancer 1998, 77:147-152. 16. Sobin H, Wittekind CH, editors: International union against cancer. UICC. In TNM classification of malignant tumors 6th edition. New York, Wiley-Liss; 2002. Discussion Preoperative staging by CT/MRI was very useful for detecting patients with local invasion and/or lymph node metastasis in the present study, but the prognosis of such patients was poor (Table 1). Although systemic M-VAC therapy reduced the tumor burden of our patients with urothelial cancer, the progno- sis was worse than we expected [17]. In the present study, M-VAC therapy was performed as adjuvant chemother- apy for the 35 patients who showed lymph node or dis- tant metastasis at surgery or during postoperative surveillance, but all of these patients died of their cancer. Page 12 of 13 Kamai et al. BMC Cancer 2010, 10:164 http://www.biomedcentral.com/1471-2407/10/164 Ito M, Nishiyama H, Kawanishi H, Matsui S, Guilford P, Reeve A, Ogawa O: P21-activated kinase 1: a new molecular marker for intravesical recurrence after transurethral resection of bladder cancer. Clin Cancer Res 2007, 178:1073-1079. 29. Holm C, Rayala S, Jirstrom K, Stal O, Kumar R, Landberg G: Association between Pak1 expression and subcellular localization and tamoxifen resistance in breast cancer patients. J Natl Cancer Inst 2006, 98:671-680. 29. Holm C, Rayala S, Jirstrom K, Stal O, Kumar R, Landberg G: Association between Pak1 expression and subcellular localization and tamoxifen resistance in breast cancer patients. J Natl Cancer Inst 2006, 98:671-680. Kamai et al. BMC Cancer 2010, 10:164 http://www.biomedcentral.com/1471-2407/10/164 Kamai et al. BMC Cancer 2010, 10:164 http://www.biomedcentral.com/1471-2407/10/164 Kamai et al. BMC Cancer 2010, 10:164 http://www.biomedcentral.com/1471-2407/10/164 27. Takahashi T, Kakehi Y, Mitsumori K, Akao T, Terachi T, Ogawa O, Habuchi T: Distinct microsatellite alterations in upper urinary tract tumors and subsequent bladder tumors. J Urol 2001, 165:672-677. 28. Ito M, Nishiyama H, Kawanishi H, Matsui S, Guilford P, Reeve A, Ogawa O: P21-activated kinase 1: a new molecular marker for intravesical recurrence after transurethral resection of bladder cancer. Clin Cancer Res 2007, 178:1073-1079. 29. Holm C, Rayala S, Jirstrom K, Stal O, Kumar R, Landberg G: Association between Pak1 expression and subcellular localization and tamoxifen resistance in breast cancer patients. J Natl Cancer Inst 2006, 98:671-680. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2407/10/164/prepub doi: 10.1186/1471-2407-10-164 Cite this article as: Kamai et al., Increased Rac1 activity and Pak1 overexpres- sion are associated with lymphovascular invasion and lymph node metasta- sis of upper urinary tract cancer BMC Cancer 2010, 10:164 27. Takahashi T, Kakehi Y, Mitsumori K, Akao T, Terachi T, Ogawa O, Habuchi T: Distinct microsatellite alterations in upper urinary tract tumors and subsequent bladder tumors. J Urol 2001, 165:672-677. 28. Ito M, Nishiyama H, Kawanishi H, Matsui S, Guilford P, Reeve A, Ogawa O: P21-activated kinase 1: a new molecular marker for intravesical recurrence after transurethral resection of bladder cancer. Clin Cancer Res 2007, 178:1073-1079. 29. Holm C, Rayala S, Jirstrom K, Stal O, Kumar R, Landberg G: Association between Pak1 expression and subcellular localization and tamoxifen resistance in breast cancer patients. J Natl Cancer Inst 2006, 98:671-680. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2407/10/164/prepub doi: 10.1186/1471-2407-10-164 Cite this article as: Kamai et al., Increased Rac1 activity and Pak1 overexpres- sion are associated with lymphovascular invasion and lymph node metasta- sis of upper urinary tract cancer BMC Cancer 2010, 10:164 27. Takahashi T, Kakehi Y, Mitsumori K, Akao T, Terachi T, Ogawa O, Habuchi T: Distinct microsatellite alterations in upper urinary tract tumors and subsequent bladder tumors. J Urol 2001, 165:672-677. 27. Takahashi T, Kakehi Y, Mitsumori K, Akao T, Terachi T, Ogawa O, Habuchi T: Distinct microsatellite alterations in upper urinary tract tumors and subsequent bladder tumors. J Urol 2001, 165:672-677. 28. References 1. Munoz JJ, Ellison LM: Upper tract urothelial neoplasms: incidence and survival during the last 2 dacades. J Urol 2000, 164:1523-1525. 25. Schnelzer A, Prechtel D, Knaus U, Dehne K, Gerhard M, Graeff H, Harbeck N, Schmitt M, Lengyel E: Rac1 in human breast cancer: overexpression, mutation analysis, and characterization of a new isoform, Rac1b. Oncogene 2000, 19:3013-3020. 2. Hall MC, Womack S, Sagalowsky AL, Carmody T, Erickstad MD, Roehrborn CG: Prognostic factors, recurrence, and survival in transitional cell carcinoma of the upper urinary tract: a 30-year experience in 252 patients. Urology 1998, 52:594-601. 26. Messing ED: Urothelial tumors of the bladder. In Campbell-Walsh Urology 9th edition. Edited by: Wein AJ, Kavoussi LR, Novick AC, Partin AW, Peters CA. Philadelphia: Saunders Elsevier; 2007:2407-2446. y 3. Quigley JP, Armstrong PB: Tumor cell intravasation elu-cidated; The chick embryo opens the window. Cell 1998, 94:281-284. 4. Kikuchi E, Horiguchi Y, Nakashima J, Hatakeyama N, Matsumoto M, Nishiyama T, Murai M: Lymphovascular invasion independently predicts Page 13 of 13 Page 13 of 13 Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2407/10/164/prepub doi: 10.1186/1471-2407-10-164 doi: 10.1186/1471 2407 10 164 Cite this article as: Kamai et al., Increased Rac1 activity and Pak1 overexpres- sion are associated with lymphovascular invasion and lymph node metasta- sis of upper urinary tract cancer BMC Cancer 2010, 10:164
https://openalex.org/W2767236639	https://europepmc.org/articles/pmc5694961?pdf=render	English	null	Data on internal cDNA amplification and color changes of the proteins derived from Pacific white leg shrimp shell	Data in brief	2,018	cc-by	1,136	https://doi.org/10.1016/j.dib.2017.11.025 2352-3409/& 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). DOI of original article: https://doi.org/10.1016/j.foodchem.2017.08.080 ⁎ Corresponding author. E-mail addresses: silverpfoxc@hotmail.com (P. Chuang), ishizak@kaiyodai.ac.jp (I. Shoichiro), yujicd@kaiyodai.ac.jp (N. Yuji), gjl@hotmail.com (G. Jialong), swatabe@kitasato-u.ac.jp (W. Shugo). Pan Chuang a, Ishizaki Shoichiro a,⁎, Nagashima Yuji a, Gao Jialong b, Watabe Shugo c a Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato, Tokyo 108-8477, Japan a Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology 4-5-7 Konan, Minato, Tokyo 108-8477, Japan b College of Food Science and Technology, Guangdong Ocean University, Haida Road 1, Mazhang, Zhanjiang 524088, Guangdong, China c S h l f M i Bi i Kit t U i it S ih 252 0373 J y J p b College of Food Science and Technology, Guangdong Ocean University, Haida Road 1, Mazhang, Zhanjiang 524088, Guangdong, China c School of Marine Biosciences, Kitasato University, Sagamihara 252-0373, Japan Contents lists available at ScienceDirect Contents lists available at ScienceDirect Data in Brief 16 (2018) 105–108 Data in Brief 16 (2018) 105–108 1. Data The data of this article provides information on the designation of primers used for different ampliﬁcations (Table 1) and the alignment of nucleotide sequences between red color-related protein derived from shrimp shell and known hemocyanin (KJ151291) (Fig. 1). Data on the color shifts of different soluble proteins under 100 °C 10 min heat treatment are presented in Table 2. Data on the effects of heating temperatures (30–100 °C) on crude water-soluble proteins are presented Table 3. Value of the data Primer data can be used for a further understanding on the ampliﬁcation of red color-related pigment-binding protein from shrimp shell. Sequence alignment data provide information on the sequence differences and is able to be compared with data from other authors when proﬁling the hemocyanins. Color shift data are valuable for the researchers interested in crustacean shell color change and color-related proteins derived from crustacean shells. Table 1 Original experimental data on the nucleotide sequences of primers used in PCR ampliﬁcation. Primera Sequence (5′–3′) Objective Inter-F TGCTCCCCACACCACTTACAAGTAC Internal ampliﬁcation Inter-R GTGGCAGTTTCRAAGTGTTCYAGCAC Gsp-R GCAATGGCATCACGAATTCG 5′-end ampliﬁcation Gsp-F TCCCAACGTGCAGTACTATG 3′-end ampliﬁcation 5′-Gsp GCACCATGAGGGTCTTAGTGGTTC ORF ampliﬁcation 3′-Gsp TCACTAATGAATGTGTTCCCCATG a Meaning of letters: Inter, internal ampliﬁcation primer; Gsp, gene-speciﬁc primer; F, forward primer; R, reverse primer. 1 al experimental data on the nucleotide sequences of primers used in PCR ampliﬁcation. a r t i c l e i n f o Article history: Received 24 August 2017 Received in revised form 5 October 2017 Accepted 8 November 2017 Available online 10 November 2017 In this article, we report original data on the designation of the primers for full-length cDNA ampliﬁcation and the internal cDNA ampliﬁcation of red color-related pigment-binding protein derived from shrimp shell. Data on the color shifts of different soluble proteins under 100 °C 10 min heat treatment and the effects of heating temperatures (from 30 to 100 °C) on the color changes of crude water-soluble proteins are also included in this report. For further details and experimental ﬁndings please refer to the article “Isolation and cDNA cloning of a novel red color-related pigment- binding protein derived from the shell of shrimp, Litopenaeus vannamei” (Chuang et al., 2017) [1]. & 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). P. Chuang et al. / Data in Brief 16 (2018) 105–108 106 Speciﬁcations Table Speciﬁcations Table Speciﬁcations Table Subject area Food science, biochemistry, molecular biology More speciﬁc subject area Food biochemistry Type of data Tables, ﬁgure How data was acquired Colorimeter (CLR-7100F, Shimadzu, Kyoto, Japan), Nucleotide sequence (ABI 3130 Genetic Analyzer, Applied Biosystems, Foster City, CA, USA), Alignment of nucleotide sequences (ClustalW program, htttp://clustalw.ddbj.nig.ac.jp) Data format Raw and analyzed data Experimental factors Protein isolation and temperature setting Experimental features The ampliﬁcation and alignment of internal cDNA sequence and the color changes of different soluble proteins were performed Data source location Tokyo, Japan Data accessibility The data are available with this article 2. Experimental design, materials and methods Hemocyanin nucleotide sequences from L. vannamei (X82502), P. monodon (JF357966), Fenneropenaeus chinensis (FJ594414), F. merguiensis (KC920897), Marsupenaeus japonicus subunit L (EF375711) and subunit Y (EF375712) were aligned. Two internal primers (Inter-F and Inter-R) were designed based on the highly Table 3 Table 3 Original experimental data on the color shifts of crude water-soluble proteins. Temperature (°C) L* a* b* 25 (control) 6.8470.000a −0.3870.005a 1.1570.005a 30 6.7570.005b −0.3470.005b 1.1170.005b 45 6.0470.002c −0.2270.004c 1.4670.009c 60 5.2070.005d 0.0570.002d 1.8370.002d 80 3.7970.005e 0.0370.000e 2.0970.005e 100 2.8470.005f 0.3370.000f 3.5270.000f Means followed by different lower-case letters within the same column differ signiﬁcantly at po0.05 versus the control. Data are expressed as mean7standard deviation (n¼3). Means followed by different lower-case letters within the same column differ signiﬁcantly at po0.05 versus the control. Data are expressed as mean7standard deviation (n¼3). conserved zone corresponding to the amino acid regions of 270–278 and 381–389 (numbering is on the basis of the amino acid sequence of LvPBP75 shown in Fig. 3) [1]. The other gene–speciﬁc primers were all designed based on the determined nucleotide sequences. conserved zone corresponding to the amino acid regions of 270–278 and 381–389 (numbering is on the basis of the amino acid sequence of LvPBP75 shown in Fig. 3) [1]. The other gene–speciﬁc primers were all designed based on the determined nucleotide sequences. Color shift of different soluble proteins was performed by heating at 100 °C for 10 min. Meanwhile, the red color change of crude-water soluble proteins were determined by heating at temperatures of 30, 45, 60, 80, and 100 °C for 10 min. Unheated samples were set as controls. Temperatures and color changes were monitored and recorded. Color changes were investigated by using the colorimeter (CLR-7100F, Shimadzu, Kyoto, Japan). The results are expressed as L* (brightness), a* (þa red, −a green), and b* (þb yellow, −b blue). Acknowledgements This work was partially funded by the Sasakawa Scientiﬁc Research Grant from The Japan Science Society (29-301). Transparency document. Supporting information Transparency data associated with this article can be found in the online version at http://dx.doi. org/10.1016/j.dib.2017.11.025. a 9a a dic-soluble heated Heated 570.005a 5.0370.045a 2970.012a −0.3070.019a 270.025a 0.9370.029a o0.05 versus unheated samples. Dat P. Chuang et al. / Data in Brief 16 (2018) 105–108 P. Chuang et al. / Data in Brief 16 (2018) 105–108 108 Fig. 1. Data on the alignment of ampliﬁed internal fragment with known hemocyanin (KJ151291). Fig. 1. Data on the alignment of ampliﬁed internal fragment with known hemocyanin (KJ151291). Reference [1] P. Chuang, I. Shoichiro, N. Yuji, G. Jialong, W. Shugo, Isolation and cDNA cloning of a novel red color-related pigment– binding protein derived from the shell of shrimp, Litopenaeus vannamei, Food Chem. 241 (2018) 104–112.
https://openalex.org/W4255666915	https://revista.spmi.pt/index.php/rpmi/article/download/227/922	Portuguese	null	SARS-CoV-2 Vaccine–Induced Immune Thrombotic Thrombocytopenia	New England journal of medicine/The New England journal of medicine	2,021	cc-by	2,803	Abstract: The first vaccines against the SARS-CoV-2 virus appea- red one year after the WHO declared COVID-19 as a pan- demic, with data from United Kingdom and United States of America (USA) reporting a reduction in hospitalization about 67% e 94%, with one or two doses of an approved vacci- ne. In February 2021, the first cases of thrombocytopenia and thrombosis at uncommon sites (cerebral venous sinus and splanchnic venous thrombosis) induced by the vaccines were described. The incidence is still unknown, but early studies in the USA suggest an incidence of 1 in 533 333/vaccinated individuals, and this is probably underestimated. Although un- common, it is a serious adverse effect with a high mortality rate, estimated at 20%. This case report highlights the need Por tudo isto, a notificação destes casos é importante pois contribui para o reconhecimento e compreensão desta síndrome.4 Introdução A pandemia pelo SARS-CoV-2 obrigou a uma rápida atuação pelas entidades de saúde sendo que em um ano, várias vacinas contra o vírus foram desenvolvidas.1 Relataram-se raros casos de efeitos adversos graves, sendo que em fevereiro de 2021 foram descritos pela primeira vez casos de trombocitopenia e trombose em locais pouco co- muns nomeadamente a nível da circulação venosa esplâncnica e nos seios venosos cerebrais.2 Estes eventos verificaram-se cerca de duas semanas após a toma da vacina, inicialmente descritos após a vacina da AstraZeneca (ChAdOx1 nCoV-19), e mais tarde, em abril de 2021, também em relação com a va- cina Johnson & Johnson (Ad26COV2).2 Dadas as semelhanças com a trombocitopenia induzida pela heparina, denominou-se esta entidade como trombocitopenia trombótica induzida pela vacina (TTIV).3 A Agência Europeia do Medicamento estima uma incidência de TIVV após a vacina da AstraZeneca entre 1 em 125 000 e 1 em 1 000 000.3 O mecanismo exato desta entidade não é ainda totalmente compreendido.3 Parece ser mais frequente em mulheres em idade jovem (20 a 55 anos).3 O tratamento, dependendo da gravidade, passa por imunoglo- bulinas intravenosas e anticoagulantes - que não a heparina.3 O reconhecimento precoce é importante na medida em que poderá minimizar a morbimortalidade a longo prazo.3 Palavras-chave: COVID/prevenção e controlo; SARS- -CoV-2; Trombocitopenia/induzida quimicamente; Trombose Venosa; Vacina contra COVID-19/efeitos adversos. Resumo: for a high level of suspicion, which may change the prognosis of some of these patients. Um ano depois de declarada pandemia pela Organiza- ção Mundial da Saúde, surgiram as primeiras vacinas contra o vírus SARS-CoV-2. Na literatura, dados referentes ao Reino Unido e aos Estados Unidos da América (EUA) sugerem que a vacinação - uma ou duas doses – reduziu as hospitalizações entre 67% e 94%. Em fevereiro de 2021 foram descritos os primeiros casos de trombocitopenia e trombose em locais atí- picos, nomeadamente a nível do seio venoso cerebral e na cir- culação venosa esplâncnica, associadas à toma da vacina. Os primeiros estudos nos EUA sugerem uma incidência (provavel- mente subestimada) de 1 em 533 333/indivíduos vacinados. Apesar de incomum, é um efeito adverso grave com alta taxa de mortalidade, estimada em 20%, em parte pelo diagnóstico tardio, pois a clínica inicial é muitas vezes inespecífica e frus- tre. Este relato de caso salienta a necessidade de um elevado índice de suspeição ab initio, que pode mudar o prognóstico destes doentes. Keywords: COVID-19 Vaccines/adverse effects; COVID- 19/prevention & control; SARS-CoV-2; Venous Thrombosis; Thrombocytopenia/chemically induced. Trombocitopenia Trombótica Imune Induzida pela Vacina Contra a COVID-19 SARS-CoV-2 Vaccine–Induced Immune Thrombotic Throm 2 Vaccine–Induced Immune Thrombotic Thrombocytopenia Diana Isabel Rocha1 (https://orcid.org/0000-0001-5641-290X), Vasco Sousa Abreu2 (https://orcid.org/0000-0001-8942- 275X), Ana Novo1 (https://orcid.org/0000-0003-4288-7423), Ana Rita Costa3 (https://orcid.org/0000-0002-1939-1043), Mónica Almeida3 (https://orcid.org/0000-0002-6276-268X) CASOS CLÍNICOS CASE REPORTS CASOS CLÍNICOS CASE REPORTS CASOS CLÍNICOS CASE REPORTS 1Serviço de Medicina Interna 2Serviço de Neurorradiologia, 3Serviço Medicina Interna e Medicina Intensiva. Centro Hospitalar e Universitário do Porto, Porto, Portugal https://doi.org/10.24950/rspmi.227 1Serviço de Medicina Interna 2Serviço de Neurorradiologia, 3Serviço Medicina Interna e Medicina Intensiva. Centro Hospitalar e Universitário do Porto, Porto, Portugal CASOS CLÍNICOS TROMBOCITOPENIA TROMBÓTICA IMUNE INDUZIDA PELA VACINA CONTRA A COVID-19 CASOS CLÍNICOS TROMBOCITOPENIA TROMBÓTICA IMUNE INDUZIDA PELA VACINA CONTRA A COVID-19 anisocoria (E>D), desvio conjugado do olhar para a esquer- da, reflexo cutâneo-plantar indiferente bilateralmente, esta- va hipertensa, normoglicemica e com sinais de má perfusão periférica. Dois dias antes havia referido queixas de mal-es- tar generalizado e vómitos. Sem internamentos prévios ou toma de anticoagulantes. para benzodiazepinas. Electrocardiograma em ritmo sinusal. Realizado TC cerebral com estudo angiográfico arterial, pela suspeita de evento vascular cerebral, que confirmou enfarte cerebral bilateral por oclusão bilateral das artérias carótidas (Figs. 1 a 4). Pela história, cedida pelo filho, de vacinação (ChAdOx1) dez dias antes, foi pedido estudo da coagulação que mostrou d-dímeros aumentados, fibrinogénio, TP e aPTT normais (Tabela 1) e anticorpos anti-PF4/heparina que foram positivos. A pesquisa de SARS-CoV-2 foi negativa. Dado o enfarte já estabelecido, com ausência de circulação anterior bilateralmente, não havia indicação para qualquer interven- ção na fase aguda, estando associado a mau prognostico vital a curto prazo. O caso foi notificado ao INFARMED. Não existia história pessoal ou familiar de trombofilia ou eventos trombóticos prévios; não teve nenhum internamen- to recente, não havia toma de anticoagulantes, nomeada- mente heparina. Não havia alterações terapêuticas prévias recentes. Analiticamente apresentava 19 000 plaquetas (com contagem de plaquetas normais em estudo analítico do início do mês), com informação adicional de ausência de agregados plaquetários. Sem alteração da função renal nem do ionograma, sem alteração do painel hepático (Ta- bela 1). Doseamento de álcool sem níveis tóxicos e pesqui- sa de drogas de abuso na urina apenas com positividade Caso Clínico Mulher de 68 anos, com contexto clínico de hipotiroidis- mo suplementado com levotiroxina e ainda medicada croni- camente com omeprazol e alprazolam, foi admitida na sala de emergência após ter sido encontrada em casa estuporo- sa. À observação na sala de emergência pontuava 6 pon- tos na escala de coma de Glasgow (O2V1M3), apresentava 1Serviço de Medicina Interna 2Serviço de Neurorradiologia, 3Serviço Medicina Interna e Medicina Intensiva. Centro Hospitalar e Universitário do Porto, Porto, Portugal 2 PUBLICAÇÃO TRIMESTRAL VOL.29 \| N.º4 \| OUT/DEZ 2022 267 Discussão A trombocitopenia trombótica imune induzida pela vacina (TTIV) é uma síndrome imune que, pelos dados disponíveis Tabela 1: Parâmetros analíticos da doente à admissão. Parâmetro analítico Resultado Valor de referência Hemoglobina 16,1 12-15 g/dL Leucócitos 20,38 x 103 4,00-11,00 x 103/µL Neutrófilos 17,71 x 103 2,00-7,50 x 103/µL Linfócitos 1,47 x 103 1,50-4,00 x 103/µL Monócitos 1,08 x 103 0,20-0,80 x 103/µL Eosinófilos 0 0,04-0,40 x 103/µL Basófilos 0,04 x 103 0,02-0,10 x 103/µL Plaquetas 19 x 103 NOTA: Sem agregados plaquetários 150-400 x 103/µL Creatinina 0,88 0,5-0,9 mg/dL Ureia 39 10-50 mg/dL Bilirrubina total 0,83 0,20-1,00 mg/dL Desidrogenase do lactato 366 135-214 U/L a 37º Creatinaquinase total 569 24-173 U/L a 37º Mioglobina 616 25-58 µg/L Proteína C reativa 133,79 0-5,0 mg/L Sódio 141 135-145 mmol/L Potássio 4,01 3,5-5,0 mmol/L Anticorpos anti PF4/heparina (ELISA) Positivo Tempo de tromboplastina parcial 33,2 29,0s Tempo de protrombina 14,4s 11,7s INR 1,24 D-dímeros 34000,0 <500 ng/mL 268 Medicina Interna REVISTA DA SOCIEDADE PORTUGUESA DE MEDICINA INTERNA 268 Í CASOS CLÍNICOS Figura 1: TC, cortes axiais, desde a base do crânio até à alta convexidade. Lesões isquémicas recentes fronto-temporo-parietais cortico-subcorticais e profundas, com envolvimento estriato-capsular e da ínsula (território carotídeo bilateral). xiais, desde a base do crânio até à alta convexidade. Lesões isquémicas recentes fronto-temporo-parietais profundas, com envolvimento estriato-capsular e da ínsula (território carotídeo bilateral). Figura 1: TC, cortes axiais, desde a base do crânio até à alta convexidade. Lesões isquémicas recentes fronto-temporo-parietais cortico-subcorticais e profundas, com envolvimento estriato-capsular e da ínsula (território carotídeo bilateral). Figura 3: Angio-TC, cortes coronais, MIP. Ausência da ACI direita [seta] e da ACI e ramos da ACE à esquerda [seta trace- jada] – estes achados podem ser melhor interpretados na reconstrução 3D; refere-se a ausência/paucidade de ramos arteriais intracranianos. até ao momento, parece ser uma variante da trombocito- penia induzida por heparina (TIH) - dada a presença de trom- bocitopenia, trombose e tendo em conta que a presença de anticorpos anti-PF4/Heparina.1,5,6 Por outro lado, e apesar da infeção pelo SARS-CoV-2 estar associada a um estado de hipercoaguabilidade, a TIIV não parece ser uma manifesta- ção da doença já que a grande maioria dos casos relatados ocorreram em doentes com teste de RNA do SARS-CoV-2 negativo. O despoletante e os fatores de risco para a TTIV são ainda desconhecidos.7 Figura 2: Angio-TC, cortes axiais, MIP. Discussão Ausência de preenchi- mento do topo das artérias carótidas internas (ACI) [setas tracejadas], artérias cerebrais médias [setas] e cerebrais ante- riores [pontas de setas], contrastando com o normal preenchi- mento da circulação vertebro-basilar. Figura 3: Angio-TC, cortes coronais, MIP. Ausência da ACI direita [seta] e da ACI e ramos da ACE à esquerda [seta trace- jada] – estes achados podem ser melhor interpretados na reconstrução 3D; refere-se a ausência/paucidade de ramos arteriais intracranianos. Esta síndrome é mais frequente em mulheres com menos de 50 anos e mais raro em doentes com mais de 70 anos.2 É uma entidade com mau prognóstico e que se associa a eventos trombóticos mais frequentemente venosos, em locais incomuns nomeadamente no leito venoso cerebral e/ou es- plâncnico, trombocitopenia moderada a severa, coagulopa- tia e anticorpos anti-PF4 positivos.2 É, até ao momento, uma reação adversa rara, e por isso os benefícios da vacina ultra- passam este risco; todavia, dado o reconhecimento tardio na maioria dos casos relatados, associou-se a uma mortalidade de 20%, sendo de cerca de 70% em doentes que se apresen- taram com trombocitopenia severa e hemorragia cerebral ou Figura 2: Angio-TC, cortes axiais, MIP. Ausência de preenchi- mento do topo das artérias carótidas internas (ACI) [setas tracejadas], artérias cerebrais médias [setas] e cerebrais ante- riores [pontas de setas], contrastando com o normal preenchi- mento da circulação vertebro-basilar. PUBLICAÇÃO TRIMESTRAL VOL.29 \| N.º4 \| OUT/DEZ 2022 269 Í TROMBOCITOPENIA TROMBÓTICA IMUNE INDUZIDA PELA VACINA CONTRA A COVID-19 Figura 4: Reconstrução 3D MIP. Ausência de preenchimento das ACI direita e esquerda, e de ramos da artéria carótida externa (ACE) esquerda - oclusão da ACI direita e da artéria carótida comum (ACC) esquerda – o potencial trajeto de am- bas as ACI encontra-se representando a tracejado; refere-se a presença de ramos da ACE direita (seta), não visíveis à es- querda (oclusão da ACC). Responsabilidades Éticas Conflitos de Interesse: Os autores declaram a inexistência de conflitos de interesse na realização do presente trabalho. Fontes de Financiamento: Não existiram fontes externas de financia- mento para a realização deste artigo. Confidencialidade dos Dados: Os autores declaram ter seguido os pro- tocolos da sua instituição acerca da publicação dos dados de doentes. Consentimento: Consentimento do doente para publicação obtido. Proveniência e Revisão por Pares: Não comissionado; revisão externa por pares. Na doente que apresentamos não havia fatores de risco cardiovascular. Também não se encontrou história pessoal ou familiar de trombofilia e também não existia história de eventos trombóticos prévios. Não teve nenhum internamento recente. Não havia toma de anticoagulantes, nomeadamen- te heparina. Conforme se pode ver na Tabela 1, não havia anemia nem parâmetros bioquímicos sugestivos de hemólise intra-vascular. A leucocitose com neutrofilia bem como o au- mento da proteína C reativa foram interpretados no contexto do evento agudo. Contributorship Statement trombose dos seios venosos.1,2,8 Os sintomas ainda que por vezes inespecíficos, dependem sobretudo do território afeta- do pelo trombo, podendo haver cefaleia, alterações de visão, dor abdominal, náuseas e vómitos, dispneia, dor ou edema do membro inferior, petéquias ou hemorragia espontânea.7,9,10 Habitualmente a sintomatologia surge entre o 6º e 14º dia após a toma da vacina.7,9,10 Para o diagnóstico são necessários qua- tro critérios: vacina da COVID-19 nos 42 dias prévios (frequen- temente apenas com 1 toma da vacina), a documentação imagiológica de evento trombótico arterial ou venoso, trombo- citopenia e anticorpos anti-PF4 positivos (método ELISA).3 A ausência de trombocitopenia não excluiu o diagnóstico, já que o doente pode encontrar-se numa fase inicial deste quadro clínico.9,10 A maioria dos doente tem d-dímeros elevados e em alguns doentes há diminuição do fibrinogénio.8,10 VSA – imaging Diagnosis and discussion of the proposed article AN - Discussion of the article ARC – Clinical diagnosis and discussion of the article ARC – Clinical diagnosis and discussion of the article MA - Clinical diagnosis and discussion of the proposed article MA - Clinical diagnosis and discussion of the proposed article All authors approved the final draft Declaração de Contribuição Declaração de Contribuição DIR – Redação do caso VSA – Diagnóstico Imagiológico e discussão do artigo proposto AN - Discussão do artigo ARC – Diagnóstico clínico e discussão do artigo MA - Diagnóstico clínico e discussão do artigo proposto Todos os autores aprovaram a versão final a ser submetida. Figura 4: Reconstrução 3D MIP. Ausência de preenchimento das ACI direita e esquerda, e de ramos da artéria carótida externa (ACE) esquerda - oclusão da ACI direita e da artéria carótida comum (ACC) esquerda – o potencial trajeto de am- bas as ACI encontra-se representando a tracejado; refere-se a presença de ramos da ACE direita (seta), não visíveis à es- querda (oclusão da ACC). Todos os autores aprovaram a versão final a ser submetida. Conclusão O conhecimento desta entidade pelos clínicos permitirá um melhor e atempado reconhecimento da mesma, o que po- derá permitir melhorar o prognóstico de alguns destes doen- tes através de uma atuação precoce. Para isto, é fundamental um alto nível de suspeição e a realização de protocolos de atuação.  Recebido / Received: 2021/12/17 Aceite / Accepted: 2022/02/18 Publicado / Published: 2022/12/19 Recebido / Received: 2021/12/17 Aceite / Accepted: 2022/02/18 Publicado / Published: 2022/12/19 Recebido / Received: 2021/12/17 7. Wolf ME, Luz B, Niehaus L, Bhogal P, Bazner H, Henkes H. Thrombo- cytopenia and intracranial venous sinus thrombosis after “COVID-19 Vac- cine AstraZeneca” exposure. J Clin Med. 2021;10:1599. doi: 10.3390/ jcm10081599 271 PUBLICAÇÃO TRIMESTRAL VOL.29 \| N.º4 \| OUT/DEZ 2022 Ethical Disclosures l Conflicts of interest: The authors have no conflicts of interest to declare. Financing Support: This work has not received any contribution, grant or scholarship Confidentiality of Data: The authors declare that they have followed the protocols of their work center on the publication of data from patients. Patient Consent: Consent for publication was obtained. A temporalidade entre a toma da vacina e a apresentação com trombocitopenia severa, anticorpos anti-PF4 positivos, d-dímeros muito aumentados e a comprovação imagiológica de enfarte cerebral bilateral e extenso da circulação anterior, culminou na assunção deste diagnóstico. Provenance and Peer Review: Not commissioned; externally peer re- viewed. © Autor (es) (ou seu (s) empregador (es)) e Revista SPMI 2022. Re- utilização permitida de acordo com CC BY-NC. Nenhuma reutilização comercial. Sugere-se que o tratamento desta entidade seja seme- lhante ao da TIH nomeadamente com imunoglobulinas e an- ticoagulantes, que não heparinas.5,6,10 Neste caso a doente apresentava-se num estado clínico grave e irreversível, sem benefícios em manter medidas de suporte avançado de órgão, tendo vindo a falecer. © Author(s) (or their employer(s)) and SPMI Journal 2022. Re-use per- mitted under CC BY-NC. No commercial re-use. Correspondence / Correspondência: Diana Isabel Rocha - dianaisabelrocha@gmail.com Correspondence / Correspondência: Diana Isabel Rocha - dianaisabelrocha@gmail.com 270 Med REVISTA DA Medicina Interna REVISTA DA SOCIEDADE PORTUGUESA DE MEDICINA INTERNA 270 Í CASOS CLÍNICOS Serviço de Medicina Interna, Centro Hospitalar e Universitário do Porto, Porto, Portugal Serviço de Medicina Interna, Centro Hospitalar e Universitário do Porto, Porto, Portugal Largo Prof. Abel Salazar, 4099-001, Porto 5. Greinacher A, Thiele T, Warkentin TE, Weisser K, Kyrle PA, Eichinger S. Thrombotic thrombocytopenia after ChAdOx1 nCov-19 vaccination. N Engl J Med. 2021;384:2092-101. doi: 10.1056/NEJMoa2104840. Largo Prof. Abel Salazar, 4099-001, Porto 6. See I, Su JR, Lale A, Woo EJ, Guh AY, Shimabukuro TT, et al. US Case Reports of Cerebral Venous Sinus Thrombosis With Thrombocyto- penia After Ad26.COV2.S Vaccination, March 2 to April 21, 2021. JAMA. 2021;325:2448-56. doi: 10.1001/jama.2021.7517. REFERÊNCIAS 1. Scully M, Singh D, Lown R, Poles A, Solomon T, Levi M, et al. Pathologic Antibodies to Platelet Factor 4 after ChAdOx1 nCoV-19 Vaccination. N Engl J Med. 2021;384:2202-11. doi: 10.1056/NEJMoa2105385. 1. Scully M, Singh D, Lown R, Poles A, Solomon T, Levi M, et al. Pathologic Antibodies to Platelet Factor 4 after ChAdOx1 nCoV-19 Vaccination. N Engl J Med. 2021;384:2202-11. doi: 10.1056/NEJMoa2105385. 8. British Society for Haematology. [Homepage na Internet]. England and Wales: News. [consultado a 23 setembro 2021]. Disponível em: https://b-s- -h.org.uk/about-us/news/high-mortality-rate-of-vaccine-induced-thrombo- cytopenia-and-thrombosis/ 2. Gowthami M. Arepally, Thomas L. Ortel; Vaccine-induced immune thrombo- tic thrombocytopenia: what we know and do not know. Blood. 2021; 138: 293–8. doi: 10.1182/blood.2021012152 9. Hematology.org [Homepage na Internet]. Washington DC: COVID-19 re- sources. [consultado a 23 setembro 2021]. Disponível em: https://www. hematology.org/covid-19/vaccine-induced-immune-thrombotic-thrombo- cytopenia 3. Aleem A, Nadeem AJ. Coronavirus (COVID-19) Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT) [Updated 2021 Jul 18]. Treasure Is- land: StatPearls Publishing; 2022. Available from: https://www.ncbi.nlm.nih. gov/books/NBK570605/ 10. Alalwan AA, Abou Trabeh A, Premchandran D, Razeem M. COVID-19 Vaccine-Induced Thrombotic Thrombocytopenia: A Case Series. Cureus. 2021;13:e17862. doi:10.7759/cureus.17862 4. Cines DB, Bussel JB. SARS-CoV-2 Vaccine-Induced Immune Thrombo- tic Thrombocytopenia. N Engl J Med. 2021;384:2254-6. doi: 10.1056/
https://openalex.org/W2275119563	https://journals.plos.org/plosntds/article/file?id=10.1371/journal.pntd.0004363&type=printable	English	null	Efficacy and Safety of Pafuramidine versus Pentamidine Maleate for Treatment of First Stage Sleeping Sickness in a Randomized, Comparator-Controlled, International Phase 3 Clinical Trial	PLoS neglected tropical diseases	2,016	cc-by	9,885	Efficacy and Safety of Pafuramidine versus Pentamidine Maleate for Treatment of First Stage Sleeping Sickness in a Randomized, Comparator-Controlled, International Phase 3 Clinical Trial Gabriele Pohlig1‡, Sonja C. Bernhard2,3‡, Johannes Blum4‡, Christian Burri3,5‡, Alain Mpanya6‡, Jean-Pierre Fina Lubaki7‡, Alfred Mpoo Mpoto8‡, Blaise Fungula Munungu9‡, Patrick Mangoni N’tombe8‡, Gratias Kambau Manesa Deo10‡, Pierre Nsele Mutantu11‡, Florent Mbo Kuikumbi6‡, Alain Fukinsia Mintwo12‡, Augustin Kayeye Munungi13‡, Amadeu Dala14‡, Stephen Macharia15‡, Constantin Miaka Mia Bilenge16†‡, Victor Kande Betu Ku Mesu17‡, Jose Ramon Franco18‡, Ndinga Dieyi Dituvanga19‡, Richard R. Tidwell20‡, Carol A. Olson21‡ 1 Swiss Tropical and Public Health Institute, Pharmaceutical Medicine Unit, Swiss Centre for International Health, Basel, Switzerland, 2 Swiss Tropical and Public Health Institute, Pharmaceutical Medicine Unit, Swiss Centre for International Health, Basel, Switzerland, 3 Pharmacy & Clinical Pharmacology at the Division of Clinical Pharmacology, University of Basel, Basel, Switzerland, 4 Swiss Tropical and Public Health Institute, Medical Services and Diagnostic, Basel, Switzerland, 5 Swiss Tropical and Public Health Institute, Department of Medicines Research, Basel, Switzerland, 6 Programme Nationale de Lutte contre la Trypanosomiase Humaine Africaine, Kinshasa, Democratic Republic of the Congo, 7 Hôpital Evangélique de Vanga, Vanga, Province of Bandundu, Democratic Republic of the Congo, 8 Mission Hospital of Vanga, Vanga, Democratic Republic of Congo, 9 Centre Hospitalier Lisungi BDOM, Kinshasa, Democratic Republic of the Congo, 10 Clinique Damas Aleka, Libreville, Gabon, 11 Institut National de Recherche Biomédicale, Kinshasa, Democratic Republic of the Congo, 12 Zone de Santé de Djuma, Djuma, Democratic Republic of the Congo, 13 Zone de Santé de Mpayi, Mpay, Democratic Republic of Congo, 14 Instituto de Combate e de Controlo das Tripanossomíases, Luanda, Angola, 15 Management Sciences for Health, Juba, South Sudan, 16 Ministry of Health, Kinshasa, Democratic Republic of the Congo, 17 Programme des Maladies Tropicales Négligées, Ministère de la Santé Publique Kinshasa, Democratic Republic of the Congo, 18 World Health Organisation Geneva, Department of Control of Neglected Diseases, Geneva, Switzerland, 19 World Health Organization, Luanda, Angola, 20 University of North Carolina, Department of Pathology and Lab Medicine, School of Medicine, Chapel Hill, North Carolina, United States of America, 21 Sapphire Oak Consultants, LLC, Lindenhurst, Illinois, United States of America OPEN ACCESS Citation: Pohlig G, Bernhard SC, Blum J, Burri C, Mpanya A, Lubaki J-PF, et al. (2016) Efficacy and Safety of Pafuramidine versus Pentamidine Maleate for Treatment of First Stage Sleeping Sickness in a Randomized, Comparator-Controlled, International Phase 3 Clinical Trial. PLoS Negl Trop Dis 10(2): e0004363. doi:10.1371/journal.pntd.0004363 Editor: Carlos Franco-Paredes, Hospital Infantil de Mexico Federico Gomez, UNITED STATES Received: September 29, 2015 Accepted: December 14, 2015 Published: February 16, 2016 Editor: Carlos Franco-Paredes, Hospital Infantil de Mexico Federico Gomez, UNITED STATES Received: September 29, 2015 Accepted: December 14, 2015 Published: February 16, 2016 Editor: Carlos Franco-Paredes, Hospital Infantil de Mexico Federico Gomez, UNITED STATES Copyright: © 2016 Pohlig et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: © 2016 Pohlig et al. This is an open access article distributed under the terms of the † Deceased. ‡ GP, SCB, JB, CB, RRT and CAO are primary authors that contributed equally to this work. AM, JPFL, AMM, BFM, PMN, GKMD, PNM, FMK, AFM, AKM, AD, SM, CMMB, VKBKM, JRF, and NDD are secondary authors that also contributed equally to this work. tidwell@med.unc.edu Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Abstract Funding: Funding for this trial was provided by a grant (Grant#38381) from the Bill and Melinda Gates Foundation (www.gatesfoundation.org) and was administered by the University of North Carolina, Chapel Hill, United States of America. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. RESEARCH ARTICLE Methods This was a Phase 3, multi-center, randomized, open-label, parallel-group, active control study where 273 male and female patients with first stage Trypanosoma brucei gambiense HAT were treated at six sites: one trypanosomiasis reference center in Angola, one hospital in South Sudan, and four hospitals in the Democratic Republic of the Congo between August 2005 and September 2009 to support the registration of pafuramidine for treatment of first stage HAT in collaboration with the United States Food and Drug Administration. Patients were treated with either 100 mg of pafuramidine orally twice a day for 10 days or 4 mg/kg pentamidine intramuscularly once daily for 7 days to assess the efficacy and safety of pafuramidine versus pentamidine. Pregnant and lactating women as well as adolescents were included. The primary efficacy endpoint was the combined rate of clinical and parasitological cure at 12 months. The primary safety outcome was the frequency and severity of adverse events. The study was registered on the International Clinical Trials Registry Platform at www.clinicaltrials.gov with the number ISRCTN85534673. Background Sleeping sickness (human African trypanosomiasis [HAT]) is a neglected tropical disease with limited treatment options that currently require parenteral administration. In previous studies, orally administered pafuramidine was well tolerated in healthy patients (for up to 21 days) and stage 1 HAT patients (for up to 10 days), and demonstrated efficacy comparable to pentamidine. 1 / 17 PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004363 February 16, 2016 Findings/Conclusions The overall cure rate at 12 months was 89% in the pafuramidine group and 95% in the pent- amidine group; pafuramidine was non-inferior to pentamidine as the upper bound of the 95% confidence interval did not exceed 15%. The safety profile of pafuramidine was supe- rior to pentamidine; however, 3 patients in the pafuramidine group had glomerulonephritis or nephropathy approximately 8 weeks post-treatment. Two of these events were judged as possibly related to pafuramidine. Despite good tolerability observed in preceding studies, the development program for pafuramidine was discontinued due to delayed post-treatment toxicity. Pafuramidine and Pentamidine for Stage 1 Sleeping Sickness Competing Interests: The Swiss Tropical and Public Health Institute (STPH) planned, managed, and monitored the trial and is a member of the Consortium for Parasitic Drug Development, which developed pafuramidine maleate with a grant from the Bill and Melinda Gates Foundation. In addition, the STPH is an academic contract research organization, which develops therapies against human African trypanosomiasis (nifurtimox- eflornithine combination and fexinidazole) and are supported on behalf of the Drugs for Neglected Diseases initiative. Carol A. Olson is now employed my Sapphire Oak Consultants. Richard R. Tidwell had stock options with Immtech Pharmaceuticals, Inc. They are a publicly help, for profit business. He no longer holds these stock options. These competing interests do not alter our adherence to all PLOS NTDs policies on sharing data and materials. PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004363 February 16, 2016 Pafuramidine and Pentamidine for Stage 1 Sleeping Sickness Introduction Sleeping sickness (human African trypanosomiasis [HAT]) is a neglected tropical disease with limited treatment options that currently require parenteral administration. Trypanosoma bru- cei (T.b.) gambiense is found in 24 countries in west and central Africa and currently accounts for over 98% of reported cases [1]. Despite the long history of the disease (first cases reported in 1373/1374), the drugs available to treat it are toxic, difficult to administer, and stage-specific [2]. First stage symptoms entail bouts of fever, headaches, joint pains, and itching, and a person can be infected for months or even years without major signs or symptoms of the disease. When more evident symptoms emerge, the patient is often already in an advanced disease stage where the central nervous system is affected (second stage). The majority of current HAT research is focused on stage 2 of the disease, which requires drugs that can cross the blood- brain barrier. Drugs for stage 2 HAT are either too toxic (melarsoprol) or have too complex a regimen (nifurtimox-eflornithine combination treatment) for use against the first stage of the disease. Only two drugs are approved for treatment of stage 1 HAT: pentamidine (only for T.b. gam- biense) and suramin (only for T.b. rhodesiense), which are both administered parenterally. Sur- amin, synthesized as a dye in 1916 [3], has been used for the treatment of sleeping sickness since 1922 [4] [5], but it can cause undesirable effects in the urinary tract and allergic reactions [1]. Pentamidine, introduced in 1937, was developed as an analog of synthalin, a hypoglycemic agent with anti-trypanosomal activity [6] [7]. Pentamidine is administered by the intramuscu- lar route and has a reported treatment failure rate after a course of five injections of approxi- mately 7% [8] [9] [10]. Though this efficacy profile is encouraging, treatment with pentamidine has limitations. It requires injection, which hampers its use in rural treatment facilities, and though adverse reactions are usually reversible and its most serious long-term consequence, diabetes, is rare, the treatment is accompanied by a high frequency of adverse events, including hypotension, nephrotoxic effects, leukopenia, and hypo- and hyperglycemia [11] [12]. There is no vaccine for T.b. gambiense HAT and there is a great need for new safe and effica- cious drugs that would be easy to use in rural health centers and affordable. Author Summary Sleeping sickness, or human African trypanosomiasis (HAT), is a neglected tropical dis- ease. Because only 2 treatment options are available to treat persons with stage 1 disease, and both require parenteral administration, oral drugs would be of great benefit to the affected population. In this Phase 3, multi-center, randomized, open-label, parallel-group study, we compared oral pafuramidine with intramuscular pentamidine in persons in sub- Sahara Africa with first stage HAT. At 12 months, the overall cure rates (combined clinical and parasitological cure) were similar: 89% in the pafuramidine group and 95% in the pentamidine group. At 24 months, the cure rates continued to be high: 84% and 89%, respectively. Pafuramidine’s safety profile was superior to the comparator drug, and it was consistent with the overall safety profile seen in previous Phase 2 studies. Upon further analysis, however, a renal safety issue was identified as being possibly related to pafurami- dine and further clinical development was halted. Nevertheless, the clinical studies con- ducted in the pafuramidine development program provide a model for future studies in rural Africa. 2 / 17 PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004363 February 16, 2016 PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004363 February 16, 2016 Methods Design This study was approved by the following independent review boards and independent eth- ics committees: Ethikkommission beider Basel, Switzerland; the institutional review board of the University of North Carolina, School of Medicine, US., IRB# 05–2062 (Formerly 05-PATH/LAB-573; the Ethics Committee for Trypanosomiasis Research of the Ministry of Health of Angola; the Ethics Committee of the Ministry of Health of the DRC; and the Ethics Committee of the Ministry of Health of the Government of Southern Sudan. Patients were randomized (1:1) to either 100 mg of pafuramidine orally twice a day (BID) for 10 days (n = 136) or 4 mg/kg pentamidine intramuscular injection once daily for 7 days (n = 137). Pafuramidine and Pentamidine for Stage 1 Sleeping Sickness collaboration with the United States (US) Food and Drug Administration (FDA). The primary objective of the study was to demonstrate the non-inferiority of oral pafuramidine versus intra- muscular pentamidine for treatment of first stage HAT caused by T.b. gambiense. Since safety and efficacy of a new drug should, if at all possible, be established in a study population repre- sentative of the target population, the secondary objective was to include pregnant and lactat- ing women as well as adolescents in the study. Reproductive studies of pafuramidine in animals have not indicated any embryo or fetal toxicity or other effects on reproductive func- tion of adult male and female rats or rabbits. Therefore, it was considered appropriate and was approved by the US FDA to proceed with studies including pregnant and lactating women. Methods Design This was a multi-center, multi-country, open-label (sponsor-blinded), parallel-group, compar- ator-controlled, randomized Phase 3 study to compare the efficacy, safety, and tolerability of pafuramidine and pentamidine in 273 patients with first stage HAT caused by T.b. gambiense. The study was conducted at six African sites where T.b. gambiense sleeping sickness is endemic: two trypanosomiasis reference centers (Angola and the Democratic Republic of the Congo [DRC]) and four hospitals (1 in South Sudan and three in the DRC) from August 2005 (first patient enrolled) to September 2009 (last patient follow-up completed). The study was registered on the International Clinical Trials Registry Platform at www. clinicaltrials.gov with the number ISRCTN85534673. International Protocol #289-C-006. Ethics statement. This will certify that the Institutional Review Boards (IRBs) at the Uni- versity of North Carolina at Chapel Hill, administered by the Office of Human Research Ethics, are organized and operate according to applicable laws and regulations governing research involving human subjects. These include, when applicable, statutes of the State of North Caro- lina and regulations of the Food and Drug Administration (21 CFR 50 and 56) and the Depart- ment of Health and Human Services [45 CFR 46 (the "Common Rule") and 45 CFR 164 (the Health Insurance Portability and Accountability Act, HIPPAA]. In addition, the IRBs conform, when applicable, to Good Clinical Practice (GCP) guidelines of the International Conference of Harmonization (ICH), to the extent these do not contradict DHHS and FDA regulations. The University of North Carolina at Chapel Hill holds a Federalwide Assurance, FWA 4801, approved by the federal Office for Human Research Protections (OHRP). pp y ( ) This study was approved by the following independent review boards and independent eth- ics committees: Ethikkommission beider Basel, Switzerland; the institutional review board of the University of North Carolina, School of Medicine, US., IRB# 05–2062 (Formerly 05-PATH/LAB-573; the Ethics Committee for Trypanosomiasis Research of the Ministry of Health of Angola; the Ethics Committee of the Ministry of Health of the DRC; and the Ethics Committee of the Ministry of Health of the Government of Southern Sudan. Introduction In 2000, the prom- ising orally administered pro-drug pafuramidine was chosen for clinical development by the Consortium for Parasitic Drug Development, which was founded in 1999 to foster the develop- ment of compounds with antiprotozoal activity [13]. Pafuramidine is the dimethoxime pro- drug of furamidine (which has demonstrated excellent efficacy in vitro against T.b. rhodesiense) [14] Pafuramidine was shown to be effective in vivo in the acute model (first stage There is no vaccine for T.b. gambiense HAT and there is a great need for new safe and effica- cious drugs that would be easy to use in rural health centers and affordable. In 2000, the prom- ising orally administered pro-drug pafuramidine was chosen for clinical development by the Consortium for Parasitic Drug Development, which was founded in 1999 to foster the develop- ment of compounds with antiprotozoal activity [13]. Pafuramidine is the dimethoxime pro- drug of furamidine (which has demonstrated excellent efficacy in vitro against T.b. rhodesiense) [14]. Pafuramidine was shown to be effective in vivo in the acute model (first stage disease) in mice [15] [16] and in monkeys (green vervet monkey [17] and rhesus monkey [18]). Preclinical evaluation in vitro as well as animal testing indicated no major safety concerns. rhodesiense) [14]. Pafuramidine was shown to be effective in vivo in the acute model (first stage disease) in mice [15] [16] and in monkeys (green vervet monkey [17] and rhesus monkey [18]). Preclinical evaluation in vitro as well as animal testing indicated no major safety concerns. In 2000, pafuramidine was further evaluated in Phase 1 studies in healthy patients after sin- gle and multiple dosing (up to 21 days) and was well tolerated [19]. The subsequent Phase 2 studies (conducted from 2001 to 2007) in patients with stage 1 sleeping sickness supported this finding [20]. Efficacy after 5 days of treatment was limited; therefore, to attain efficacy compa- rable to that of pentamidine, the treatment duration was prolonged to 10 days. The pharmaco- kinetic properties of pafuramidine (in particular, the lack of proportional conversion of DB289 to DB75 at therapeutic doses) precluded using a higher dose to improve efficacy [19] [20] [21] [22]. This single, confirmatory, pivotal Phase 3 study was developed to support the registration of pafuramidine for treatment of stage 1 HAT under a Special Protocol Assessment in PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004363 February 16, 2016 3 / 17 Pafuramidine and Pentamidine for Stage 1 Sleeping Sickness Study Patients Male and female patients were eligible to participate if they were 12 years of age, weighed 30 kg, had first stage T.b. gambiense infection documented by the presence of trypanosomes in the blood and/or lymph, and had no evidence of second stage disease (no trypanosomes detected in the cerebrospinal fluid [CSF] and 5 white blood cells [WBCs]/mm3 in CSF). Patients were also excluded if tested positive for malaria or helminth infections. Patients were not tested for HIV prior to treatment. Patients were treated at two trypanosomiasis reference centers (Angola and the DRC) and four hospitals (1 in South Sudan and three in the DRC). Written informed consent was obtained from each patient. If the patient was a minor or men- tally impaired, a legal guardian also signed the consent form and if a patient was illiterate, an impartial witness assisted in the consent process. Pregnant and lactating female patients as well as adolescents 12 to 15 years could be enrolled. Adolescents underwent additional safety laboratory testing at the 3-month post-treat- ment visit. Eligible pregnant and lactating female patients could participate with the under- standing that additional safety measurements regarding course and outcome of the pregnancy and/or the health of their infant would be performed. Patients were excluded if they had a possible or confirmed second stage T.b. gambiense infection (ie, presence of parasite in the CSF upon microscopic examination or a WBC count in the CSF of >5 mm3); any active, clinically relevant medical conditions that in the investiga- tor’s opinion might jeopardize patient safety or interfere with study participation; presented with a score of less than 9 on the Glasgow Coma Scale; were previously treated for HAT; or dis- played other conditions that would compromise participation. Changes to Trial Design There was one protocol amendment that provided detailed microscopy instructions for exam- ining blood and CSF for the presence of trypanosomes and determining WBC count in CSF. The amendment also detailed randomization of pregnant and lactating women in a separate strata, and provided additional clarifications and administrative changes. 4 / 17 PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004363 February 16, 2016 PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004363 February 16, 2016 Pafuramidine and Pentamidine for Stage 1 Sleeping Sickness of treatment and at 3, 6, 12, and 18 months post-treatment [26]. Lumbar puncture was per- formed for microscopic examination of CSF fluid for WBCs and trypanosomes at baseline and 6, 12, and 18 months post-treatment, and at any other evaluation where relapse was suspected or trypanosomes were demonstrated in blood or lymph nodes. Additional assessments of clini- cal efficacy were performed at 24 months post-treatment. During the treatment and post-treatment period, safety evaluations included vital signs, physical examination, adverse event monitoring, laboratory tests, electrocardiogram (ECG) monitoring to the extent possible at each site, and documentation of concomitant medications. Signs or symptoms of HAT were queried and graded. Laboratory tests assessed clinical chemis- try (aspartate aminotransferase, alanine aminotransferase, total bilirubin, glucose, and creati- nine) and hemoglobin. Electrocardiograms were performed at baseline, 1 hour prior to dosing, and 1 hour after dosing for all patients. An additional ECG was obtained on Day 7 post-treat- ment for pafuramidine-treated patients. Interventions Screening occurred within 7 weeks prior to dosing with pafuramidine or pentamidine (within 6 weeks prior to the baseline evaluation) using the card agglutination test for trypanosomes [11] [12] and microscopic examination (thin and/or thick smear) of blood and lymph node aspirate for trypanosomes either at the trypanosomiasis treatment centers or by mobile diag- nostic units. All diagnostic tests performed by mobile teams were repeated in the treatment centers. Lumbar puncture was performed at the treatment centers in all trypanosome-positive cases detected by any method and the disease stage was determined by microscopic examina- tion of CSF for trypanosomes and by WBC counts. If the result was negative, a blood sample was examined (including hematocrit centrifugation [23] and miniature anion exchange centri- fugation technique [m-AECT] [24]). All patients were tested for malaria, and filaria using thick and thin blood smears and for diarrhea. If necessary, malaria treatment was given before enrol- ment; filariasis therapy was administered after study treatment when necessary. Patients were admitted as in-patients to the clinical site for the full duration of the treatment/observation period (11 days for pafuramidine or 7 days for pentamidine). Other baseline documentation included demographics, medical history, signs and symptoms of HAT, and concomitant dis- ease(s) and medication(s). Clinical supplies of pafuramidine were provided to the sites in bottles (50 tablets of 100 mg) labeled to indicate study drug, strength, expiration date, protocol number, and other informa- tion according to local regulations. Pentamidine was provided locally by the agency (generally the national HAT control programs) responsible for each center in the form of pentamidine isethionate for injection in single-dose vials at 200 mg/vial. For efficacy assessments, patients underwent microscopic examination of blood (thin and/ or thick smear), hematocrit centrifugation of blood [25], microscopic examination of lymph node aspirate, and microscopic examination of blood after m-AECT concentration at the end 5 / 17 PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004363 February 16, 2016 Outcomes Clinical response definitions are listed in Table 1. The primary efficacy endpoint was the com- bined rate of cure and probable cure at the 12-month follow-up in the per-protocol data set. The overall cure rate was defined as the proportion of treated patients with no clinical signs and symptoms of HAT, no evidence of trypanosomes in any body fluid examined, and no treatment with other trypanocidal agents for any reason; in addition, 5 WBCs/mm3 in CSF obtained from a lumbar puncture was required. Secondary efficacy endpoints were cure, clinical cure, probable relapse, relapse, and death rates at the end of treatment and all follow-up visits. Parasitological cure, probable relapse, relapse, and death rates were also assessed at the 12-month test of cure evaluation and at the 24-month evaluation; the clinical cure was considered equivalent to the parasitological cure at the 24-month evaluation. Study efficacy parameters and timing of post-treatment evaluations were based on WHO recommendations for clinical product development for HAT [27]. Although 18 months post- Table 1. Clinical response definitions. Category WHO Term Characteristics Parasitological cure Cure Lumbar puncture performed: no evidence for parasitological relapse and 5 WBCs/mm3 in CSF Clinical cure Probable cure No evidence for parasitological relapse in absence of lumbar puncture (no clinical signs; symptoms/signs attributable to other disease; investigator decides no retreatment necessary) or No parasitological evidence of relapse with 6–0 WBCs/mm3 in CSF Action: No retreatment Probable relapse Probable relapse No evidence of parasitological relapse and >20 WBCs/mm3 in CSF or No evidence of parasitological relapse in a patient who refuses lumbar puncture and who presents with clinical signs of HAT and/or marked deterioration of clinical condition relative to previous evaluations that is unlikely due to another disease. In addition, in the investigator’s opinion, all other reasons for the patient’s clinical status have been excluded and rescue treatment is required. Action: Retreatment Relapse Relapse Trypanosomes have been detected in any body ﬂuid Action: Retreatment Death Death Death of patient during treatment or follow-up; death will be categorized based on likely or deﬁnite cause of death as HAT; adverse event related to treatment of HAT; causes unrelated to HAT and treatment; unknown causes CSF = cerebrospinal ﬂuid, HAT = human African trypanosomiasis, WBCs = white blood cells, WHO = World Health Organization doi:10.1371/journal.pntd.0004363.t001 PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004363 February 16, 2016 6 / 17 Table 1. Clinical response definitions. Sample Size A total of 250 patients, 125 patients per treatment group, were originally expected to be treated in order to include 100 patients per treatment group in the per-protocol. This sample size pro- vided more than 90% power to demonstrate non-inferiority of pafuramidine to pentamidine for the primary endpoint, when the study drugs have equivalent probable cure rates of 95% in the per-protocol analysis. Non-inferiority comparison was conducted with an alpha equal to 0.048 and non-inferiority margin (ie, delta) of 0.15. Pafuramidine and Pentamidine for Stage 1 Sleeping Sickness treatment is recommended to assess clinical cure in HAT control programs due to anticipated increased drop-out rates from follow-up after 6 to 12 months, the 12-month evaluation was chosen as the primary endpoint in this study in order to maintain a robust data set for the pri- mary analysis. Safety was assessed through the end of treatment evaluation and included adverse events, laboratory results, vital sign measurements, physical examinations, and use of any concomitant medications. The term “adverse event” included any of the following events that developed or increased in severity during the study: 1) any signs or symptoms whether thought to be related or unrelated to HAT; 2) any clinically significant laboratory abnormality; or 3) any abnormal- ity detected during physical examination. Adverse events were graded by the investigator (1 = mild, 2 = moderate, 3 = severe, 4 = intolerable). Adverse events were assessed at every study visit and were classified according to the terms found in the Medical Dictionary for Regu- latory Activities (MedDRA). A serious adverse event was defined as any event that suggested a significant hazard, con- traindication, side effect, or precaution, it included any event that: 1) is fatal; 2) is life threatening; 3) is a persistent or significant disability/incapacity; 4) requires or prolongs in- patient hospitalization; 5) is a congenital anomaly/birth defect; or 6) is an important medi- cal event, based upon appropriate medical judgment, that may jeopardize the patient or may require medical or surgical intervention to prevent one of the other outcomes defining serious. Changes to Outcomes There were no changes to any of the outcomes. Outcomes OS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004363 February 16, 2016 6 / 17 PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004363 February 16, 2016 Statistical Methods The primary efficacy analysis, demonstrating the non-inferiority of pafuramidine to pentami- dine for the combined rate of cure and probable cure, was conducted with an alpha equal to 0.048 and non-inferiority margin (ie, delta) of 0.15. The comparison was made with a 1-sided 97.6% confidence interval (CI) for the treatment difference in parasitological cure rate. The normal approximation to the binomial distribution with continuity correction was used to con- struct the CI. The primary data set for efficacy analysis was the per-protocol data set. The efficacy analysis was carried out for the per-protocol data set (primary analysis), the intention-to-treat (ITT) data set, and the modified ITT (mITT) data set (supportive analyses). The per-protocol data set was defined as patients who received a minimum of 7 days of pafura- midine or five injections of pentamidine and who attended the test of cure assessment at Month 12 or reached an efficacy endpoint (death, non-response, or relapse) at an earlier time. Patients without lumbar puncture at Month 12 were included and their outcome was assessed based on clinical signs and symptoms and parasitological findings from any body fluid examined. The mITT data set consisted of all patients who received the minimum amount of random- ized study drug (7 or 5 days) and for whom an end-of-treatment assessment and at least one follow-up efficacy assessment were available. Patients who had received at least one dose of study drug were included in the ITT data set and patients who were lost to follow-up or discon- tinued from the study for any reason were considered treatment failures in the ITT analysis. The last observation carried backward was used to account for missing data at an earlier evalu- ation (in case of cure at a later evaluation). For the mITT analysis, missing data were addressed according to the last observation carried forward principle. The secondary efficacy variables were summarized at all time points with point estimates and 1-sided 97.5% CIs for the difference between treatments. The safety data set consisted of all patients who received at least one dose of study drug and had at least one safety evaluation after dosing. Treatment group differences in the proportion of patients who reported treatment-emergent adverse events for Day 1 through Day 11 were assessed with Fisher’s exact test. Blinding The study was open-label, since pafuramidine is administered orally and pentamidine is administered intramuscularly. However, the sponsor was blinded to treatment assignments. Pafuramidine and Pentamidine for Stage 1 Sleeping Sickness Randomization Patients were randomly assigned by the local investigators to receive either pafuramidine or pentamidine in the order in which they were enrolled. Randomization was carried out in blocks of variable size following a randomization schedule prepared by the sponsor; randomization of pregnant and lactating women was stratified. Each study site was provided with series of indi- vidual envelopes each containing a card with the treatment assignment for 1 patient and a con- trol number. After a patient signed the informed consent and inclusion/exclusion criteria were confirmed, the investigator opened the next envelope in the randomization list to obtain treat- ment assignment for that patient and then transferred the control number to the patient’s case report form. PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004363 February 16, 2016 Stopping Guidelines and Interim Analysis The sponsor may have terminated this study prematurely, either in its entirety or at a particular site, for reasonable cause or safety concerns. The sponsor remained blinded and the data were provided to the data safety monitoring board (DSMB) for evaluation. Based on these data, the DSMB made recommendations to the sponsor regarding continuation of the study. The study could have been stopped if: 1) any new untoward safety issues were identified in the pafurami- dine treatment group such that pafuramidine was significantly less safe than pentamidine; 2) the re-estimated sample size exceeded 500 patients to achieve 90% power for the primary effi- cacy endpoint; or 3) efficacy analysis indicated that pafuramidine was significantly more effec- tive than pentamidine (p<0.002). An interim analysis was to be conducted when half of the enrolled patients reached the 12-month post-treatment endpoint, however, this was not done because the pafuramidine development program was discontinued due to delayed post-treatment toxicity (details are provided in the Harms section). 7 / 17 PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004363 February 16, 2016 Numbers Analyzed and Excluded As shown in Fig 1, 133 of 136 patients (97.8%) in the pafuramidine group and 129 of 137 patients (94.2%) in the pentamidine group were included in the efficacy analysis. Three patients in the pafuramidine group and 8 patients in the pentamidine group were excluded because they were lost to follow-up. Pafuramidine and Pentamidine for Stage 1 Sleeping Sickness Any clinically significant physical examination changes from baseline were captured as an adverse event. Participant Flow First stage HAT patients rarely present at a hospital or a treatment center. Therefore, intense screening activities were necessary. Between July 2005 and March 2007, a total of 234,919 patients were screened to find 839 individuals affected with HAT (Fig 1). The exclusion rate was high (566 of 839 patients, 67.5%); primary reasons were that patients had stage 2 HAT and did not meet inclusion criteria. A total of 273 patients were randomized: 136 patients received pafuramidine and 137 received pentamidine; all 273 completed the study. Most of the patients (91%) were enrolled in the DRC (248 of 273 patients); 5.5% were enrolled in Angola (15 of 273 patients), and 3.7% (10 of 273 patients) were enrolled in South Sudan. As shown in Fig 1, follow-up attendance at Month 24 was good; only 2 patients in the pafur- amidine group and 8 patients in the pentamidine group were lost to follow-up. Baseline Data As seen in Table 2, baseline demographic characteristics between the two treatment groups were similar. The median age of patients in both treatment groups was approximately 30 years, and the majority of patients in both groups were female (70% and 66%, respectively). Statistical Methods The number and percentage of patients who reported treatment-emergent adverse events were summarized for each treatment group at the system organ class, high-level group terms, and preferred term level. Treatment group differences in the proportion of patients who reported each high-level group term were assessed with Fisher’s exact test. 8 / 17 PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004363 February 16, 2016 PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004363 February 16, 2016 Outcomes and Estimations As shown in Table 3 at the test of cure evaluation (12 months post-treatment), the combined rate of cure and probable cure was 89% (118 of 133 patients) in the pafuramidine group and 95% (123 of 129 patients) in the pentamidine group in the per-protocol population. Pafuramidine was non-inferior to pentamidine as the upper bound of the 95% CI did not exceed 15%. This finding was supported by the 24-month follow-up data, where cure rates of 84% for the pafuramidine group and 89% for the pentamidine group were maintained (Table 4). Supportive analysis in the ITT and mITT populations generated similar results. Table 5 summarizes the secondary efficacy variables for each follow-up visit, including the cumulative number of cures, probable cures, probable relapses, relapses, and deaths for each treatment group. There were no deaths in either treatment group during the active study period, and all patients responded to treatment. Relapses in the pafuramidine treatment group appeared to be evenly distributed over the whole follow-up period, whereas a trend for late relapses was observed in the pentamidine treatment group. The numbers of adolescents and pregnant women (8 and 10, respectively) were too small to make any definitive conclusions about efficacy in these patients. For lactating women, the over- all cure rates of pafuramidine and pentamidine at the test of cure evaluation were the same (23 of 26 patients [88.4%] in each group). However, the low number of lactating women also did not allow for definitive conclusions. 9 / 17 PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004363 February 16, 2016 Pafuramidine and Pentamidine for Stage 1 Sleeping Sickness Fig 1. Pafuramidine (DB289) Phase 3 study CONSORT flowchart. doi:10.1371/journal.pntd.0004363.g001 Harms No patients prematurely discontinued due to an adverse event during the study. As shown in Table 6, the most commonly reported adverse events were injection site pain, pyrexia, hypoglycemia, and hypotension. These events occurred more frequently in the Fig 1. Pafuramidine (DB289) Phase 3 study CONSORT flowchart. doi:10.1371/journal.pntd.0004363.g001 PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004363 February 16, 2016 10 / 17 Fig 1. Pafuramidine (DB289) Phase 3 study CONSORT flowchart. Fig 1. Pafuramidine (DB289) Phase 3 study CONSORT flowchart. doi:10.1371/journal.pntd.0004363.g001 doi:10.1371/journal.pntd.0004363.g001 Harms No patients prematurely discontinued due to an adverse event during the study. As shown in Table 6, the most commonly reported adverse events were injection site pain, pyrexia, hypoglycemia, and hypotension. These events occurred more frequently in the 10 / 17 PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004363 February 16, 2016 Pafuramidine and Pentamidine for Stage 1 Sleeping Sickness Table 2. Baseline demographics. Table 2. Baseline demographics. Table 2. Baseline demographics. Pafuramidine 100 mg BID (N = 136) n (%) Pentamidine 4 mg/kg QD (N = 137) n (%) Age (years) Mean (SD) 33.4 (13.66) 33.7 (14.10) Median 30 31 Min, max 12, 64 13, 75 n (%) >64 years 0 5 (3.6) Sex n (%) male 41 (30.1) 47 (34.3) n (%) female 95 (69.9) 90 (65.7) Status Adolescents 8 (5.9) 8 (5.8) Pregnant 4 (2.9) 8 (5.8) Lactating 28 (20.6) 26 (19.0) Pregnant & lactating 1 (0.7) 1 (0.7) Weight Mean (SD) 44.7 (7.90) 45.7 (7.82) Median 44 46 Min, max 30, 64 30, 69 Height (cm) N = 135 N = 137 Mean (SD) 160.4 (9.41) 160.6 (9.38) Median 160 160 Min, max 139, 187 138, 185 BID = twice a day, QD = once a day, SD = standard deviation Pentamidine 4 mg/kg QD (N = 137) n (%) Pafuramidine 100 mg BID (N = 136) n (%) pentamidine group than the pafuramidine group, with the exception of pyrexia, which occurred more frequently in the pafuramidine group. The incidence of patients with at least one adverse event overall for Day 1 through Day 11 was statistically significantly less in the pafuramidine treatment group (82%, 111 of 136) than in the pentamidine group (99%, 135 of 137) (p<0.05). Among high-level group terms, there were statistically significant differences in favor of the pafuramidine treatment group versus the pentamidine group for hepatobiliary investigations (7% vs. 77%, respectively); renal and urinary tract investigations and urinalyses (2% vs. 9%, respectively); glucose metabolism disorders (including diabetes mellitus) (6% vs. 18%, respectively); and decreased nonspecific blood pressure disorders and shock (44% vs. 62%, respectively) (p<0.05 for all). Table 3. Combined rate of cure and probable cure at 12 months. Patient Group Pafuramidine Pentamidine 95% CIa 100 mg BID 4 mg/kg QD n/N (%) n/N (%) Per-protocol population 118/133 (88.7) 123/129 (95.3) -0.1, 13.1 ITT population 118/136 (86.8) 123/137 (89.8) -4.6, 10.6 mITT population 121/136 (89.0) 131/137 (95.6) 0.4, 12.9 BID = twice a day, CI = conﬁdence interval, ITT = intent-to-treat population, mITT = modiﬁed intent-to-treat population, QD = once a day a Conﬁdence intervals are based on normal approximation to the binomial for pentamidine–pafuramidine. doi:10.1371/journal.pntd.0004363.t003 PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004363 February 16, 2016 11 / 17 Table 3. Pafuramidine and Pentamidine for Stage 1 Sleeping Sickness Table 4. Combined rate of cure and probable cure at 24 months. Pafuramidine Pentamidine 95% CIa 100 mg BID 4 mg/kg QD n/N (%) n/N (%) Per-protocol population 113/135 (83.7) 116/130 (89.2) -2.7, 13.7 ITT population 113/136 (83.1) 116/137 (84.7) -7.1, 10.3 mITT population 114/136 (83.8) 123/137 (89.8) -2.0, 14.0 BID = twice a day, CI = conﬁdence interval, ITT = intent-to-treat population, mITT = modiﬁed intent-to-treat population, QD = once a day a Conﬁdence intervals based on normal approximation to the binomial for pentamidine–pafuramidine Table 4. Combined rate of cure and probable cure at 24 months. Table 4. Combined rate of cure and probable cure at 24 months. BID = twice a day, CI = conﬁdence interval, ITT = intent-to-treat population, mITT = modiﬁed intent-to-treat population, QD = once a day a Conﬁdence intervals based on normal approximation to the binomial for pentamidine–pafuramidine A statistically significantly greater percentage of pafuramidine patients than pentamidine patients experienced epidermal and dermal conditions (5% vs. 1%, respectively) (p<0.05). The majority of the adverse events were mild or moderate in severity and typical for patients recovering from first stage HAT. The ECG results from this study were included in a separately published study on cardiac alterations in HAT [28]. In brief, the mean PQ and QTc intervals did not increase during treat- ment of first stage disease in either treatment group. The appearance and disappearance of repolarization changes at the end of treatment were comparable between groups. A total of 43 patients experienced serious adverse events during the study (including the fol- low-up period): 19 of 136 patients (14.0%) in the pafuramidine group and 24 of 137 patients (17.5%) in the pentamidine group. Of these, only 3 patients had serious adverse events while on treatment: 1 in the pafuramidine group (cellulitis considered probably not related to study drug) and 2 in the pentamidine group (hypersensitivity considered not related to study drug and subcutaneous abscess considered probably related to the study drug). All other serious adverse events occurred during the follow-up period. Of the 43 patients with serious adverse events, it was initially considered probable that only one was related to the study drug (subcutaneous abscess in the pentamidine group). Re-evalua- tion of the relatedness of these events to the study drug was subsequently performed when seri- ous renal and hepatic post-treatment toxicity was observed in 3 patients in a supportive Phase 1 study of pafuramidine, which was conducted in South Africa (in December 2007), during the follow-up period of the current Phase 3 study. The Phase 1 study included 175 male and female volunteers taking oral pafuramidine 100 mg BID for 14 days [19]. Retrospectively, the glomer- ulonephritis reported for the 2 pafuramidine patients in the current Phase 3 study was consid- ered to be possibly related to the study drug. Thirteen patients (6 in the pafuramidine group and 7 in the pentamidine group) died during the follow-up period. All deaths were considered not related or probably not related to the study drug. Two deaths in the pafuramidine group were considered to be related to HAT; one death was considered to be related to relapse of HAT and another was considered to be associ- ated with treatment of a HAT relapse with melarsoprol. Safety data for adolescent as well as pregnant and lactating women were similar to the obser- vations in the general population. Combined rate of cure and probable cure at 12 months. Table 3. Combined rate of cure and probable cure at 12 months. BID = twice a day, CI = conﬁdence interval, ITT = intent-to-treat population, mITT = modiﬁed intent-to-treat population, QD = once a day a Conﬁdence intervals are based on normal approximation to the binomial for pentamidine–pafuramidine. 11 / 17 Pafuramidine and Pentamidine for Stage 1 Sleeping Sickness Table 5. Secondary efficacy variables at months 3, 6, 12, 18, and 24 (per-protocol dataset). Pafuramidine Pentamidine WHO Efﬁcacy Criteria 100 mg BID 4 mg/kg QD Time Point n/N (%) n/N (%) 95% CIa Cure Month 3 2/135 (1.5) 1/137 (0.7) -3.2, 1.7 Month 6 124/135 (91.9) 131/137 (95.6) -2.0, 9.5 Month 12 117/133 (88.0) 122/129 (94.6) -0.2, 13.4 Month 18 99/131 (75.6) 109/129 (84.5) -0.7, 18.6 Month 24 86/135 (63.7) 83/130 (63.8) -11.4, 11.7 Probable cure Month 3b 131/135 (97.0) 135/137 (98.5) -2.0, 5.0 Month 6b 5/135 (3.7) 3/137 (2.2) -5.5, 2.5 Month 12b 4/133 (3.0) 4/129 (3.1) -4.1, 4.3 Month 18b 16/131 (12.2) 13/129 (10.1) -9.8, 5.5 Month 24c 28/135 (20.7) 34/130 (26.2) -4.8, 15.6 Probable relapse Month 3 0 0 -- Month 6 2/135 (1.5) 1/137 (0.7) -3.2, 1.7 Month 12 5/133 (3.8) 1/129 (0.8) -6.6, 0.6 Month 18 7/131 (5.3) 1/129 (0.8) -8.7, -0.4 Month 24 11/135 (8.1) 3/130 (2.3) -11.1, -0.6 Relapsed Month 3 2/135 (1.5) 1/137 (0.7) -3.2, 1.7 Month 6 2/135 (1.5) 1/137 (0.7) -3.2, 1.7 Month 12 3/133 (2.3) 1/129 (0.8) -4.4, 1.5 Month 18 4/131 (3.1) 3/129 (2.3) -4.7, 3.2 Month 24 5/135 (3.7) 3/130 (2.3) -5.5, 2.7 Death Month 3 0 0 -- Month 6 2/135 (1.5) 1/137 (0.7) -3.2, 1.7 Month 12 4/133 (3.0) 1/129 (0.8) -5.5, 1.0 Month 18 5/131 (3.8) 3/129 (2.3) -5.7, 2.7 Month 24e 5/135 (3.7) 7/130 (5.4) -3.3, 6.7 BID = twice a day, CI = conﬁdence interval, QD = once a day, WHO = World Health Organization a Conﬁdence intervals based on normal approximation to the binomial. b Includes cases of “uncertain evolution” [27] c Includes cases classiﬁed as probable cure based on oral information d One patient in the pentamidine group was classiﬁed as a treatment failure at end of treatment and is included in Relapse at Month 3. e One death was reported in the pafuramidine group subsequent to the last patient visit and is not included in the efﬁcacy endpoints. Table 5. Secondary efficacy variables at months 3, 6, 12, 18, and 24 (per-protocol dataset). with Good Clinical Practice and regulatory standards. In addition, this was the first Phase 3 study of a new drug intended for treatment of sleeping sickness conducted under a US FDA Investigational New Drug Application that followed contemporary International Conference on Harmonisation guidance. Discussion Limitations Although this study was conducted in rural conditions in Angola, South Sudan, and the DRC with local teams that had limited experience in clinical studies, this study was fully compliant 12 / 17 PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004363 February 16, 2016 doi:10.1371/journal.pntd.0004363.t005 Pafuramidine and Pentamidine for Stage 1 Sleeping Sickness Table 6. Treatment-emergent adverse events experienced by 5% of patients in either treatment group (treatment period). Pafuramidine 100 mg BID Pentamidine 4 mg/kg QD SOC (N = 136) (N = 137) HLGT n (%) n (%) Preferred Term All Adverse Events Treatment-Related Adverse Eventsa All Adverse Events Treatment-Related Adve Eventsa Total patients with at least 1 adverse eventb 111 (81.6) 54 (39.7) 135 (98.5) 127 (92.7) Gastrointestinal disorders 21 (15.4) 10 (7.3) 23 (16.8) 12 (8.8) Gastrointestinal signs and symptoms 18 (13.2) 9 (6.6) 19 (13.9) 12 (8.8) Abdominal pain 7 (5.1) 1 (0.7) 4 (2.9) 2 (1.5) Nausea 4 (2.9) 2 (1.5) 11 (8.0) 9 (6.6) General disorders and administration site conditions 46 (33.8) 10 (7.4) 61 (44.5) 41 (29.9) Administration site reactions 0 0 35 (25.5) 35 (25.5) Injection site pain 0 0 33 (24.1) 33 (24.1) Body temperature conditions 43 (31.6) 8 (5.9) 33 (24.1) 10 (7.3) Pyrexia 42 (30.9) 8 (5.9) 31 (22.6) 10 (7.3) Investigations 33 (24.3) 10 (7.4) 111 (81.0) 108 (78.8) Haematology investigations (incl blood groups) 17 (12.5) 5 (3.7) 11 (8.0) 6 (4.4) Haemoglobin decreased 7 (5.1) 2 (1.5) 6 (4.4) 3 (2.2) Haemoglobin increased 10 (7.4) 3 (2.2) 5 (3.6) 3 (2.2) Hepatobiliary investigationsb 10 (7.4) 4 (2.9) 106 (77.4) 105 (76.6) Alanine aminotransferase increased 2 (1.5) 2 (1.5) 72 (52.6) 71 (51.8) Aspartate aminotransferase increased 7 (5.1) 4 (2.9) 104 (75.9) 103 (75.2) Metabolic, nutritional and blood gas investigations 9 (6.6) 3 (2.2) 13 (9.5) 10 (7.3) Blood glucose decreased 5 (3.7) 2 (1.5) 12 (8.8) 10 (7.3) Renal and urinary tract investigations and urinalysesb 3 (2.2) 0 12 (8.8) 6 (4.4) Blood creatinine increased 3 (2.2) 0 11 (8.0) 5 (3.6) Metabolism and nutrition disorders 11 (8.1) 1 (0.7) 30 (21.9) 24 (17.5) Glucose metabolism disorders (including diabetes mellitus)b 8 (5.9) 0 25 (18.2) 21 (15.3) Hypoglycaemia 8 (5.9) 0 25 (18.2) 21 (15.3) Nervous system disorders 24 (17.6) 8 (5.9) 26 (19.0) 19 (13.9) Headaches 19 (14.0) 5 (3.7) 13 (9.5) 4 (2.9) Headache 19 (14.0) 5 (3.7) 13 (9.5) 4 (2.9) Neurological disorders NEC 9 (6.6) 4 (2.9) 16 (11.7) 15 (10.9) Dizziness 9 (6.6) 4 (2.9) 6 (4.4) 5 (3.6) Dysgeusia 0 0 11 (8.0) 11 (8.0) Skin and subcutaneous tissue disorders 7 (5.1) 2 (1.5) 1 (0.7) 0 Epidermal and dermal conditions2 7 (5.1) 2 (1.5) 1 (0.7) 0 Vascular disorders 64 (47.1) 29 (21.3) 86 (62.8) 79 (57.7) Decreased and nonspeciﬁc blood pressure disorders and shock2 60 (44.1) 28 (20.6) 85 (62.0) 78 (56.9) Hypotension 60 (44.1) 28 (20.6) 85 (62.0) 78 (56.9) BID = twice a day, HLGT = high-level group terms, NEC = not elsewhere classiﬁed, QD = once a day, SOC = system organ class a Considered at least possibly related to study drug by the Investigator. doi:10.1371/journal.pntd.0004363.t006 with Good Clinical Practice and regulatory standards. In addition, this was the first Phase 3 study of a new drug intended for treatment of sleeping sickness conducted under a US FDA Investigational New Drug Application that followed contemporary International Conference on Harmonisation guidance. with Good Clinical Practice and regulatory standards. In addition, this was the first Phase 3 study of a new drug intended for treatment of sleeping sickness conducted under a US FDA Investigational New Drug Application that followed contemporary International Conference on Harmonisation guidance. PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004363 February 16, 2016 PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004363 February 16, 2016 13 / 17 p y y g y g tically signiﬁcant (p<0.05) difference between treatment groups for overall incidence based on Fisher’s exact test. = high-level group terms, NEC = not elsewhere classiﬁed, QD = once a day, SOC = system organ class ibl l d d d b h I i sibly related to study drug by the Investigator. BID = twice a day, HLGT = high-level group terms, NEC = not elsewhere classiﬁed, QD = once a day, SOC = sys a Considered at least possibly related to study drug by the Investigator. Interpretation The results demonstrate the efficacy of pafuramidine in the treatment of first stage HAT, with an overall cure rate that was statistically non-inferior to that observed for pentamidine (89% vs. 95%, respectively) at 12 months post-treatment. The results obtained in the per-protocol set were confirmed in the ITT and mITT analysis populations, which included missing follow-up visits and patients lost to follow-up. The Month 24 results in all populations corroborate the 12-month results and demonstrate the robustness of the primary efficacy analysis. Compared with patients who received pentamidine, pafuramidine-treated patients (total population including the subpopulations of adolescents and pregnant and lactating women) had lower rates of treatment-related adverse events (93% vs. 40%, respectively) and lower rates of adverse events related to hepatic, renal, and metabolic toxicity. An ECG analysis revealed no cardiotoxicity for either drug [28]. These data are consistent with the good tolerability observed for pafuramidine in the previous Phase 2 studies [20]. This study was also designed to evaluate efficacy and safety of pafuramidine and pentami- dine in subpopulations that are particularly vulnerable to the long-term socioeconomic bur- dens associated with HAT, mainly pregnant and lactating women. However, the number of participating pregnant and lactating women was too low for a thorough separate analysis. The low number may be a result of social pressures and fear of treatment, which could be a detri- ment to seeking HAT treatment and going to a hospital. Low fertility and amenorrhea, which are often associated with HAT, may also have contributed [29]. From the limited number of relevant patients, there was no evidence for reduced efficacy or additional safety issues relating to pafuramidine compared with those observed in the overall study population. Numeric cure rates were similar and no specific safety issues were identified. The initial safety profile observed for pafuramidine in this study was consistent with the results of preceding studies in the pafuramidine clinical development program [19] [20]. How- ever, 3 patients in the pafuramidine group exhibited glomerulonephritis or nephropathy approximately 8 weeks post-treatment. On further examination, these events appeared to be similar to events that occurred in the previously mentioned supportive South African Phase 1 safety study. After re-evaluation, 2 of the 3 patients were considered to have events that were possibly related to pafuramidine by the principal investigator. Pafuramidine and Pentamidine for Stage 1 Sleeping Sickness Interpretation It should be noted that the analysis of safety data, particularly of the serious adverse events that occurred in the HAT study reported here, did not reveal any apparent negative long-term effects of pafuramidine. The patients who experienced renal toxicity recovered without sequelae, and the additional safety data obtained during the follow-up revealed no differences in abnormal biochemistry values between pafuramidine and pentamidine groups. Eventually, clinical development of pafuramidine was discontinued in early 2008, since the renal toxicity observed in the additional Phase 1 study was considered to be an unacceptable risk. Preliminary evidence of the involvement of the kidney injury molecule (KIM-1) was only very recently provided through the use of a mouse diversity panel [30]. b Statistically signiﬁcant (p<0 05) difference between treatment groups for overall incidence based on Fisher’s exact test se events experienced by 5% of patients in either treatment group (treatment period). ment-emergent adverse events experienced by 5% of patients in either treatment group (treatment period) PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004363 February 16, 2016 14 / 17 Acknowledgments Medical writing assistance was provided by Larry W. Hancock, Ph.D. (Sapphire Oak Consul- tants, LLC) and Flowers C Lovern, MA (Medical Writing Innovations, LLC). We would like to sincerely acknowledge the tremendous work of the laboratory technicians, nurses, and mem- bers of mobile teams in the participating centers. Supporting Information S1 File. CONSORT checklist. (DOC) S1 File. CONSORT checklist. 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https://openalex.org/W2008415685	https://europepmc.org/articles/pmc3050434?pdf=render	English	null	Simultaneous quantification of human herpesvirus 8 DNA by real time PCR in different tissues of HIV infected cuban patients with Kaposi's sarcoma	Herpesviridae	2,010	cc-by	4,496	* Correspondence: vkouri@ipk.sld.cu † Contributed equally 1Institute of Tropical Medicine “Pedro Kourí”, Havana City, Cuba Full list of author information is available at the end of the article Abstract In Cuba, previous reports have shown an increase of epidemic KS, reaching a total of 120 cases by the end of 2007, despite the use of HAART. To evaluate and compare the role of human herpes virus 8 (HHV-8) viral loads in different compartments of AIDS-related Kaposi’s sarcoma (AIDS-KS) patients real-time polymerase chain reaction (RT-PCR) was used to determine the genome copy number of HHV-8 in plasma, saliva, tissue and peripheral blood mononuclear cells (PBMC) of 49 AIDS-KS patients. Overall, 98% of AIDS-KS patients harbored detectable HHV-8. HHV-8 could be detected in 91.6% of KS tissue lesions showing the highest viral load (median log = 3.14 copies/ 100 ng DNA) followed by saliva and PBMC which were positive in 78%, and 69.2%; respectively. In contrast, HHV-8 was detected in only 37% of plasma samples, which also showed lower viral loads. Men who had sex with men (MSM) were more likely to have three-times higher HHV-8 genome copies in KS lesions when compared with tis- sues from heterosexuals individuals (OR 3; 95% CI 1.1 to 12.5). These results emphasize the systemic nature of HHV- 8-infection and demonstrate the possible role of saliva in HHV-8 transmission among MSM. performed using the QIAamp® DNA Mini Kit (QIAGEN, Germany) and the genomic DNA (gDNA) concentration was determined using spectrophotometer (GeneQuant II, Pharmacia Biotech, EUA) and adjusted to 100 ng, with the exception of plasma where 10 uL were directly used since it was not possible to quantify the gDNA. In order to obtain the standard DNA for absolute quantifi- cation, gDNA was extracted from the BCBL-1 cell line and the DNA copy numbers was determined by spectro- photometer as well. Then, the OD concentration was converted to DNA copy number following methods published elsewhere [2]. Once the gDNA copy number from BCBL-1 cell line was calculated, it was adjusted to 1 million copies and a standard curve with serial ten- fold dilutions (from 106 to 10 copies) assayed in tripli- cates was prepared. Once the run finished, the standard curve was automatically generated by the LightCycler software version 3.3, using the Second Derivative Maxi- mum (SDMM) method. In additon, the detection of Human b globin gene was used as internal control in © 2010 Kourí 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. Simultaneous quantification of human herpesvirus 8 DNA by real time PCR in different tissues of HIV infected cuban patients with Kaposi’s sarcoma Vivian Kourí1†, Pedro A Martínez1†, Orestes Blanco1, Virginia Capó1, María E Rodríguez1, María del C Dovigny1, Lidia Cardellá2, Angela Gala1, Narciso A Jiménez1, Consuelo Correa1, Yoan Alemán1, Lissette Pérez1, Alina Álvarez1, Ulrich Hengge3 Kourí et al. Herpesviridae 2010, 1:3 http://www.herpesviridae.org/content/1/1/3 Open Access Findings One hundred and forty-two clinical samples belonging to 49 patients with epidemic KS histologically diagnosed in the Pathology Department at the Institute of Tropical Medicine Pedro Kourí between 2004-2007 were included. The research was approved by local and national ethics committees; all subjects provided their written informed consent. Clinical, immunological and epidemiological data from each patient are depicted in Table 1. Different types of samples (41 saliva, 48 tissues, 26 PBMC, and 27 plasma) obtained from each individual were tested at the same time. PBMC and plasma were obtained by separation of 20 mL of citrated whole blood using a Ficoll separation gradient (SIGMA, UK). Paraffin was removed from tissues by xylene treatment according to published protocols [1]. DNA extraction was Correspondence: vkouri@ipk.sld.cu † Contributed equally 1Institute of Tropical Medicine “Pedro Kourí”, Havana City, Cuba Full list of author information is available at the end of the article Page 2 of 7 Kourí et al. Herpesviridae 2010, 1:3 http://www.herpesviridae.org/content/1/1/3 Table 1 Demographical, epidemiological and clinical characteristic of the study population Demographical, epidemiological and clinical variables AIDS-KS N = 49 Mean Age 38.2 years (Range: 22-57) Gender Female 2 (4.1%) Male 47 (95.9%) Race White 35 (71.4%) Mulatto 9 (18.4%) Black 5 (10.2%) Sexual orientation Heterosexual 5 (10.2%) Homosexual 44 (89.8%) Type of KS according to macroscopic classification Cutaneous 35 (71.4%) Mucocutaneous 7 (14.3%) Disseminated 7 (14.3%) Histological classification Macular 16 (32.6%) Patch 7 (14.3%) Tumor 14 (28.6%) Not classified 12 (24.5%) Mean HIV viral load* (copies/mL) 65 135 (Range: < 50-580 000) Mean T CD4+ cell count (cel/mm3) 295 (Range: 8-974) T CD4+ cell count < 200 21 (42.9%) 200-499 21 (42.9%) > 500 7 (14.2%) Mean number of years after KS diagnosis 1.6 years Data not available from 7 patients. ble 1 Demographical, epidemiological and clinical characteristic of the study population Table 1 Demographical, epidemiological and clinical characteristic of the study population Demographical epidemiological and clinical variables AIDS-K detection of HHV-8 in tissue and the detection in saliva or PBMC (p < 0.01) that was not observed for plasma (Table 2). Although PCR inhibitions could be a possible explanation for the 4 negative results from KS lesions, several other factors that may have limited KSHV detec- tion, like lesion sampling, since the lesion sample was not always the same for molecular detection and diag- nosis. Findings In addition, a possible histological inaccuracy could not be excluded because some patients were diag- nosed as early stage of KS [5]. However, the percentage of KSHV DNA detected in the present study is similar to previous results published by Mendez and colleagues [6] and higher than the 87% of KSHV DNA detection reported by Kennedy and co-authors [7]. each clinical sample (with exception of plasma) for mea- suring of the exact amount of input DNA [3]. RT-PCR primers and conditions were described pre- viously by Watzinger et al [4] with minor modifications adapted for the LightCycler 1.5 [4]. Samples were con- sidered negative if the Ct value exceeded cycle 40, or if the copy number was below 10 copies. All patients or samples with more than 10 copies were considered posi- tive, thus infected with HHV-8. For statistical analysis SPSS 11,5 (Inc. SPSS, Chicago, IL, the USA) and Stat- graphic were used. Tests for comparison of proportions between the averages were performed using the Chi- square test and the Pearson coefficient of correlation for 95% confidentiality. To our knowledge, this is the first report, where HHV-8 viral load has been simultaneously determined in four different fluids and cells (affected tissue, saliva, plasma and PBMC) from the same AIDS-KS patients. Forty-eight (98%) of the 49 AIDS-KS patients had detectable levels of HHV-8 DNA at least in one of the samples studied, (67% of patients had more than 2 sam- ples positives), with the virus being more frequently detected in KS lesions (44 tissues, 91.6%) followed by saliva in 78% and PBMC in 69.2%. The detection prob- ability and viral load being significantly lower in plasma (37%). There was a positive correlation between the All fresh frozen tissues, PBMC and saliva samples, were confirmed to have the same amount of the input gDNA in100 ng, by detecting similar crossing point (Cp) ampli- fication signal of the Human b globin gene among them (average Cp: 24.08, range: 22-25); in contrast, paraffin- embedded tissues showed a gDNA amplification signal after cicle 29 (range:27-32), probably due to degradation [8]. This result was consistent with the detection of HHV-8 load significantly higher in fresh frozen tissue than in paraffin-embedded tissue (p < 0.003) (Table 2). Despite the difference detected between fresh frozen and paraffin-embedded tissue, when copy numbers from Page 3 of 7 Kourí et al. Findings Herpesviridae 2010, 1:3 http://www.herpesviridae.org/content/1/1/3 Table 2 KSHV load levels in different fluid and cells from each KS patient (Continued) ADS-KS48 28680 (fresh frozen tissue) NS 230 NS ADS-KS49 58 (paraffin embedded) NS NS NS Abbreviations: Ndt: non detectable; NS: no sample available. HHV-8 load in saliva and PBMC was significantly lower than in tissue (p < 0.05) and significantly higher than in plasma (p < 0.05; Figure 1), however there were no statistic differences between the viral load in saliva and PBMC (p > 0.05). Of note, saliva represented the second most frequent source for HHV-8 detection (Fig- ure 1). In the light of previous reports [11-13] and con- sistent with the present results transmission by saliva may contribute to the spread of HHV-8 infection among the HIV seropositive population besides sexual intercourse [14]. It still remains controversial why, if sal- iva is the main source of virus, HHV-8 infection shows a sexual pattern of transmission. Nevertheless, saliva is the only mucosal fluid in which infectious HHV-8 has been different samples (tissue-plasma, tissue-saliva, tissue- PBMC, plasma-saliva, PMBC-saliva, and PBMC-plasma) within a given individual were compared, a significantly higher HHV-8 viral load in KS lesions compared to all the other samples (p < 0.05) was detected (Figure 1). One of the reasons that may explain why the higher HHV-8 load is detected in tissue, specifically in the case of HIV infection, is that the HIV tat protein promotes the replication of HHV-8-stimulated cell proliferation and inhibits apoptosis of spindle cells infected with HHV-8 [9,10]. Recent reports have described a higher HHV-8 load of AIDS-KS lesions compared with ende- mic KS, although the differences were not statistically significant. different samples (tissue-plasma, tissue-saliva, tissue- PBMC, plasma-saliva, PMBC-saliva, and PBMC-plasma) within a given individual were compared, a significantly higher HHV-8 viral load in KS lesions compared to all the other samples (p < 0.05) was detected (Figure 1). One of the reasons that may explain why the higher HHV-8 load is detected in tissue, specifically in the case of HIV infection, is that the HIV tat protein promotes the replication of HHV-8-stimulated cell proliferation and inhibits apoptosis of spindle cells infected with HHV-8 [9,10]. Recent reports have described a higher HHV-8 load of AIDS-KS lesions compared with ende- mic KS, although the differences were not statistically significant. Findings Herpesviridae 2010, 1:3 http://www.herpesviridae.org/content/1/1/3 Table 2 KSHV load levels in different fluid and cells from each KS patient ID Tissue copies/100 ng gDNA PBMC copies/100 ng gDNA Saliva copies/100 ng gDNA Plasma copies/uL ADS-KS1 293 (fresh frozen tissue) 41 71 ndt ADS-KS2 28200 (fresh frozen tissue) 32 193 ndt ADS-KS3 17670 (fresh frozen tissue) 22 134 ndt ADS-KS4 51610 (fresh frozen tissue) 130 62 ndt ADS-KS5 294 (fresh frozen tissue) ndt 269 92 ADS-KS6 167 (paraffin embedded) 40 130 ndt ADS-KS7 15850 (fresh frozen tissue) ndt ndt ndt ADS-KS8 30000 (fresh frozen tissue) ndt 145 ndt ADS-KS9 ndt (paraffin embedded) ndt ndt ndt ADS-KS10 78 (paraffin embedded) 119 121 239 ADS-KS11 1952 (fresh frozen tissue) 38 191 39 ADS-KS12 21840 (fresh frozen tissue) 390 96 49 ADS-KS13 529 (paraffin embedded) ndt ndt 2452 ADS-KS14 37630 (fresh frozen tissue) ndt 394 ndt ADS-KS15 26090 (fresh frozen tissue) 19 ndt ndt ADS-KS16 57 (paraffin embedded) ndt 77 ndt ADS-KS17 23 (paraffin embedded) ndt 49 68 ADS-KS18 477 (paraffin embedded) 307 23 30 ADS-KS19 42 (paraffin embedded) 31 160 ndt ADS-KS20 1725 (fresh frozen tissue) ndt 145 ndt ADS-KS21 ndt (fresh frozen tissue) 42 2482 ndt ADS-KS22 0 (fresh frozen tissue) 40 584 ndt ADS-KS23 22 (paraffin embedded) 26 343 20 ADS-KS24 1418000 (fresh frozen tissue) 19 236 65 ADS-KS25 36460 (fresh frozen tissue) 167 32 55 ADS-KS26 749 (fresh frozen tissue) NS 172 NS ADS-KS27 47050 (fresh frozen tissue) NS NS NS ADS-KS28 58 (fresh frozen tissue) NS ndt NS ADS-KS29 389 (fresh frozen tissue) NS ndt NS ADS-KS30 39830 (fresh frozen tissue) NS 89 NS ADS-KS31 10040 (paraffin embedded) NS NS NS ADS-KS32 819 (fresh frozen tissue) NS NS NS ADS-KS33 128800 (fresh frozen tissue) NS 445 NS ADS-KS34 ndt (fresh frozen tissue) NS 366 NS ADS-KS35 206 (fresh frozen tissue) NS ndt NS ADS-KS36 68280 NS 114 ndt ADS-KS37 877 (paraffin embedded) NS NS NS ADS-KS38 30730 (fresh frozen tissue) NS 131 NS ADS-KS39 NS 53 9447 ndt ADS-KS40 867 (paraffin embedded) NS ndt NS ADS-KS41 30520 (fresh frozen tissue) NS 148 NS ADS-KS42 35 (fresh frozen tissue) NS NT NS ADS-KS43 31740 (fresh frozen tissue) NS 177 NS ADS-KS44 10120 (fresh frozen tissue) NS 132 NS ADS-KS45 139800 (fresh frozen tissue) NS NS NS ADS-KS46 2483 (fresh frozen tissue) NS ndt NS ADS-KS47 163 (fresh frozen tissue) NS NS NS Table 2 KSHV load levels in different fluid and cells from each KS patient Page 4 of 7 Kourí et al. Findings Figure 1 Comparison of the logarithmic KSHV load detected in different samples of epidemic KS patient using the RT-PCR assay of a conserved region of ORF-26. Page 5 of 7 Page 5 of 7 Kourí et al. Herpesviridae 2010, 1:3 http://www.herpesviridae.org/content/1/1/3 find association between the HHV-8 viral load in all the samples analyzed with: CD4 cell counts, HIV viral load, type of KS (cutaneous, mucocutaneous and systemic), the histological stage (macular, patch and tumoral) nor the number of years elapsed between the KS diagnosis and death (p > 0.05). identified, although factors associated with HHV-8 sali- vary shedding remain unclear [15]. Moreover, future research will need to assess the possible impact of saliva in HHV-8 transmission among Cuban HIV seronegative individuals. Most authors agree that the viral genome present in PBMC of AIDS-KS patients occurs in a latent state, but the role of HHV-8 persistence in PBMC with regard to the pathogenesis of KS remains unclear [16]. The lower copy number of HHV-8 detected in PBMC in compari- son to KS tissue, could be due to its latent state within this compartment, in contrast to viral expression present in lesional spindle cells [17]. p In our study, Caucasian AIDS-KS patients were more likely to be positive for HHV-8 infection than black or mulatto KS patients (p = 0.006). There is no previous report regarding the association of HHV-8 levels with skin color. However, it has been described several times that AIDS-KS is more frequent in individuals with white skin [24]. Some authors have proposed a genetic predis- position to HHV-8 infection in individuals with certain HLA types (A6801 and DRB1*04) that tend to have increased viral excretion in saliva e.g. in African women [25]. MSM were more likely to have three-times higher HHV-8 genome copies in KS lesions when compared with tissues from heterosexuals individuals (OR 3; 95% CI 1.1 to 12.5). This seems to be a very important find- ing that confirms what has been reported by other authors regarding sexual risk behaviors that are more prevalent in homosexual intercourse [26]. With the pre- sent study we confirm that HHV-8 produces a systemic infection in different body compartments and that the highest HHV-8 levels were produced in lesional KS tis- sue. Furthermore, saliva has been recognized as an important reservoir for HHV-8 transmission. Harrington and colleagues demonstrated that the pre- sence of HHV-8 in plasma is intermittent [18]. References 20. Garrigue I, Doussau A, Asselineau J, Bricout H, Couzi L, Rio C, Merville P, Fleury H, Lafon ME, Thiebaut R: Prediction of cytomegalovirus (CMV) plasma load from evaluation of CMV whole-blood load in samples from renal transplant recipients. J Clin Microbiol 2008, 46:493-498. 1. Volkenandt M, Dicker A, Fanin R, Banerjee D, Albino S, Bertino JR: Polymerase Chain Reaction Analysis of DNA from Paraffin-Embedded Tissue. In Methods in Molecular Biology PCR protocols: Current Methods and Applications Edited by: Inc HP EUA 1993, 81-87. 1. Volkenandt M, Dicker A, Fanin R, Banerjee D, Albino S, Bertino JR: Polymerase Chain Reaction Analysis of DNA from Paraffin-Embedded Tissue. In Methods in Molecular Biology PCR protocols: Current Methods and Applications Edited by: Inc HP EUA 1993, 81-87. 21. Brantsaeter AB, Holberg-Petersen M, Jeansson S, Goplen AK, Bruun JN: CMV quantitative PCR in the diagnosis of CMV disease in patients with HIV- infection-a retrospective autopsy based study. BMC Infect Dis 2007, 7:127. 2. Yin JL, Shackel NA, Zekry A, McGuinness PH, Richards C, Putten KV, McCaughan GW, Eris JM, Bishop GA: Real-time reverse transcriptase- polymerase chain reaction (RT-PCR) for measurement of cytokine and growth factor mRNA expression with fluorogenic probes or SYBR Green I. Immunol Cell Biol 2001, 79:213-221. 2. Yin JL, Shackel NA, Zekry A, McGuinness PH, Richards C, Putten KV, McCaughan GW, Eris JM, Bishop GA: Real-time reverse transcriptase- polymerase chain reaction (RT-PCR) for measurement of cytokine and growth factor mRNA expression with fluorogenic probes or SYBR Green I. Immunol Cell Biol 2001, 79:213-221. 22. Polstra AM, Cornelissen M, Goudsmit J, van der Kuyl AC: Retrospective, longitudinal analysis of serum human herpesvirus-8 viral DNA load in AIDS-related Kaposi’s sarcoma patients before and after diagnosis. J Med Virol 2004, 74:390-396. 3. Schmitz M, Scheungraber C, Herrmann J, Teller K, Gajda M, Runnebaum IB, Durst M: Quantitative multiplex PCR assay for the detection of the seven clinically most relevant high-risk HPV types. J Clin Virol 2009, 44:302-307. 3. Schmitz M, Scheungraber C, Herrmann J, Teller K, Gajda M, Runnebaum IB, Durst M: Quantitative multiplex PCR assay for the detection of the seven clinically most relevant high-risk HPV types. J Clin Virol 2009, 44:302-307. 23. Moore PS, Chang Y: Kaposi’s Sarcoma-Associated Herpesvirus. In Fields Virology. Volume 2. 4 edition. Edited by: Knipe DM, Howley PM, Griffin DE, Lamb RAM MA, Roizman B, Straus SE. USA: LIPPINCOTT WILLIAMS 2001:2803-2833. 4. Competing interests The authors declare that they have no competing interests. Received: 7 July 2010 Accepted: 7 December 2010 Published: 7 December 2010 19. Kuo CP, Wu CL, Ho HT, Chen CG, Liu SI, Lu YT: Detection of cytomegalovirus reactivation in cancer patients receiving chemotherapy. Clin Microbiol Infect 2008, 14:221-227. Author details 1 Author details 1Institute of Tropical Medicine “Pedro Kourí”, Havana City, Cuba. 2Latin- American School of medicine, Havana City, Cuba. 3Skin Center, Düsseldorf, Germany. 15. Gasperini P, Barbierato M, Martinelli C, Rigotti P, Marchini F, Masserizzi G, Leoncini F, Chieco-Bianchi L, Schulz TF, Calabro ML: Use of a BJAB-derived cell line for isolation of human herpesvirus 8. J Clin Microbiol 2005, 43:2866-2875. Findings For other viral infections, plasma viral load has been used as the best marker to estimate disease progression [19-21]. However, this does not seem to be true for KS and HHV-8, as has been proposed by Polstra and colleagues [22]. Hundred percent of Cuban AIDS-KS patients, in whom HHV-8 was detected in plasma also tested posi- tive in other clinical samples (saliva, KS tissue, PBMC). Thus, viremia could occur in those patients with active viral replication, alternating with latency periods where the virus is not detected in plasma, as has been reported for other members of the Herpesviridae family [23]. There was a significant Pearson’s correlation when each patient’s tissue HHV-8 levels were compared with saliva and PBMC (p < 0.01); however, this correlation did not exist for plasma samples (Figure 2). We did not One of the main challenges for researchers is to iden- tify the exact mode of virus transmission following Figure 2 Graphic representation of KSHV loads in different samples per each AIDS-KS patient. Figure 2 Graphic representation of KSHV loads in different samples per each AIDS-KS patient. Page 6 of 7 Kourí et al. Herpesviridae 2010, 1:3 http://www.herpesviridae.org/content/1/1/3 herpesvirus-8 infection and oral shedding in amerindian and non- amerindian populations in the brazilian Amazon region. J Infect Dis 200 196:844-852. sexual intercourse, as the presence of HHV-8 in semen, anal secretions or vaginal fluid has not been consistently demonstrated [27-30]. 12. Al-Otaibi LM, Ngui SL, Scully CM, Porter SR, Teo CG: Salivary human herpesvirus 8 shedding in renal allograft recipients with Kaposi’s sarcoma. J Med Virol 2007, 79:1357-1365. Authors’ contributions 16. Boivin G, Cote S, Cloutier N, Abed Y, Maguigad M, Routy JP: Quantification of human herpesvirus 8 by real-time PCR in blood fractions of AIDS patients with Kaposi’s sarcoma and multicentric Castleman’s disease. J Med Virol 2002, 68:399-403. All authors read and approved the final manuscript. VK and PAM participated in the conception and design, acquisition of data, analysis and interpretation of data, drafted the manuscript. OB, MER, MdCD and NAJ made the clinical diagnosis and patients follow up, sampling, clinical analysis and interpretation of data. LC and AG participated in the design of the study and performed the statistical analysis. CC, YA, LP and AÁ carried out the molecular genetic studies. Finally, UH participated in the design of the study and drafted the manuscript. 17. Martro E, Cannon MJ, Dollard SC, Spira TJ, Laney AS, Ou CY, Pellett PE: Evidence for both lytic replication and tightly regulated human herpesvirus 8 latency in circulating mononuclear cells, with virus loads frequently below common thresholds of detection. J Virol 2004, 78:11707-11714. 18. Harrington WJ Jr, Bagasra O, Sosa CE, Bobroski LE, Baum M, Wen XL, Cabral L, Byrne GE, Pomerantz RJ, Wood C: Human herpesvirus type 8 DNA sequences in cell-free plasma and mononuclear cells of Kaposi’s sarcoma patients. J Infect Dis 1996, 174:1101-1105. References Watzinger F, Suda M, Preuner S, Baumgartinger R, Ebner K, Baskova L, Niesters HG, Lawitschka A, Lion T: Real-time quantitative PCR assays for detection and monitoring of pathogenic human viruses in immunosuppressed pediatric patients. J Clin Microbiol 2004, 42:5189-5198. 4. Watzinger F, Suda M, Preuner S, Baumgartinger R, Ebner K, Baskova L, Niesters HG, Lawitschka A, Lion T: Real-time quantitative PCR assays for detection and monitoring of pathogenic human viruses in immunosuppressed pediatric patients. J Clin Microbiol 2004, 42:5189-5198. 24. Nawar E, Mbulaiteye SM, Gallant JE, Wohl DA, Ardini M, Hendershot T, Goedert JJ, Rabkin CS: Risk factors for Kaposi’s sarcoma among HHV-8 seropositive homosexual men with AIDS. Int J Cancer 2005, 115:296-300. 5. Calonje E, Wilson Jones E: Tumors and Tumor-like conditions of blood vessels and lymphatics. In Lever’s Histopathology of the skin. 8 edition. Edited by: Elder D, Elenitsas R, Jaworsky C, Jhonson BJ. USA: Lippincott Williams 1999:623-651. 5. Calonje E, Wilson Jones E: Tumors and Tumor-like conditions of blood vessels and lymphatics. In Lever’s Histopathology of the skin. 8 edition. Edited by: Elder D, Elenitsas R, Jaworsky C, Jhonson BJ. USA: Lippincott Williams 1999:623-651. 25. Alkharsah KR, Dedicoat M, Blasczyk R, Newton R, Schulz TF: Influence of HLA Alleles on Shedding of Kaposi Sarcoma-Associated Herpesvirus in Saliva in an African Population. J Infect Dis 2007, 195:809-816. 6. Mendez JC, Procop GW, Espy MJ, Paya CV, Smith TF: Detection and semiquantitative analysis of human herpesvirus 8 DNA in specimens from patients with Kaposi’s sarcoma. J Clin Microbiol 1998, 36:2220-2222. 26. Casper C, Carrell D, Miller KG, Judson FD, Meier AS, Pauk JS, Morrow RA, Corey L, Wald A, Celum C: HIV serodiscordant sex partners and the prevalence of human herpesvirus 8 infection among HIV negative men who have sex with men: baseline data from the EXPLORE Study. 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Kouri V, Eng SM, Rodriguez ME, Resik S, Orraca O, Moore PS, Chang Y: Seroprevalence of Kaposi’s sarcoma-associated herpesvirus in various populations in Cuba. Rev Panam Salud Publica 2004, 15:320-325. References Coombs NJ, Gough AC, Primrose JN: Optimisation of DNA and RNA extraction from archival formalin-fixed tissue. Nucleic Acids Res 1999, 27:e12. 9. Pyakurel P, Pak F, Mwakigonja AR, Kaaya E, Biberfeld P: KSHV/HHV-8 and HIV infection in Kaposi’s sarcoma development. Infect Agent Cancer 2007, 2:4. 9. Pyakurel P, Pak F, Mwakigonja AR, Kaaya E, Biberfeld P: KSHV/HHV-8 and HIV infection in Kaposi’s sarcoma development. Infect Agent Cancer 2007, 2:4. 28. Viviano E, Gallo E, Bongiorno MR, Romano N: [The genomic sequences of the human herpesvirus 8 in biological samples from HIV-positive and- negative subjects in Sicily]. Ann Ig 1999, 11:507-509. 10. Zeng Y, Zhang X, Huang Z, Cheng L, Yao S, Qin D, Chen X, Tang Q, Lv Z, Zhang L, Lu C: Intracellular Tat of human immunodeficiency virus type 1 activates lytic cycle replication of Kaposi’s sarcoma-associated herpesvirus: role of JAK/STAT signaling. J Virol 2007, 81:2401-2417. 11. de Souza VA, Sumita LM, Nascimento MC, Oliveira J, Mascheretti M, Quiroga M, Freire WS, Tateno A, Boulos M, Mayaud P, Pannuti CS: Human 10. Zeng Y, Zhang X, Huang Z, Cheng L, Yao S, Qin D, Chen X, Tang Q, Lv Z, Zhang L, Lu C: Intracellular Tat of human immunodeficiency virus type 1 activates lytic cycle replication of Kaposi’s sarcoma-associated herpesvirus: role of JAK/STAT signaling. J Virol 2007, 81:2401-2417. 29. Engels EA, Atkinson JO, Graubard BI, McQuillan GM, Gamache C, Mbisa G, Cohn S, Whitby D, Goedert JJ: Risk Factors for Human Herpesvirus 8 Infection among Adults in the United States and Evidence for Sexual Transmission. J Infect Dis 2007, 196:199-207. 11. de Souza VA, Sumita LM, Nascimento MC, Oliveira J, Mascheretti M, Quiroga M, Freire WS, Tateno A, Boulos M, Mayaud P, Pannuti CS: Human Page 7 of 7 Page 7 of 7 Page 7 of 7 Kourí et al. Herpesviridae 2010, 1:3 http://www.herpesviridae.org/content/1/1/3 Kourí et al. Herpesviridae 2010, 1:3 http://www.herpesviridae.org/content/1/1/3 30. Taylor MM, Chohan B, Lavreys L, Hassan W, Huang ML, Corey L, Ashley Morrow R, Richardson BA, Mandaliya K, Ndinya-Achola J, et al: Shedding of human herpesvirus 8 in oral and genital secretions from HIV-1- seropositive and-seronegative Kenyan women. J Infect Dis 2004, 190:484-488. doi:10.1186/2042-4280-1-3 Cite this article as: Kourí et al.: Simultaneous quantification of human herpesvirus 8 DNA by real time PCR in different tissues of HIV infected cuban patients with Kaposi’s sarcoma. Herpesviridae 2010 1:3. 30. Taylor MM, Chohan B, Lavreys L, Hassan W, Huang ML, Corey L, Ashley Morrow R, Richardson BA, Mandaliya K, Ndinya-Achola J, et al: Shedding of human herpesvirus 8 in oral and genital secretions from HIV-1- seropositive and-seronegative Kenyan women. J Infect Dis 2004, 190:484-488. doi:10.1186/2042-4280-1-3 Cite this article as: Kourí et al.: Simultaneous quantification of human herpesvirus 8 DNA by real time PCR in different tissues of HIV infected cuban patients with Kaposi’s sarcoma. Herpesviridae 2010 1:3. doi:10.1186/2042-4280-1-3 Cite this article as: Kourí et al.: Simultaneous quantification of human herpesvirus 8 DNA by real time PCR in different tissues of HIV infected cuban patients with Kaposi’s sarcoma. Herpesviridae 2010 1:3. 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https://openalex.org/W3151628534	https://plantmethods.biomedcentral.com/counter/pdf/10.1186/s13007-021-00738-1	English	null	A stable isotope dilution method for a highly accurate analysis of karrikins	Plant methods	2,021	cc-by	11,256	© The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco mmons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background: Karrikins (KARs) are recently described group of plant growth regulators with stimulatory effects on seed germination, seedling growth and crop productivity. So far, an analytical method for the simultaneous targeted profiling of KARs in plant tissues has not been reported. Results: We present a sensitive method for the determination of two highly biologically active karrikins (KAR1 and KAR2) in minute amounts of plant material (< 20 mg fresh weight). The developed protocol combines the optimized extraction and efficient single-step sample purification with ultra-high performance liquid chromatography-tandem mass spectrometry. Newly synthesized deuterium labelled KAR1 was employed as an internal standard for the valida- tion of KAR quantification using a stable isotope dilution method. The application of the matrix-matched calibration series in combination with the internal standard method yields a high level of accuracy and precision in triplicate, on average bias 3.3% and 2.9% RSD, respectively. The applicability of this analytical approach was confirmed by the suc- cessful analysis of karrikins in Arabidopsis seedlings grown on media supplemented with different concentrations of KAR1 and KAR2 (0.1, 1.0 and 10.0 µmol/l). Conclusions: Our results demonstrate the usage of methodology for routine analyses and for monitoring KARs in complex biological matrices. The proposed method will lead to better understanding of the roles of KARs in plant growth and development. Keywords: Karrikins, Smoke water, Stable isotope labelled standard, Stable isotope dilution method, Ultra-high performance liquid chromatography (UHPLC), Tandem mass spectrometry (MS/MS) prepared saturated extracts (so-called smoke water, SW). The SW prepared from smoke produced by con- trolled combustion devices can be used as an afford- able biostimulant in agriculture and horticulture [2, 5, 6]. The stimulatory effect of KARs is independent from plant reproductive strategy, seed size and/or plant mor- phology or ecology. They are effective not only on plants from fire-prone areas, but also on various species from different families and environments [5]. There are six known KARs (Fig. 1a), of which KAR1 is most active on seed germination of fire-following plants, but the model plant Arabidopsis responds more strongly to KAR2 [7]. In parallel, a structurally and functionally similar sub- stance, named strigolactone (SL, Fig. 1b), has been shown to trigger germination of parasitic plants [8, 9]. KARs Hrdlička et al. Plant Methods (2021) 17:37 https://doi.org/10.1186/s13007-021-00738-1 Hrdlička et al. Plant Methods (2021) 17:37 https://doi.org/10.1186/s13007-021-00738-1 Plant Methods Plant Methods Open Access Background Karrikins (KARs) are small organic compounds derived from butenolide molecules (Fig. 1a) with their principal effect on seed germination [1, 2]. Details of their origin is still unknown, but their formation requires oxygen and a pyran ring derived from the heating of polysaccharides and sugars [3, 4]. KARs are produced during wild fires and play a key role in the restoration of destroyed areas. Moreover, they can also be components of artificially *Correspondence: novako@ueb.cas.cz 1 Laboratory of Growth Regulators, Institute of Experimental Botany, The Czech Academy of Sciences and Faculty of Science, Palacký University, Šlechtitelů 27, 78371 Olomouc, Czech Republic Full list of author information is available at the end of the article Hrdlička et al. Plant Methods (2021) 17:37 Page 2 of 13 Page 2 of 13 Hrdlička et al. Plant Methods spectrometry (MS/MS) has been widely used to deter- mine levels of various plant hormones [reviewed in 12– 17]. The complexity of plant matrix, where minor PGRs are present in the background of more abundant primary and secondary metabolites, requires a combination of proper sample preparation and high instrument perfor- mance (robustness and sensitivity). For assessment of the accuracy and precision of MS-based methods, the con- centration of each analyte should be calculated using the stable isotope dilution method (SIDM; [16]). In general, SIDM is used to determine the quantity of a chemical substance in a sample based on internal standard (IS) to analyte ratio. Moreover, IS labelled with stable isotopes such as deuterium (2H), 13C, 15 N, and/or 18O atoms bal- ances the inefficiencies and/or losses within the process of sample preparation as well as the ion suppression effects during the MS analysis [18]. In the case of using deuterium-labelled IS, three or more 2H are used. One reason is the stronger binding of 2H isotopes to car- bons than 1H isotopes, which can lead to small physico- chemical differences between the analyte and IS and thus potentially better chromatographic separation. In addition, a higher number of 2H prevents interferences of naturally occurring analyte isotopes with IS [19]. In plant hormone profiling methods, the use of IS for tar- geted quantification analysis has become the primarily performed technique [showed in 20–23]. Fig. 1 The structures of six naturally occurring karrikins (a) and strigolactones (b). The structural similarity of KARs and SLs, naturally occurring strigol and synthetic analogue GR24, is due to the presence of a 2-furanone moiety (red part) Fig. 1 The structures of six naturally occurring karrikins (a) and strigolactones (b). The structural similarity of KARs and SLs, naturally occurring strigol and synthetic analogue GR24, is due to the presence of a 2-furanone moiety (red part) are perceived by KARRIKIN-INSENSITIVE2 (KAI2), a homolog of SLs receptor. In addition, both signaling pathways share the same or similar components, such as the F-box protein MORE AXILARY GROWTH2 (MAX2) and the transcriptional corepressors SUPPRES- SOR OF MAX2 1 (SMAX1)-LIKE proteins (SMXL) [10, 11]. A better understanding of plant growth regulators’ (PGRs) biosynthesis, metabolism and mode of action requires analysis of changes in their levels in various plant organs and tissues in different physiological processes. Moreover, the biological activity of PGR compounds is dependent on their concentration levels both in the whole plant and in individual organs. It is therefore of great importance to develop highly sensitive and robust analytical methods for monitoring the endogenous lev- els of PGRs in various plant tissues. Such analyses still remain challenging, since PGRs are present at very low concentrations (pmol/g fresh weight) in a complex bio- logical matrix [12]. Typical analysis of plant hormones usually includes a multi-step sample pre-treatment, such as solid phase extraction (SPE) and/or immunoaf- finity extraction followed by subsequent instrumental measurements of individual metabolites [13]. SPE is the most frequently used method to isolate compounds from complex matrices. By removing interfering compounds such as salts, pigments, polysaccharides, lipids and pro- teins from the sample, the chemical noise is reduced and potential co-eluting analytes are eliminated. In addition, less complex samples are more gentle on highly sensitive instrumentation such as mass spectrometers. Very recently, ultra-high performance liquid chroma- tography combined with tandem mass spectrometry (UHPLC–MS/MS) was used for KAR quantification [24]. A standard dilution method (SDM) using a KAR struc- tural analogue and a standard addition method (SAM) were compared. The SAM was successfully validated and applied in the determination of KARs in eight smoke water samples of various origins and ages. However, the SIDM was not tested due to the absence of an isotopically labelled standard. In this study, we have developed a com- plex analytical protocol suitable for the SPE-based isola- tion of KARs supplemented by sensitive and selective quantification using the UHPLC–MS/MS method. After synthesizing a new deuterium-labelled internal stand- ard, we were able to apply the SIDM as a quantification approach for KAR analyses. Finally, we demonstrated utility of our MS-based method for KARs profiling in samples of ten-day-old seedlings of Arabidopsis thaliana treated with KAR1 and KAR2, which are commercially available and commonly used in plant research. Development of extraction and purification protocol Development of extraction and purification protocol been validated, its application for the analysis of bio- logical samples is time consuming [25]. Moreover, the methodology can be less suitable for tissue specific experiments due to the requirement for a large amount of plant material. The targeted profiling of KARs in minute plant tissues using a new deuterium-labelled internal standard was an essential requirement in the field of karrikin research. Therefore, the development of a stable isotope dilution method was initiated by the synthesis of a new IS. p pi p Due to the expected extremely low concentration of KARs in plants, it has been necessary to develop an effi- cient extraction step with a suitable solvent under opti- mized conditions. The intriguing similarity between two germination stimulants, karrikins and strigolactones (Fig. 1) inspired us to develop extraction approaches for KARs isolation from complex plant matrices. Both groups of substances are soluble in water and/or in mix- tures of semi-polar organic solvents [24, 27]. This fea- ture can be used to advantage in the extraction step, as decreasing concentrations of organic solvents generally decreases the extraction efficiency of interfering sub- stances such as plant pigments [23]. As shown previ- ously, KAR1 and KAR2 are stable in both weakly acidic and neutral conditions (pH 5.0 and 7.0) [24]. Moreover, the use of acidic conditions at low temperature mini- mizes enzymatic activity during plant tissue extraction [28]. Similarly to SL extraction [27], the percentage of the organic component in the solvent was chosen to pro- vide efficient solubility of KARs, as well as to meet com- patibility with loading conditions of SPE sorbents used afterwards. Therefore, four polar solvents (10% metha- nol, 10% acetonitrile, 10% methanol acidified with 0.1% formic acid and 10% acetonitrile acidified with 0.1% for- mic acid) were tested as suitable solvents and the stabil- ity of KARs was evaluated (Additional file 1). The solvent mixtures were spiked with known amounts of KAR1 and KAR2, and then analysed by UHPLC–MS/MS. Recovery of each compound was calculated as its peak area rela- tive to the corresponding peak’s area in control samples. The approximately twofold lower yield of KARs in ace- tonitrile compared to methanol probably indicates their different solubility. The results also showed lower con- centrations of KAR1 and KAR2 quantified in spiked non- acidified solvents. Results and discussion Preparation of stable isotope labelled standard [2H3]KAR1fi A previously published study showed the difficulties of KAR quantification due to the complexity of smoke water matrices [24]. Although the SAM method has During the last decade, liquid chromatography (LC) or gas chromatography (GC) coupled to tandem mass Page 3 of 13 Hrdlička et al. Plant Methods (2021) 17:37 Hrdlička et al. Plant Methods (2021) 17:37 Development of extraction and purification protocol Interestingly, the maximum recovery The isotopically labelled 3-(2H3)methyl-2H-furo[2,3- c]pyran-2-one ([2H3]KAR1) was prepared from 3-bromo-2H-furo[2,3-c]pyran-2-one (KAR-Br) by the coupling reaction with trideuteromethylboronic acid (Fig. 2a). The reaction was performed analogously to the previously reported preparation of 3-methyl- 2H-furo[2,3-c]pyran-2-one (KAR1) [26]. We did not use a greater excess of trideuteromethylboronic acid due to its limited availability and probably for this reason the yield of the reaction was significantly decreased by the formation of side product, furo[2,3-c]pyran-2-one, in approximately 35% yield. The crude product was then purified twice by column chromatography. The identity and purity of prepared [2H3]KAR1 was examined using 1H and 13C NMR spectrometry, HPLC–DAD-(ESI +) MS and GC-(EI)MS methods. The isotopic purity was calculated from LC–MS data of the product after sub- traction of a theoretical isotope model (Fig. 2b). The molar content of (2H3]KAR1) was at least 99.0%. How- ever, the content of non-deuterated KAR1 was also detected, not more than 1.0%. Importantly, dideu- tero- and monodeutero-derivatives were not detected (Fig. 2b). The isotopic purity is sufficient for the pro- posed use of the newly synthesized compound ([2H3] KAR1) as an internal standard for the quantification of karrikins in various (non-)biological materials. Fig. 2 Preparation scheme and isotope distribution of a newly synthesized internal standard. a Stable isotope labelled standard ([2H3]KAR1) was prepared from KAR bromoderivative (KAR-Br) under the following conditions (Cond.): [2H3]CB(OH)2, Pd(OAc)2, S-Phos, K3PO4, toluene. b Full-scan (m/z 150–160) positive-ion mass spectrum of [2H3]KAR1 (right), theoretical isotope model of [2H3]KAR1 (Molecular Formula: C8H3D3O3, left). R.A. – relative abundance (%) Fig. 2 Preparation scheme and isotope distribution of a newly synthesized internal standard. a Stable isotope labelled standard ([2H3]KAR1) was prepared from KAR bromoderivative (KAR-Br) under the following conditions (Cond.): [2H3]CB(OH)2, Pd(OAc)2, S-Phos, K3PO4, toluene. b Full-scan (m/z 150–160) positive-ion mass spectrum of [2H3]KAR1 (right), theoretical isotope model of [2H3]KAR1 (Molecular Formula: C8H3D3O3, left). R.A. – relative abundance (%) Fig. 2 Preparation scheme and isotope distribution of a newly synthesized internal standard. a Stable isotope labelled standard ([2H3]KAR1) was prepared from KAR bromoderivative (KAR-Br) under the following conditions (Cond.): [2H3]CB(OH)2, Pd(OAc)2, S-Phos, K3PO4, toluene. b Full-scan (m/z 150–160) positive-ion mass spectrum of [2H3]KAR1 (right), theoretical isotope model of [2H3]KAR1 (Molecular Formula: C8H3D3O3, left). R.A. – relative abundance (%) Fig. 2 Preparation scheme and isotope distribution of a newly synthesized internal standard. Development of extraction and purification protocol a Stable isotope labelled standard ([2H3]KAR1) was prepared from KAR bromoderivative (KAR-Br) under the following conditions (Cond.): [2H3]CB(OH)2, Pd(OAc)2, S-Phos, K3PO4, toluene. b Full-scan (m/z 150–160) positive-ion mass spectrum of [2H3]KAR1 (right), theoretical isotope model of [2H3]KAR1 (Molecular Formula: C8H3D3O3, left). R.A. – relative abundance (%) Hrdlička et al. Plant Methods (2021) 17:37 Hrdlička et al. Plant Methods (2021) 17:37 Page 4 of 13 Page 4 of 13 C18). The average recoveries of KARs in the elution frac- tion ranged from 18 to 28% and from 30 to 37% for C8 and C18, respectively. The use of ISOLUTE multimode sorbent combining of non-polar (C18), strong cation exchange (SO3 –) and strong anion exchange (–NR3 +) retention mechanisms showed higher recoveries of KAR1 and KAR2 (63 ± 18 and 59 ± 10%, respectively). Similarly, application of polymer-based RP columns with a hydro- philic-lipophilic-balance (HLB) water-wettable sorbent resulted in 60% yield of KAR1 and almost 80% yield of KAR2 (Fig. 3a). To determine losses during the purifica- tion process, we also monitored the loading capacity and extraction recovery in different steps of the tested proto- col (flow through, wash and elution). For example, using the HLB resin, no KARs were eluted from the sorbents during sample application and washing steps (Fig. 3a). Under our experimental conditions, all tested KAR standards were mostly eluted with 2 ml of 80% methanol with average recovery 66%. The second elution step (2 ml 80% methanol) did not significantly increase the yield of KARs (Fig. 3b). was not achieved with any of the solvents studied, which could also be due to losses during the evaporation step and/or the analyte adsorption to plastic or glass contain- ers [29]. Moreover, Scaffidi et al. [30] showed ultraviolet- dependent degradation of KAR1 to head-to-head cage photodimers upon irradiation with a solar light source. It can be expected that KARs decay rapidly in natural sun- light [3]. However, these possible factors contributing to KAR losses were not investigated. In summary, the high- est recovery was obtained with acidified 10% methanol (Additional file 1), therefore, this solution was used for extraction in all subsequent optimization procedures. q p p The next step in the method optimization was focused on the selection of SPE sorbents suitable for the subse- quent pre-concentration of KARs from plant samples. Development of extraction and purification protocol As mentioned above, the combination of an optimized extraction protocol with a simple one-step purification allows the reduction of a complex plant matrix resulting in sensitivity and selectivity enhancements of the final MS-based analysis [13]. Moreover, structurally similar SLs can be isolated using reverse phase [31], polymeric [32] or ion-exchange sorbents [33]. To maximize the yield of the SPE step and to reduce the effect of the plant matrix, four different SPE sorbents based on reverse phase (RP) or multiple-mode interactions were tested. The overall process efficiency (PE) of each analyte was then compared (Fig. 3b). Due to their polar character (log P < 0), KARs were weakly retained on silica-based RP resins with short and long carbon alkyl chains (C8 and To evaluate the behaviour of sorbents in the presence of a complex matrix, a test of four sorbents was also per- formed with plant extract spiked with known amounts of KARs. As shown in Fig. 3c, the average recoveries of KARs were two-fold lower for C8 and C18 sorbents com- pared to two other SPE columns. Moreover, C8 and C18 resins do not efficiently remove interfering substances from complex biological matrices that can contaminate Fig. 3 Process efficiency of the SPE-based method for KAR isolation. a, c Recovery (%) of KAR1 and KAR2 purified by four different SPE sorbents (C8, C18, Isolute M-M and Oasis HLB) without (a, 0 mg FW) and with plant matrix (c, 10 mg FW). b Representative test of loading capacity and extraction recoveries at different steps during the purification protocol described in Fig. 4. For all experiments, 10 pmol of each analyte was added to 1 ml of 10% methanol acidified with 0.1% formic acid without and with the presence of a plant tissue. The samples were then extracted and applied on SPE columns. After UHPLC–MS/MS analysis, the KARs’ peak areas were compared to the peak’s areas of the original stock and expressed as a percentage recovery. Values are means ± SD (n = 3) Fig. 3 Process efficiency of the SPE-based method for KAR isolation. a, c Recovery (%) of KAR1 and KAR2 purified by four different SPE sorbents (C8, C18, Isolute M-M and Oasis HLB) without (a, 0 mg FW) and with plant matrix (c, 10 mg FW). b Representative test of loading capacity and extraction recoveries at different steps during the purification protocol described in Fig. 4. Optimization of UHPLC–MS/MS method p Over the last decade, UHPLC has been the most com- monly used rapid LC technique in bioanalysis, includ- ing methods described for various PGRs [23, 34–40]. In order to quantify KARs, a one-step purification method was combined with a fast chromatographic run based on sub-2-μm ethylene-bridged hybrid (BEH) polymer-based particles. Initially, purified plant tissue samples were separated on a short reversed-phase BEH column with a total run time of 7.0 min, including equilibration [24]. However, in the absence of adequate chromatography to separate the endogenous metabolites from the interfering compounds, the LC–MS/MS method will not be specific to the analyte of interest [41]. Due to the co-elution of [2H3]KAR1 with interfering substances originating from a complex multi-component plant matrix (Additional file 2), the previously published separation method had to be modified. Instead of a 5 cm-length column, a three times longer BEH Shield RP18 column with the same particle size (1.7 µm) and the same diameter (2.1 mm) was applied. In this case, extending the column length increased the number of theoretical plates and therefore improved the chromatographic resolution [42]. Thus, Fig. 4 Optimized protocol for KAR determination in plant tissues. a Scheme of sample microextraction and purification using one-step solid phase extraction with Oasis HLB columns (30 mg/1 ml). b Representative multiple-reaction monitoring chromatograms of KAR1 and KAR2 with appropriate internal standard [2H3]KAR1 containing 1 pmol of each derivative per injection separated by optimized UHPLC–MS/MS method. FA – formic acid, MeOH – methanol, MRM – multi reaction monitoring Fig. 4 Optimized protocol for KAR determination in plant tissues. a Scheme of sample microextraction and purification using one-step solid phase extraction with Oasis HLB columns (30 mg/1 ml). b Representative multiple-reaction monitoring chromatograms of KAR1 and KAR2 with appropriate internal standard [2H3]KAR1 containing 1 pmol of each derivative per injection separated by optimized UHPLC–MS/MS method. FA – formic acid, MeOH – methanol, MRM – multi reaction monitoring without changing the mobile phase composition (metha- nol and water acidified with 0.1% formic acid), the linear gradient was only slightly modified (see chromatographic parameters in the chapter UHPLC–MS/MS Conditions). As shown in Additional file 2, the use of a larger amount of BEH sorbent together with a two-fold extension of the chromatographic run provided better base-line separa- tion from the sample background interferences. Moreo- ver, there was no visible deterioration in the peak shape of [2H3]KAR1. Development of extraction and purification protocol For all experiments, 10 pmol of each analyte was added to 1 ml of 10% methanol acidified with 0.1% formic acid without and with the presence of a plant tissue. The samples were then extracted and applied on SPE columns. After UHPLC–MS/MS analysis, the KARs’ peak areas were compared to the peak’s areas of the original stock and expressed as a percentage recovery. Values are means ± SD (n = 3) Fig. 3 Process efficiency of the SPE-based method for KAR isolation. a, c Recovery (%) of KAR1 and KAR2 purified by four different SPE sorbents (C8, C18, Isolute M-M and Oasis HLB) without (a, 0 mg FW) and with plant matrix (c, 10 mg FW). b Representative test of loading capacity and extraction recoveries at different steps during the purification protocol described in Fig. 4. For all experiments, 10 pmol of each analyte was added to 1 ml of 10% methanol acidified with 0.1% formic acid without and with the presence of a plant tissue. The samples were then extracted and applied on SPE columns. After UHPLC–MS/MS analysis, the KARs’ peak areas were compared to the peak’s areas of the original stock and expressed as a percentage recovery. Values are means ± SD (n = 3) Hrdlička et al. Plant Methods (2021) 17:37 Hrdlička et al. Plant Methods (2021) 17:37 Page 5 of 13 Fig. 4 Optimized protocol for KAR determination in plant tissues. a Scheme of sample microextraction and purification using one-step solid phase extraction with Oasis HLB columns (30 mg/1 ml). b Representative multiple-reaction monitoring chromatograms of KAR1 and KAR2 with appropriate internal standard [2H3]KAR1 containing 1 pmol of each derivative per injection separated by optimized UHPLC–MS/MS method. FA – formic acid, MeOH – methanol, MRM – multi reaction monitoring the MS instrument and interfere with column binding, elution and ionization [34]. Our findings showed simi- lar KAR1 pre-concentration process efficiencies for ISO- LUTE multimode and HLB sorbents, in average 34 and 38%, respectively. However, the difference is evident in the higher reproducibility of the results obtained using the Oasis HLB column (RSD% < 20; Fig. 3c). These find- ings were in good agreement with previously described SPE method for SLs determination [27]. Additionally, comparable results were achieved with only one-fifth amount of sorbent required to perform the clean-up pro- cedure (150 mg vs. 30 mg of the Oasis HLB cartridges for SL vs. KAR isolations, respectively). Development of extraction and purification protocol Moreover, poly- mer-based SPE is widely used for extraction of plant hor- mones from minute samples due to higher stability and sample capacity [23, 34, 35]. Based on the obtained data, we selected the Oasis HLB columns packed with 30 mg of m-divinylbenzene and N-vinylpyrrolidone copolymer for further characteriza- tion of the extraction protocol (Fig. 4a). As mentioned above, optimized extraction in acidified 10% methanol stabilized KAR metabolites and also reduced the con- centrations of interfering compounds such as lipids and plant pigments. Moreover, one-step purification proto- col including washing (water) and elution (80% metha- nol) steps pre-concentrated the KAR metabolites in the purified plant extracts. All steps together combine approaches suitable for KAR isolation before subsequent UHPLC–MS/MS analysis (Fig. 4b). Optimization of UHPLC–MS/MS method All analytes, including the newly synthetized internal stand- ard [2H3]KAR1, gave a strong signal from the protonated precursor [M + H]+ and two most abundant product ions [24]. Therefore, quantification and confirmation MRM transitions for each compound were selected and used to determine KARs under the optimized MS conditions listed in Additional file 3. As mentioned above, the MRM channels were time sectored to increase the cycle time for each analyte and acquired sufficient sensitivity (Fig. 4b). In addition, the automatically calculated dwell time pro- vided at least 16 data points per chromatographic peak to ensure reliable integration [23]. Under these param- eters, the limits of detection (LOD) and quantification (LOQ), defined as signal-to-noise ratios (3:1 and 10:1, respectively), were determined for each analyte. Simi- larly to the previous published study [24], the minimum detectable amounts of KAR1 and KAR2 were close to 0.1 fmol. To test the method linearity, a 14-point calibration curve (Cal 1) was constructed for each target analyte by plotting a known concentration of non-labelled analyte ranging from 0.25 fmol to 1000 pmol and a fixed amount of a deuterium labelled IS (0.5 pmol of [2H3]KAR1). The curves had a linear range spanning at least 4 orders of magnitude from 0.01 to 25 pmol with a coefficient of determination R2 ≥ 0.999 (Additional file 4). The opti- mized analytical method (isolation and quantification) was validated to further allow the analysis of KAR con- centrations in plant tissues. i Hereafter for method validation shown in Fig. 5 and Additional files 5, 6, the parameters of recovery (RE), matrix effect (ME) and process efficiency (PE) were deter- mined using three sets of samples spiked with 10 pmol of KAR1 and KAR2 as described in the chapter Method development and validation. First, we compared the absolute peak areas obtained for neat solution standards with the corresponding peak areas for standards spiked into plant extracts after purification into plant extracts and peak areas for standards spiked before the SPE step [46]. Moreover, the retention capacity of the Oasis HLB sorbent was also tested with increasing amounts of plant matrix (5, 10 and 20 mg FW of Arabidopsis seedlings). In general, REs express the proportion of analytes obtained from the sample during its purification by SPE [47]. Optimization of UHPLC–MS/MS method Under our chromatographic conditions, KAR2 and KAR1 compounds were reproducibly eluted Hrdlička et al. Plant Methods (2021) 17:37 Page 6 of 13 solvent-only calibration (Cal 1) for the determination of KAR2 based on the SIDM. This difficulty could be solved by the second stable isotope labelled IS for KAR2 or by spiking target analytes into biological matrix spanning the intended calibration range [44]. Therefore, the una- vailability of deuterium labelled KAR2 was replaced by adding known concentrations of the reference standard into a qualified batch of sample matrix. Every effort was made to prepare the standard calibrators in a biological matrix, which matched the chemical background with respect to species, composition, and sample pre-pro- cessing [45]. Two matrix-matched calibration sets (Cal 2 and 3) were prepared for each KAR analyte and further investigated. Similar to Cal 1, the calibration solutions contained various concentrations of each unlabelled KAR metabolite and a defined concentration of the stable iso- tope labelled IS. The first calibration series (Cal 2) was dissolved in plant matrix samples obtained after the SPE step (10 mg FW of Arabidopsis seedlings pre-extracted in acidified 10% methanol and pre-purified on an HLB col- umn). The second matrix calibration curve (Cal 3) was constructed using KAR standards added to the crude Arabidopsis extract and then purified by SPE (Fig. 4a). Following regulatory guidelines on bioanalytical method validation [18], all calibration standards at six concentra- tion levels were analysed in duplicate to generate a linear calibration curve. Both matrix calibrations showed a lin- ear range extending from 0.05 to 10 pmol with R2 ≥ 0.999 (Additional file 4). in 7.02 ± 0.03 min and 11.79 ± 0.04 min, respectively (Fig. 4b). Due to the chromatographic isotope effect [43], deuterated analogue was eluted slightly earlier than cor- responding authentic standards at a constant time dif- ference 0.12 min. Finally, to maximize the MS signal intensity for each compound, two time scan segments were used for analysis of KAR2 and KAR1 (5.5–9.0 and 10.0–13.0 min, respectively), see Fig. 4b. The high selectivity of MS/MS instruments based on specific data acquisition modes, such as multiple reaction monitoring (MRM), allows precise quantification of trace analytes in complex matrices [12]. In accordance with the previously published method, karrikins were detected by a triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) in positive mode. Optimization of UHPLC–MS/MS method Sur- prisingly, the recovery of KAR metabolites was not influ- enced by higher sample weights, and the use of 30 mg cartridges was sufficient to maximize the yield of the SPE step (Fig. 5a). On the other hand, the negative effect of the sample matrix was evident from the values of ME and PE, reaching on average only 30 and 50% for KAR1 and KAR2, respectively (Additional file 5). Our results showed a combined effect of possible losses during sample prep- aration and suppression of analyte ionization in the ion Method validationh Matrix effect (%) and process efficiency (%) were calculated with solvent-only calibration in methanol (Cal 1), calibration dissolved in the plant matrix blanks obtained after the SPE step (Cal 2), and matrix-matched calibration prepared similarly to the sample according to developed purification protocol (Cal 3) IS (10 pmol of [2H3]KAR1) was also added to apply the SIDM approach. After extraction in acidified 10% metha- nol and subsequent SPE purification, the samples were analysed by optimized UHPLC–MS/MS method (Fig. 4). The concentration of both KARs was determined using three different calibration series (Cal 1, Cal 2 and Cal 3) and the methods’ precision and accuracy were calculated (Table 1). Similar to the results of ME and PE assays, the final validation experiment indicates the requirement for a matrix-matched calibration passed through the SPE cartridge. The method’s precision was quantified by eval- uating the closeness of a series of replicate samples, and was expressed in terms of the relative standard deviation (RSD%). The RSD% were below 5% for all tested levels of both KAR compounds (see Table 1). The accuracy of the analytical method, defined as the difference between the levels obtained in an analytical run and the accepted ref- erence value, was estimated by percentage bias (%Bias). In general, analysis of KAR1 was accurate applying the Cal 1 and Cal 3 calibration series (bias below 27% and 13%, respectively). The accuracy of KAR2 quantification was insufficient when the matrix-free calibration (Cal 1) was applied. The acquired data showed that the use of matrix calibrators improved the method accuracy, how- ever, a combination of the stable isotope dilution method with a matrix-matched calibration prepared similarly to the sample (Cal 3) was only applicable (Table 1). The accuracy means for KAR2 were 132.0%, 58.5% and –3.7% for the Cal 1, Cal 2 and Cal 3 curves, respectively. Hence, accuracy of the developed analytical approach is satisfac- tory for the detection of trace components within ± 15% of the true amounts in a complex plant matrix [49]. All validation parameters of the developed method were comparable to the results reported by authors using LC–MS/MS for plant hormones analysis in plant tissue source by co-eluting compounds originating from the sample matrix. Method validationh The developed method combines a convenient sam- ple purification process based on one-step SPE with UHPLC–MS/MS and enables precise quantification by stable isotope dilution method (SIDM). As shown in Fig. 3c, the pre-concentration process efficiency of KAR2 was almost two-fold higher in the presence of Arabidop- sis matrix compared to the yield of KAR1. These results indicated the future problems in the application of Hrdlička et al. Plant Methods (2021) 17:37 Page 7 of 13 Hrdlička et al. Plant Methods Fig. 5 Validation of karrikin quantification method using the parameters of recovery (a), matrix effect (b) and process efficiency (c). The recovery (%) was calculated from the peak area of each compound (10 pmol of KAR1 and KAR2) added to the plant extract (5, 10 and 20 mg FW of ten-day-old Arabidopsis seedlings). Matrix effect (%) and process efficiency (%) were calculated with solvent-only calibration in methanol (Cal 1), calibration dissolved in the plant matrix blanks obtained after the SPE step (Cal 2), and matrix-matched calibration prepared similarly to the sample according to developed purification protocol (Cal 3) Fig. 5 Validation of karrikin quantification method using the parameters of recovery (a), matrix effect (b) and process efficiency (c). The recovery (%) was calculated from the peak area of each compound (10 pmol of KAR and KAR ) added to the plant extract (5 10 and 20 mg FW of ten-day-old Fig. 5 Validation of karrikin quantification method using the parameters of recovery (a), matrix effect (b) and process efficiency (c). The recovery (%) was calculated from the peak area of each compound (10 pmol of KAR1 and KAR2) added to the plant extract (5, 10 and 20 mg FW of ten-day-old Arabidopsis seedlings). Matrix effect (%) and process efficiency (%) were calculated with solvent-only calibration in methanol (Cal 1), calibration dissolved in the plant matrix blanks obtained after the SPE step (Cal 2), and matrix-matched calibration prepared similarly to the sample according to developed purification protocol (Cal 3) Fig. 5 Validation of karrikin quantification method using the parameters of recovery (a), matrix effect (b) and process efficiency (c). The recovery (%) was calculated from the peak area of each compound (10 pmol of KAR1 and KAR2) added to the plant extract (5, 10 and 20 mg FW of ten-day-old Arabidopsis seedlings). Method validationh In an additional step, we determined the concentration of each analyte in all enriched samples using the ratio of the analyte signal to the internal standard signal and the corresponding calibration curve (Additional file 4). Solvent-only calibration (Cal 1) and two matrix-matched calibration series (Cal 2 and Cal 3) were compared to demonstrate the equivalence of qualitative results. The KAR concentrations in Arabidopsis samples were then used to calculate IS-normalized ME and PE values [48], see chapter Method development and validation. Over- all, the IS-normalized values of KAR1 determined by the Cal 1–3 curves were quite similar, ranging from 87 to 98% for ME and from 90 to 102% for PE. Both values confirmed that the use of [2H3]KAR1 made it possible to correct for the ME and PE observed for the target ana- lyte. Conversely, the results of IS-normalized ME and PE for KAR2 indicated incorrect quantification applying the Cal 1 and Cal 2 curves (Fig. 5b, c). The calculated values of ME and PE were in the range 139% to 153% and 141% to 164%, respectively (Additional file 6). The use of the SIDM in combination with the Cal 3 calibration achieved a successfully valid quantification of KAR2 (Fig. 5b, c). On average, the IS-normalized ME was only 112% and the IS-normalized PE was 114% (see Additional file 6). Our findings confirmed the need for standard solutions pre- pared in plant extracts similar to that of the sample [18]. The use of Cal 3 can compensate for most of the errors obtained during the whole procedure (Fig. 4).f Finally, the effectiveness of the presented method was demonstrated by measurement of spiked samples of ten-day-old Arabidopsis (10 mg FW) with a standard mixture containing 1, 5 and 10 pmol of authentic KAR standards. The newly synthesized isotopically labelled Hrdlička et al. Plant Methods (2021) 17:37 Page 8 of 13 Hrdlička et al. Plant Methods Table 1 Method validation Analytical precision (RSD%) and accuracy (%bias) of whole procedure shown for different amounts of karrikins (1, 5 and 10 pmol). The extract of 10 mg (FW) Arabidopsis sample was spiked from 1 to 10 pmol of authentic KAR standards, purified by SPE and analysed by UHPLC-MS/MS. Concentrations of KARs were quantified using the standard isotope dilution method combined with calibration curves without (Cal 1) and with (Cal 2 and 3) plant matrix. Method validationh Values are means ± SD (n = 3) Compound Calibration curve Determinated spiked KARs content [pmol] Method precision [RSD%] Method accuracy [%bias] 1 5 10 1 5 10 1 5 10 KAR1 Cal 1 1.27 ± 0.03 5.60 ± 0.05 11.09 ± 0.49 2.4 1.0 4.5 27.1 12.0 10.9 Cal 2 1.58 ± 0.04 6.89 ± 0.07 13.57 ± 0.60 2.4 1.0 4.4 58.0 37.7 35.7 Cal 3 1.08 ± 0.03 5.39 ± 0.06 11.29 ± 0.54 2.6 1.0 4.8 8.2 7.7 12.9 KAR2 Cal 1 2.79 ± 0.12 10.50 ± 0.28 20.72 ± 0.74 4.2 2.6 3.6 178.8 110.1 107.2 Cal 2 1.72 ± 0.08 7.39 ± 0.21 15.61 ± 0.61 4.6 2.9 3.9 71.5 47.8 56.1 Cal 3 1.06 ± 0.05 4.47 ± 0.13 9.34 ± 0.36 4.5 2.9 3.9 6.0 − 10.6 − 6.6 Analytical precision (RSD%) and accuracy (%bias) of whole procedure shown for different amounts of karrikins (1, 5 and 10 pmol). The ex Arabidopsis sample was spiked from 1 to 10 pmol of authentic KAR standards, purified by SPE and analysed by UHPLC-MS/MS. Concentr using the standard isotope dilution method combined with calibration curves without (Cal 1) and with (Cal 2 and 3) plant matrix. Values Analytical precision (RSD%) and accuracy (%bias) of whole procedure shown for different amounts of karrikins (1, 5 and 10 pmol). The extract of 10 mg (FW) Arabidopsis sample was spiked from 1 to 10 pmol of authentic KAR standards, purified by SPE and analysed by UHPLC-MS/MS. Concentrations of KARs were quantified using the standard isotope dilution method combined with calibration curves without (Cal 1) and with (Cal 2 and 3) plant matrix. Values are means ± SD (n = 3) Table 2 Karrikin quantification in ten-day-old Arabidopsis thaliana seedlings grown on media supplemented with KAR1 and KAR2 Values are means ± SD (n = 3) Concetration of KARs in growing media [µmol/l] KAR content [pmol/g FW] KAR1 KAR2 0.1 1.8 ± 0.2 5.5 ± 0. 4 1 60.9 ± 8.9 66.1 ± 11.4 10 1577.9 ± 111.4 1065.9 ± 101.5 1 µM concentrations, respectively. Interestingly, elevated karrikin levels were determined in Arabidopsis samples treated with the highest concentration (10 µM of each compound). Our results suggest that this approach ena- bles the targeted and sensitive determination of karrikin levels and thus allows the detailed study of the physi- ological roles and modes of action of KAR in plants. Method validationh Interestingly, our findings indicate a different accumu- lation rate of KARs in plant tissue after treatment with their low or high concentrations, however, the mecha- nism of this process is still unknown. This effect could be further investigated in further attempts to modify plant development by exogenously applied karrikins in order to improve crop yields [51]. In addition, our quantita- tive data on KAR levels in plant tissues cannot be directly compared with previous reports, since KARs have been only quantified in smoke water samples so far [6, 24]. Values are means ± SD (n = 3) samples [23, 34, 35, 38–40, 50]. In conclusion, applying the Cal 3 calibration, the precision and accuracy demon- strate the methods’ reliability and usefulness for routine KAR analysis in plant material. Conclusions P i It is one of a fundamental biological interests to improve our knowledge about how small signalling molecules, such as karrikins, regulates vital processes in plants. The study of KARs’ mode of action should include not only the signalling pathways, transcription factors and respon- sive genes, but also knowledge of their concentration lev- els in various plant organs. The use of modern analytical tools allows accurate detection and quantification of low abundant compounds. Precise measurements of karrikins are technically highly challenging and seed-germination bioassays are mainly used to detect activity [3]. In the presented study, we have developed a new sensitive and specific method for isolation and analysis of karrikin compounds in small amounts of plant tissue samples. The protocol is based on a solid phase extraction combined with a sensitive UHPLC–MS/MS method. Quantification of the ana- lytes was performed by a stable isotope dilution method employing a newly synthetized isotopically labelled inter- nal standard. This new method was fully validated and successfully applied for KAR analysis in treated Arabi- dopsis samples. Our results demonstrate the applicabil- ity of the developed methodology for routine analyses and for monitoring KARs in complex biological matri- ces. Based on the synthesis of new standards, potential applications of this approach in analyses of all described KARs in one-step SPE/UHPLC–MS/MS runs are under To assess the applicability of the newly developed approach, we quantified KAR levels in ten-day-old Arabidopsis seedlings (10 mg FW) grown on media supplemented with different concentrations of KAR compounds (Table 2). After extraction in acidified 10% methanol and subsequent SPE purification, the samples were analysed by optimized UHPLC–MS/MS method (Fig. 4). Units and tens pmol/g FW of KAR1 and KAR2 were detected in samples grown on medium enriched with a mixture of pure karrikin standards at 100 nM and Hrdlička et al. Plant Methods (2021) 17:37 Page 9 of 13 developed. We are also aware that employment of novel atmospheric pressure ionization interfaces can also lead to further improvements in our quantitative method. and methylboronic acid (methyl-d3) (25.0 mg, 0.44 mmol) in toluene (2.0 ml) was added and to the reac- tion mixture was heated at 100 °C with stirring for 48 h in a sealed tube under an argon atmosphere. After cooling to room temperature, the reaction mixture was filtered through celite and washed with toluene (15.0 ml). Conclusions P i The filtrate was then evaporated under reduced pressure and the residue was suspended in dichloromethane (20.0 ml). The organic phase was washed with water, brine, dried over anhydrous sodium sulphate and evaporated under reduced pressure. The crude product was puri- fied by column chromatography on silica using mobile phase petroluem ether – ethylacetate – triethylamine (3:1:0.025) and subsquently by column chromatography on silica using mobile phase chloroform-triethylamine (97.5:2.5) and Merck silica gel Kieselgel 60 (230–400 mesh). Yield: 22 mg (49%). Reagents and materials Methanol (gradient grade for liquid chromatography), acetonitrile (gradient grade for liquid chromatogra- phy) and water (for chromatography) were obtained from Merck (Darmstadt, Germany). Oasis® HLB (RP, polymer-based SPE cartridges, 30 mg/1 ml) were pur- chased from Waters (Milford, MA, USA), Isolute® M-M (100 mg/1 ml) from Biotage (Uppsala, Sweden), Bond Elut-C8 (500 mg/3 ml) from Agilent Technologies (Santa Clara, CA, USA), and Spe-ed SPE C18 (100 mg/1 ml) from Applied Separations (Allentown, PA, USA). The methylboronic acid (methyl-d3) was obtained from Cam- bridge Isotope Laboratories Inc. (Tewksbury, MA, USA) Formic acid and other reagents for chemical synthe- sis were purchased from Sigma-Aldrich (St. Louis, MI, USA). KAR1 and KAR2 were synthesized as described previously by Hrdlička et al. (2019) [24]. The solid sub- stances of authentic KAR standards were dissolved in methanol to a concentration 10–3 mol/l and then gradu- ally diluted to lower concentrations. The GC–MS analyses were performed on GC–MS QP2010 Ultra (Shimadzu, Kyoto, Japan). The separa- tion was performed on a Zebron ZB-5MS capillary col- umn, length: 30 m, inner diameter 0.32 mm, thin layer 0.25 µm. Helium was used as a carrier gas at constant flow 1.20 mL.min-1. The injection volume of the samples was 1 µl, sample concentration 1.0 µg.mL-1 in splitless mode. The injector temperature was 260 °C, sampling time 1 min, solvent cut time was 1.5 min. The temper- ature programme started at 60 °C held for 1 min and was followed by temperature rate 20 °C.min-1 to 280 °C which was held for 5 min. The interface temperature was 280 °C. The ion source operated with collision energy 70 eV at temperature 250 °C and detector voltage 0,7 kV. The mass spectra were scanned in a range 50–650 m/z in a speed 2000 scans.s-1. GC–MS (EI, 70 eV): retention time 8,27 min; m/z (rel.int.): 123.10 (100), 153.05 (69), 96.10 (26.5), 68.10 (24.5), 97.1 (16.5). Biological material Arabidopsis thaliana (ecotype Col-0) seedlings were grown on full MS medium with 1% sacharose and 1% agar (Duchefa Biochemie, Haarlem, Netherlands) at pH 5.7 in a growth chamber under long-day conditions at 23 °C under a 16-h photoperiod. Stock solution of kar- rikin compounds (KAR1 and KAR2) was dissolved in deionized water and applied to cultivation media at the final concentration 0.1, 1 and 10 µmol/l. The ten-day-old plants were harvested, carefully rinsed in distilled water (three times) and subsequently dried with filter paper to avoid contamination of plant surface. The samples were immediately plunged into liquid nitrogen, weighed and stored at -80 °C until extraction and purification before analysis. Untreated Arabidopsis plants were grown under the same conditions as described above and used for method development and validation or as controls for KAR quantification. The analysis of HPLC–PDA-(ESI +)MS purity was per- formed on an Acquity UPLC® H-Class System combined with Acquity PDA and Acquity QDa detectors (all from Waters) with detection at wavelengths of 210–400 nm and electrospray ionization in positive mode in m/z 50–1000 range, respectively. Briefly, 10 μl of the IS (con- cetration 10 μg/ml) was injected onto a thermostated (25 °C) RP column (150 mm × 2.1 mm, 5 μm C18 Sym- metry, Waters) and eluted at a flow rate of 0.3 ml/min using a linear gradient of 15 mM ammonium formate at pH 4.0 (A) and pure methanol (B) as follow: 0 min, 10% B; 0–25 min, 10–90% B, 25–35 min, 90% B. The column was then re-equilibrated under the initial conditions (10% B) for 10 min. The MS conditions were set for source/probe temperatures at 120/600 °C and capillary/cone voltages of + 800/ + 15 V. Nitrogen was used the desolvation gas. HPLC–DAD–(ESI +)MS: m/z 154.1 (purity 96.2%). Extraction and purification optimization All samples were homogenized and weighed under liquid nitrogen into 2 ml plastic microtubes (Eppendorf, Ger- many) containing three 2 mm ceria-stabilized zirconium oxide beads. 3-(2H3)methyl-2H-furo[2,3-c]pyran-2-one ([2H3]-KAR1, 10 pmol) was used as internal standard to check the recovery during purification and to validate the determination of KAR1 and KAR2. The frozen plant material (5–20 mg FW) was extracted in 1 ml of ice-cold extraction solution (0.1% formic acid in 10% methanol, v/v) using vibration mill MM 301 (27 Hz, 3 min; Retsch GmbH & Co. KG, Haan, Germany). Samples were soni- cated (4 °C, 3 min; Elma, Germany) and subsequently incubated using a benchtop laboratory rotator Stuart SB3 (4 °C, 30 min; Bibby Scientific Ltd., Staffordshire, UK). After centrifugation (14,000 rpm, 15 min; Beckman Coulter, Brea, CA, USA), the supernatants were purified by RP polymer-based solid phase extraction Oasis® HLB columns (1 cc per 30 mg, Waters). The SPE sorbent was activated sequentially by 1 ml of 100% methanol and 1 ml of deionized water, then equilibrated with 1 ml extraction solution (0.1% formic acid in 10% methanol, v/v). After sample loading, the HLB column was washed with 1 ml of deionized water and analytes were eluted with 2 ml of 80% methanol (v/v). The eluted samples were evaporated to dryness at 30 °C under a stream of nitrogen (TurboVap LV, Biotage) and stored at -20 °C until UHPLC-MS/MS analysis. For the method validation, three different calibration series (Cal 1, 2 and 3) were used. Solvent-only calibra- tion curve (Cal 1) was constructed using serial dilutions of authentic standards and known concentrations of internal labelled standards in methanol. Furthermore, two matrix-matched calibrations, Cal 2 and Cal 3, were prepared using 10 mg FW of Arabidopsis seedling per calibration point. For calibration 2, KAR standards were dissolved in the plant matrix blanks obtained after the SPE step. Calibration curve 3 was constructed using plant extract spiked with a known amount of KARs purified by developed purification protocol. All calibration curves were analysed in duplicate and constructed using least square linear regression analysis method (Additional file 4). i To validate the isolation protocol (Fig. 4), three sets of samples were prepared in triplicate and analysed by the UHPLC–MS/MS system. Extraction and purification optimization In the first set, Arabidop- sis seedlings (5, 10 and 20 mg, FW) were extracted by acidified 10% methanol spiked with 10 pmol of KAR1 and KAR2 and stable isotope labelled IS (10 pmol of [2H3] KAR1), and subsequently purified by the SPE protocol. In the second set, the same plant extracts passed through SPE sorbent and then were spiked with the analytes and IS (10 pmol of each compound). Third set consisted non- matrix samples representing a standard mixture (10 pmol of authentic compounds and IS) purified by the SPE step without plant extract. Non-normalized recovery (in per- centages) was calculated as a ratio of average peak areas of a non-labelled analytes spiked before and after SPE purification [46]. Non-normalized matrix effect and pro- cess efficiency of the method were then expressed as the ratio of average peak areas of KARs spiked before and after extraction to average peak area of the same ana- lyte standards, respectively (Additional file 5). Futher- more, IS-normalized PE a ME were calculated as a Synthesis of isotope labelled standardh The mixture of palladium diacetate (16.0 mg, 72.5 µmol), S-Phos (2-dicyclohexylphosphino-2′,6′- dimethoxybiphenyl) (90.0 mg, 0.218 mmol) and potas- sium phosphate (70.0 mg, 0.60 mmol) in toluene (3.0 ml) was stirred at room temperature in a sealed tube for 30 min under an argon atmosphere. The solution of 3-bromo-furo[2,3-c]pyran-2-one (63.0 mg, 0.29 mmol) Hrdlička et al. Plant Methods (2021) 17:37 Hrdlička et al. Plant Methods (2021) 17:37 Page 10 of 13 Page 10 of 13 and KAR2 without (0 mg FW) and/or with plant matrix. Briefly, 10 mg FW of Arabidopsis seedlings was extracted in ice-cold acidified 10% methanol spiked with known amounts of KARs. All tested sorbents were activated with 1 ml of 100% methanol and 1 ml of deionized water, and equilibrated with 1 ml of acidified 10% methanol (Fig. 4a). After samples loading (1 ml of plant extract or neat standard), each sorbent was washed with 1 ml of deionized water and analytes were then eluted by two- step elution using 2 × 1 ml of 80% methanol. The sam- ples thus prepared were evaporated to dryness under a stream of nitrogen, re-suspended in 100 μl of 10% MeOH and then analysed by UHLC-MS/MS method (10 μl per injection). The performance efficiency of four different cartridges was calculated as percentages of average peak areas relative to the corresponding peak areas in control samples. All experiments were performed in triplicates. 1H and 13C-NMR spectra were recorded on a Jeol 500 ECA instrument operating at 500 MHz for 1H and 126 MHz for 13C. Chemical shifts are reported in ppm. Coupling constants (J) are reported in Hertz (Hz), and the following abbreviations are used: singlet (s), doublet (d). 1H NMR (CDCl3): 6.509 (d, J = 5.50 Hz, 1H, CH), 7.322 (d, J = 5.50 Hz, 1H, CH); 13C NMR (CDCl3): 7.58, 101.30, 105.00, 128.64, 141.75, 143.28, 150.41, 172.38. Availability of data and materials All relevant data can be found within the manuscript and its additional files. Acknowledgements The authors thank Ioanna Antoniadi and Barbora Pařízková for careful revision of the article. Method development and validationh The stability of KARs was tested in triplicate by adding 10 pmol of KAR1 and KAR2 to 1 ml of 10% methanol or 10% acetonitrile (non-acidified and/or acidified with 0.1% formic acid). The samples were thoroughly mixed, evaporated to dryness under a stream of nitrogen, re- suspended in 100 μl of 10% MeOH and then analysed by UHLC-MS/MS method (10 μl per injection). Finally, recoveries of each compound (percentages of average peak areas in each solvent relative to respective peak areas obtained from analyses of reference samples) were calculated (Fig. 3). To develop an isolation protocol, four SPE sorbents (Bond Elut-C8, Spe-ed C18, Isolute M-M and Oasis HLB) were tested using a mixture of 10 pmol of KAR1 Hrdlička et al. Plant Methods (2021) 17:37 Page 11 of 13 Hrdlička et al. Plant Methods (2021) 17:37 Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s13007-021-00738-1. Additional file 1. KARs stability in different extraction solutions. Additional file 2. Optimization of chromatographic separation of an isotope labelled standard. Additional file 3. Optimized parameters for the quantification of karrikins by UHPLC-MS/MS. Additional file 4. Calibration curves used to validate the method. Additional file 5. Non-normalized recovery, matrix effect and process efficiency. Additional file 6. Internal standard-normalized recovery, matrix effect and process efficiency. concentration ratio of Set1 and Set 2 to Set 3, respectively [48]. Supplementary Information Finally, 10 mg FW of Arabidopsis seedlings was extracted in ice-cold acidified 10% methanol spiked with 1, 5 and 10 pmol of KARs and 10 pmol of stable isotope- labelled IS, and subsequently purified by the SPE proto- col (Fig. 4). Concentrations of karrikins were quantified by UHPLC-MS/MS method using the standard isotope dilution method [52] in combination with three calibra- tion series (Cal 1, Cal 2 and Cal 3). The precision of the method was expressed as the relative standard deviation (RSD%) of three replicate measurements. The method accuracy was expressed as a relative bias of the deter- mined analyte concentrations compared with the spiked amounts of KAR standards (Table 2). All experiments were done in triplicates. Funding g This work was financially supported by the Ministry of Education, Youth and Sport of the Czech Republic, ERDF project “Plants as a tool for sustainable global development” (No. CZ.02.1.01/0.0/0.0/16_019/0000827) and Palacky University Olomouc (IGA_PrF_2021_011). Authors’ contributions Karrikins were analysed by an Acquity UPLC® I-Class System combined with a Xevo™ TQ-XS triple quadrupole mass spectrometer (Waters). The samples were dissolved in 100 μL of 10% methanol (v/v), filtered using modified nylon 0.2-μm Centrifugal Filters and then transferred to insert-equipped vials. 10 μl of each sample was injected onto an Acquity UPLC® BEH C18 reversed-phase col- umn (1.7 µm, 2.1 × 50 mm) and/or Acquity UPLC® BEH Shield RP18 column (1.7 µm, 2.1 × 150 mm). The col- umn temperatures were held at 40 °C. The compounds of interest were separated by a 5-min gradient elution with the flow 0.4 ml/min using acidified methanol (A, 0.1% formic acid in methanol) and acidified water (B, 0.1% for- mic acid in water), as follow 0–1 min isocratic elution by 5% A, 1–3 min linear gradient to 20% A and 3–5 min iso- cratic elution by 20% A. After this, column was washed with 100% A for 1 min and re-equilibrated to the initial conditions (5% A) for 1 min. Using a 150 mm-length col- umn, the modified gradient included a flow 0.2 ml/min and an extension of the linear gradient from 5 to 20% A in 1–6 min and an isocratic elution by 20% A in 6–10 min, followed by washing step with 100% methanol for 1 min and re-equilibration to initial conditions (2 min). KD and ON designed the research with the help of JvS. JH performed the experiments. TG synthesised the karrikin standards. JH and ON analysed data for analytical method validation. JH, KD and ON wrote the manuscript. All authors read and approved the final manuscript. The authors declare that they have no competing interests. The authors declare that they have no competing interests. Author details 1 Laboratory of Growth Regulators, Institute of Experimental Botany, The Czech Academy of Sciences and Faculty of Science, Palacký University, Šlechtitelů 27, 78371 Olomouc, Czech Republic. 2 Department of Chemical Biology, Faculty of Science, Palacký University, Šlechtitelů 27, 78371 Olomouc, Czech Republic. 3 Department of Experimental Biology, Faculty of Science, Palacký University, Šlechtitelů 27, 78371 Olomouc, Czech Republic. 4 Research Centre for Plant Growth and Development, School of Life Sciences, University of KwaZulu- Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa. During the UHPLC-MS/MS acquisition, the effluent was introduced into the electrospray ion source of a triple quadrupole mass spectrometer operating in positive mode under the following conditions: capillary voltage, 0.5 kV; source/desolvation temperature, 120 °C/600 °C; cone/des- olvation gas flow, 150/1,000 l/h; collision gas flow (argon), 0.15 ml/min. Karrikins were quantified in MRM mode using dwell time in automatic mode for 16 scan points per peak and optimized MS conditions (Additional file 3). Acquired data were processed by MassLynx™ MS Software with TargetLynx™ program (version 4.2, Waters, Milford, MA, USA). Received: 8 January 2021 Accepted: 23 March 2021 Received: 8 January 2021 Accepted: 23 March 2021 Received: 8 January 2021 Accepted: 23 March 2021 Declarations Ethics approval and consent to participate Not applicable. 1. Flematti GR, Ghisalberti EL, Dixon KW, Trengove RD. A compound from smoke that promotes seed germination. Science. 2004;305:977. 2. 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Profiling ABA metabolites in Nicotiana tabacum L. leaves by ultra-performance liquid chromatography-electro- spray tandem mass spectrometry. Talanta. 2009;80:390–9. 20. Chiwocha SDS, Abrams SR, Ambrose SJ, Cutler AJ, Loewen M, Ross ARS, et al. A method for profiling classes of plant hormones and their metabo- lites using liquid chromatography-electrospray ionization tandem mass spectrometry: An analysis of hormone regulation of thermodormancy of lettuce (Lactuca sativa L.) seeds. Plant J. 2003;35:405–17. 41. Jemal M. High-throughput quantitativ Biomed Chromatogr. 2000;14:422–9. 41. Jemal M. High-throughput quantitative bioanalysis by LC/MS/MS. Biomed Chromatogr. 2000;14:422–9. 21. Pan X, Welti R, Wang X. Simultaneous quantification of major phyto- hormones and related compounds in crude plant extracts by liquid chromatography-electrospray tandem mass spectrometry. Phytochemis- try. 2008;69:1773–81. 42. Nováková L, Svoboda P, Pavlík J. Ultra-high performance liquid chroma- tography. In: Fanali, S., Haddad, P.R., Poole, C.F., Riekkola, M.-L., editors. Liquid Chromatography. Elsevier Inc.; 2017. p. 719–69. g y 43. Pratt JJ. Isotope dilution analysis using chromatographic separation of isotopic forms of the compound to be measured. Ann Clin Biochem. 1986;23:251–76. 22. Farrow SC, Emery RJN. Concurrent profiling of indole-3-acetic acid, absci- sic acid, and cytokinins and structurally related purines by high-perfor- mance-liquid-chromatography tandem electrospray mass spectrometry. Plant Methods. 2012;8:42. 44. Fu Y, Li W, Flarakos J. Recommendations and best practices for calibration curves in quantitative LC–MS bioanalysis. Bioanalysis. 2019;11:1375–7. 23. Šimura J, Antoniadi I, Široká J, Tarkowská D, Strnad M, Ljung K, et al. Plant hormonomics: multiple phytohormone profiling by targeted metabo- lomics. Plant Physiol. 2018;177:476–89. 45. Azadeh M, Gorovits B, Kamerud J, Macmannis S, Safavi A, Sailstad J, et al. Calibration curves in quantitative ligand binding assays: recommenda- tions and best practices for preparation, design, and editing of calibration curves. AAPS J. 2017;20:22. 24. Hrdlička J, Gucký T, Novák O, Kulkarni M, Gupta S, Van Staden J, et al. Quantification of karrikins in smoke water using ultra-high performance liquid chromatography–tandem mass spectrometry. Plant Methods. 2019;15:81. 46. Matuszewski BK, Constanzer ML, Chavez-Eng CM. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal Chem. 2003;75:3019–30. 47. Poole CF. New trends in solid-phase extraction. Trends Anal Chem. 2003;22:362–73. 25. Ito S, Tsukada K. References Matrix effect and correction by standard addition in quantitative liquid chromatographic–mass spectrometric analysis of diar- rhetic shellfish poisoning toxins. J Chromatogr A. 2001;943:39–46. 48. Caban M, Migowska N, Stepnowski P, Kwiatkowski M, Kumirska J. Matrix effects and recovery calculations in analyses of pharmaceuticals based on the determination of β-blockers and β-agonists in environmental samples. J Chromatogr A. 2012;1258:117–27. 26. Sun K, Chen Y, Wagerle T, Linnstaedt D, Currie M, Chmura P, et al. Syn- thesis of butenolides as seed germination stimulants. Tetrahedron Lett. 2008;49:2922–5. 49. Van Rhijn JA, Heskamp HH, Davelaar E, Jordi W, Leloux MS, Brinkman UAT. Quantitative determination of glycosylated and aglycon isoprenoid Hrdlička et al. Plant Methods (2021) 17:37 Hrdlička et al. Plant Methods (2021) 17:37 Page 13 of 13 Page 13 of 13 52. Rittenberg D, Foster GL. A new procedure for quantitative analysis by isotope dilution, with application to the determination of amino acids and fatty acids. J Biol Chem. 1940;133:737–44. 52. Rittenberg D, Foster GL. A new procedure for quantitative analysis by isotope dilution, with application to the determination of amino acids and fatty acids. J Biol Chem. 1940;133:737–44. cytokinins at sub-picomolar levels by microcolumn liquid chromatogra- phy combined with electrospray tandem mass spectrometry. J Chroma- togr A. 2001;929:31–42. 50. Urbanová T, Tarkowská D, Novák O, Hedden P, Strnad M. Analysis of gibberellins as free acids by ultra performance liquid chromatography- tandem mass spectrometry. Talanta. 2013;112:85–94. 51. Kulkarni MG, Ascough GD, Van Staden J. Smoke-water and a Smoke- isolated Butenolide improve growth and yield of tomatoes under green- house conditions. HortThechnology. 2008;18:449–54. cytokinins at sub-picomolar levels by microcolumn liquid chromatogra- phy combined with electrospray tandem mass spectrometry. J Chroma- togr A. 2001;929:31–42. 52. Rittenberg D, Foster GL. A new procedure for quantitative analysis by isotope dilution, with application to the determination of amino acids and fatty acids. 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https://openalex.org/W2795901721	https://www.e-compos.org.br/e-compos/article/download/213/269	Portuguese	null	Modos de compreendeer imagens: questões de método sobre a análise textual de representações visuais	E- compós	2,009	cc-by	9,012	www.e-compos.org.br \| E-ISSN 1808-2599 \| www.e-compos.org.br \| E-ISSN 1808-2599 \| Resumo Propomos um panorama breve dos pontos de contato entre as abordagens semióticas e estéticas da análise das imagens, tendo em vista o regime discursivo que as demarca, especialmente no contexto da comunicação: reconhecendo a predominância histórica das perspectivas que a semiologia aportou à discussão sobre as regências textuais das representações visuais, procuramos introduzir ao debate o ponto de vista que as ciências da arte (muito especialmente as questões de método da abordagem histórica dos fatos artísticos), supondo encontrar aí certos modos de aproximação à estrutura psicológica da experiência dos ícones e de sua interpretação e fruição que não poderiam ser negligenciadas por uma teoria da compreensão da imagem enquanto forma textual. Palavras-chave d Pó G d ã C i ã \| E ó B íli 11 2 i / 2008 Há um registro determinado no qual algo a que podemos chamar de “questão da imagem” atormenta os espíritos, em especial quando é evocada em nosso campo de estudos, com algum grau de centralidade. Trata-se, em primeiro lugar, de um gênero de interrogações que, a título da abordagem problemas atinentes a outras áreas de conhecimento, ainda assim aporta questões muito peculiares a esse universo de fenômenos: assim sendo, ao se perguntar sobre a imagem, a filosofia da arte se interessa sobretudo pelo suposto status representacional dos ícones visuais; por seu turno, a história da arte se pergunta sobre as matrizes do problema da evolução dos estilos pictóricos, em diferentes épocas. Análise de imagens. Semiótica. Ciências da arte. Revista da Associação Nacional dos Program Entretanto, como já dissemos, algo de muito próprio inspira os estudos sobre fenômenos e processos comunicacionais, quando neles a imagem é interpelada enquanto objeto de atenção: a finalidade comunicacional das representações visuais e o diagnóstico sobre a imensa impregnação da cultura de nossos dias Benjamim Picado \| jbpicado@hotmail.com Doutor em Comunicação e Semiótica pela Pontifícia Universidade Católica de São Paulo – PUCSP. Professor da Faculdade de Comunicação e do Programa de Pós-Graduação em Comunicação e Cultura Contemporâneas da Universidade Federal da Bahia – UFBA. Benjamim Picado Benjamim Picado Benjamim Picado \| jbpicado@hotmail.com Doutor em Comunicação e Semiótica pela Pontifícia Universidade Católica de São Paulo – PUCSP. Professor da Faculdade de Comunicação e do Programa de Pós-Graduação em Comunicação e Cultura Contemporâneas da Universidade Federal da Bahia – UFBA. Doutor em Comunicação e Semiótica pela Pontifícia Universidade Católica de São Paulo – PUCSP. Professor da Faculdade de Comunicação e do Programa de Pós-Graduação em Comunicação e Cultura Contemporâneas da Universidade Federal da Bahia – UFBA. www.e-compos.org.br \| E-ISSN 1808-2599 \| linguagem infundiram na compreensão sobre o fato de que as imagens preenchem funções próprias do discurso (seja do ponto de vista de sua expressão, seja na perspectiva de sua apreciação e compreensão): caso exemplar aqui é o dos primeiros ensaios de uma semiologia visual, encarnada nas formulações originárias de Roland Barthes (1964) e no modo como nela se estabelecem os princípios da redução da iconicidade das representações visuais às matrizes lingüísticas da compreensão e da expressão (PICADO, 2003). pelas imagens são ambas decorrentes da ênfase com a qual nossa área de estudos associa os ícones visuais com formas discursivas, estratégias retóricas e funções narrativas que lhe seriam próprias. O prolongamento desse tipo de interesse pelos regimes discursivos da imagem parece-nos haver chegado a um tal ponto de resolução que poderíamos inclusive ensaiar certos modelos de uma hipotética redução das formas da representação visual aos princípios de organização de uma ordem discursiva determinada. Em nosso percurso de pesquisa e estudos, mencionamos essa idéia de uma possível matriz da discursividade visual, o resultado mais evidente da sedimentação do interesse das teorias da comunicação pela questão da imagem. Revista da Associação Nacional dos Programas de Pós-Graduação em Comunicação \| E-compós, Brasília, v.11, n.2, maio/ago. 2008 Na perspectiva de uma “semiologia de primeira geração”, o aspecto de manifestação propriamente icônico (ou analógico) das representações visuais ficaria sempre legado ao que chamamos alhures (ecoando uma sugestão do próprio Barthes) de uma certa insignificância originária do status semiológico da imagem. Nessa perspectiva de análise (prolongada por muitos dos estudos comunicacionais), a regência discursiva das imagens implicaria sempre o concurso de estruturas lingüísticas, às quais a expressão e fruição das formas visuais ficaria necessariamente subordinada (PICADO, 2004). Supomos que muitos dos aspectos dessa discussão são, na verdade, decorrentes de uma má-apreensão quanto ao caráter das proposições originárias de uma semiologia visual, mas que não devem ser combatidas antes de uma avaliação do terreno heurístico de base dessas mesmas proposições. vínculo com as estratégias retóricas e narrativas dos meios de comunicação massivos. primeira semiologia, preferimos, portanto, adotar uma postura um pouco diferente daquela que caracteriza nosso campo de estudos: em geral, discussões dessa natureza apenas aprofundam uma certa impressão de um debate entre escolas semióticas, mais do que propriamente uma discussão sobre esse fenômeno determinado que é o das regências textuais próprias aos materiais visuais no contexto da comunicação. Se pudéssemos conjurar as interessantes questões que todas essas teorias devotadas ao campo do artístico trouxeram para uma aproximação metodologicamente rentável à dimensão discursiva da imagem, poderíamos decerto nos preparar para superar essa espécie de “fosso disciplinar” que, por vezes, parece dificultar que falemos do universo iconográfico de nossa época, especificando nele aspectos de sua realização enquanto acervo de estratégias e de mensagens estruturadas na forma de textos e, na mesma medida, organizadas a partir de materiais que foram elaborados tendo em vista sua possível fruição sensível por parte de sua recepção. Em nosso entendimento, um caminho alternativo é, antes de mais nada, o de um aggiornamento de alguns outros endereços teóricos da análise de materiais visuais: nos definimos mais especificamente pelas questões de método das abordagens de materiais expressivos da comunicação visual, com especial ênfase nas vizinhanças possíveis entre a abordagem textual da análise da imagem e as contribuições aportadas pelo campo das teorias da arte, em geral. Nesse caso, teríamos que considerar forçosamente o papel de uma abordagem dos materiais da cultura mediática mais inspirada por questões oriundas, por exemplo, das teorias estéticas, de um modo geral. Nesses termos, a questão do valor textual da imagem passaria a ter uma menor correlação com o problema de sua hipotética redução às estruturas lingüística do discurso enunciativo, e mais com aspectos d provocação que as imagens podem suscitar de nossos juízos perceptivos e de nossos modos de contemplação e fruição propriamente visuais. Revista da Associação Nacional dos Programas de Pós Graduação em Comunicação \| E compós Brasíli Nesse caso, teríamos que considerar forçosamente o papel de uma abordagem dos materiais da cultura mediática mais inspirada por questões oriundas, por exemplo, das teorias estéticas, de um modo geral. Ao avaliarmos as teses desta Por mais de uma vez, em nossas próprias investigações, procuramos explorar os eixos teórico-metodológicos das abordagens que privilegiaram, no estudo das representações visuais, seus aspectos de regência por variadas formas da expressão discursiva: dado de pressuposto dessa exploração é o reconhecimento de que o problema dessa regência mesma é que caracterizou a pertinência com a qual a questão da função das representações visuais foi historicamente tematizada em nosso campo de estudos (PICADO, 2005) Supomos que muitos dos aspectos dessa discussão são, na verdade, decorrentes de uma má-apreensão quanto ao caráter das proposições originárias de uma semiologia visual, mas que não devem ser combatidas antes de uma avaliação do terreno heurístico de base dessas mesmas proposições. Ao avaliarmos as teses desta Com esse mesmo fim, já identificamos, em vários capítulos de nossas explorações, uma espécie de contaminação que as ciências da www.e-compos.org.br \| E-ISSN 1808-2599 \| ligadas à modelação icônica das representações visuais: desse modo, a questão da significação textual das imagens visuais não estará restrita àqueles aspectos que definem a redução de suas propriedades visuais às funções do discurso enunciativo ou narrativo, mas também incorporaria algum discurso teoricamente bem fundado (e capaz de impregnar amplas regiões do fenômeno representacional na imagem) sobre os aspectos propriamente plastico- icônicos de sua manifestação. e frases, em contextos narrativos, reportativos ou retóricos, por exemplo) deverá fazer- nos vislumbrar a dimensão estética dessa manifestação na sua estrita correlação com as finalidades comunicacionais desses gêneros de ícones. Nesse ponto, o caráter propriamente estético de sua manifestação encontra-se delimitado pelos aspectos inevitavelmente discursivos do agenciamento no qual encontramos submetido o regime da produção e da recepção da imagem visual na contemporaneidade. E, nesse sentido, finalmente, podemos dizer que uma interrogação às regências discursivas das imagens, em contextos mediáticos (e sob uma ênfase nos aspectos propriamente visuais da manifestação desta regência), concerniria possivelmente ao universo daquilo que muitos autores chamam de uma “estética da comunicação”.1 Em determinados ramos da investigação sobre a imagem, é o problema mais elementar da presença de formas textuais no corpo icônico das representações visuais que motivou uma indagação sobre as relações entre imagem e discurso. Esse problema nem sempre foi exclusivo das abordagens semiológicas, muito embora o campo da comunicação tenha se comportado como se esse fôsse o caso (assumindo inclusive um certo tipo de perplexidade da qual boa parte dos semiólogos parecem sofrer ao tratar da questão); fora de nosso campo de estudos, não encontramos esse mesmo tipo de perplexidade e de necessidade de estrita demarcação (ou, pior ainda, de redução) entre domínios como os da imagem e do discurso. R i t d A i ã N i l d 1 O acordo acerca do real significado de um sintagma como estética da comunicação ainda parece longe de nosso alcance, ao menos naquilo que podemos designar como uma disciplina ou um saber de limites mais ou menos claros: na perspectiva sugerida por autores como Herman Parret (1997), o que se chama por esse nome concerne à questão da essencial comunicabilidade de nossos afetos, e o modo como o domínio da sensibilidade deve ser tomado como matricial da (ou, num linguajar mais próprio, transcendente à) experiência cooperativa na qual as teorias pragmáticas fixaram o problema da compreensão textual, como um todo. Numa outra vertente à qual se faz apelo à idéia de uma estética da comunicação, é a indagação sobre os meios de comunicação que está, por sua vez, no centro das preocupações de uma disciplina como a estética e, nesse caso, trata-se de dimensionar, nos limites próprios a essa disciplina filosófica, toda ordem de interrogações que se lançaram sobre as estratégias expressivas e às estruturas prévias da compreensão mobilizadas por esses mesmos media (CAUNE, 1997). Nesses termos, a questão do valor textual da imagem passaria a ter uma menor correlação com o problema de sua hipotética redução às estruturas lingüísticas do discurso enunciativo, e mais com aspectos de provocação que as imagens podem suscitar de nossos juízos perceptivos e de nossos modos de contemplação e fruição propriamente visuais. Ainda assim, entretanto, o fato de que estamos tratando de imagens que se definem pelo seu valor discursivo (pelo modo como se coordenam com funções próprias à compreensão de sentenças Ressentimo-nos, por vezes, do fato de que a aproximação metodológica de nosso próprio campo de estudos ao universo da produção, da fruição e da interpretação das imagens não pareceu valorizar suficientemente a importante fortuna nocional e metodológica da análise que as chamadas ciências da arte (aí compreendidas as teorias poéticas, estéticas e a própria história da arte) aportaram de maneira permanente e continuada para essa discussão sobre o valor comunicacional intrínseco ao universo dos ícones visuais, sobretudo em suas manifestações de www.e-compos.org.br \| E-ISSN 1808-2599 \| Tomemos em tela o caso dos historiadores da arte, por exemplo: para eles, em geral, a questão da presença de segmentos textuais no corpo da imagem não é suscitada pelas mesmas obsessões teóricas que ainda afligem os teóricos da significação, mas obviamente por questões próprias ao campo da investigação histórica das formas da expressão visual. Mas, nem mesmo por isso, estão eles privados de um acesso fenomenologicamente interessante ao caso em questão, isto é, às relações entre formas textuais e a matéria icônica da qual se compõe necessarimente uma representação pictórica. constituiu, em geral, uma lacuna na exploração dos historiadores das formas visuais do passado e do nosso tempo.2 O caso de Meyer Schapiro (2000) é uma exceção a esse diagnóstico e um caso particularmente ilustrativo dos pontos de contato entre uma abordagem ao mesmo tempo histórica e semiótica do problema das regências textuais da imagem e, em particular, da questão da presença de segmentos verbais, a título de quaisquer funções que sejam (e, no caso da história da arte, eles nem sempre são da ordem de um favorecimento de funções narrativas ou ficcionais, por exemplo): em primeiro lugar, a questão da presença do texto na imagem é (ao menos para o historiador das imagens) uma questão ligada às exigências às quais a arte da representação esteve submetida, em determinados períodos, sobretudo quando consideramos sua relação com certas instituições como a Igreja, o Estado, o mecenato e mesmo o campo da arte, este considerado como espaço de disputas e de negociação de prestígios. Mesmo para um autor como Gombrich (tão afeito, por seu turno, aos problemas e métodos mais tradicionais do historiador da arte), a questão das relações entre imagem e texto também perpassou seus interesses de especulação, em determinados instantes (GOMBRICH, 1985): em seu caso em especial, essa preocupação se deu a título de uma caracterização das estratégias de programação de efeitos na arte do século XX (portanto, como uma questão ligada à definição dos estilos de certos períodos da modernidade artística). 2 Palavra, imagem e a iconicidade das formas textuais Propomos assim que uma abordagem desses aspectos de regência textual das imagens no contexto da cultura mediática deverá ser complementada por um discurso teórico (e de resultantes metodológicas mais claras) sobre as funções propriamente comunicacionais 1 O acordo acerca do real significado de um sintagma como estética da comunicação ainda parece longe de nosso alcance, ao menos naquilo que podemos designar como uma disciplina ou um saber de limites mais ou menos claros: na perspectiva sugerida por autores como Herman Parret (1997), o que se chama por esse nome concerne à questão da essencial comunicabilidade de nossos afetos, e o modo como o domínio da sensibilidade deve ser tomado como matricial da (ou, num linguajar mais próprio, transcendente à) experiência cooperativa na qual as teorias pragmáticas fixaram o problema da compreensão textual, como um todo. Numa outra vertente à qual se faz apelo à idéia de uma estética da comunicação, é a indagação sobre os meios de comunicação que está, por sua vez, no centro das preocupações de uma disciplina como a estética e, nesse caso, trata-se de dimensionar, nos limites próprios a essa disciplina filosófica, toda ordem de interrogações que se lançaram sobre as estratégias expressivas e às estruturas prévias da compreensão mobilizadas por esses mesmos media (CAUNE, 1997). 1 O acordo acerca do real significado de um sintagma como estética da comunicação ainda parece longe de nosso alcance, ao menos naquilo que podemos designar como uma disciplina ou um saber de limites mais ou menos claros: na perspectiva sugerida por autores como Herman Parret (1997), o que se chama por esse nome concerne à questão da essencial comunicabilidade de nossos afetos, e o modo como o domínio da sensibilidade deve ser tomado como matricial da (ou, num linguajar mais próprio, transcendente à) experiência cooperativa na qual as teorias pragmáticas fixaram o problema da compreensão textual, como um todo. Numa outra vertente à qual se faz apelo à idéia de uma estética da comunicação, é a indagação sobre os meios de comunicação que está, por sua vez, no centro das preocupações de uma disciplina como a estética e, nesse caso, trata-se de dimensionar, nos limites próprios a essa disciplina filosófica, toda ordem de interrogações que se lançaram sobre as estratégias expressivas e às estruturas prévias da compreensão mobilizadas por esses mesmos media (CAUNE, 1997). www.e-compos.org.br \| E-ISSN 1808-2599 \| www.e-compos.org.br \| E-ISSN 1808-2599 \| caso das abordagens classicamente semiológicas. No caso da exploração histórica da arte cristã do medievo, o estudo sobre a incidência de formas de escitura na matéria visual de pinturas é uma peculiar decorrência do problema mais geral (e muito característico de uma história da cultura), a saber, o da introdução de meios de divulgação como o livro impresso; é precisamente em tais condições que Schapiro (2000) nos introduz a questão das relações entre imagem e texto na arte paleo-cristã do alto medievo; neste mesmo sentido, as questões semiológicas estarão sempre submetidas às condicionantes de uma descrição histórica do fenômeno. contexto temporal: nesse sentido, Schapiro é reconhecidamente um dos historiadores da arte que, desde muito cedo, mais familiaridade manifestou com respeito aos instrumentais da semiótica e da semiologia para o trabalho de análise histórica das formas representacionais, e sua obra exibe claramemente as provas dessa sensibilidade semiológica requerida ao trabalho do historiador (SCHAPIRO, 1969). Para além das separações apressadas entre o texto e a imagem, tomadas como naturezas distintas, Schapiro sempre procurou compreender o fenômeno da representação visual a partir da admissão de um regime de integração entre a formas da escritura e da imagem, tomando essa mesma síntese no contexto da forma propriamente visual da representação pictórica (e, nesse sentido, é que pôde assim acolher, com a devida propriedade, os instrumentais de uma semiologia aplicada ao estudo das formas visuais). Se desejamos assimilar a fortuna dessa exploração teórica, incorporando-a ao acervo das manifestações mais recentes de uma retórica visual, devemos ser capazes de isolar do trabalho de autores como Schapiro aquilo que é da ordem dos operadores metodológicos de sua análise histórica, de modo a nos reportarmos às estratégias textuais da imagem contemporânea, a partir das mesmas chaves que o permitiram descrever esse fenômeno da relação entre texto e imagem em ícones da arte da cristandade. Muito embora tenham se interrogado sobre a incidência de formas textuais no corpo da imagem, a indagação sobre os regimes estruturais de funcionamento textual da representação pictórica 2 No caso de Gombrich (1983), questões dessa natureza apenas emergem quando ele precisa lidar com as manifestações mais contemporâneas da expressão visual, muito especialmente no campo das artes gráficas e nas modalidades de expressão visual, próprias do universo dos meios de comunicação de massa. As manifestações desse interesse são esparsas pela obra gombricheana, mas destacamos aqui uma referência à análise do trabalho do cartunista Saul Steinberg, objeto da admiração do historiador e assunto de alguns de seus ensaios, mesmo aqueles que encontramos em suas obras mais conhecidas, como Art and Illusion. (GOMBRICH, 1959; 1983). 2 No caso de Gombrich (1983), questões dessa natureza apenas emergem quando ele precisa lidar com as manifestações mais contemporâneas da expressão visual, muito especialmente no campo das artes gráficas e nas modalidades de expressão visual, próprias do universo dos meios de comunicação de massa. As manifestações desse interesse são esparsas pela obra gombricheana, mas destacamos aqui uma referência à análise do trabalho do cartunista Saul Steinberg, objeto da admiração do historiador e assunto de alguns de seus ensaios, mesmo aqueles que encontramos em suas obras mais conhecidas, como Art and Illusion. (GOMBRICH, 1959; 1983). implicam sua assimilação prévia ao plano icônico da representação visual. Esse mesmo fenômeno é evidente, por exemplo, no emprego que fazem dela certas escolas pictóricas do século XIX: os exemplos de Schapiro (bem díspares, por sinal) vêm de Courbet, Manet e de Homer, e dos modos como o recurso à assinatura do pintor é plasticamente integrado às funções figurativas da pintura, muitas vezes até tomado como elemento complementar do fundo da composição, da paisagem ou do cenário interior, por exemplo (SCHAPIRO, 2000). Quando examinamos a questão da presença do texto no interior da imagem, num caso como o dos quadrinhos, verificamos que a incidência das formas de uma escritura não possui apenas a função de indicar a fala ou o pensamento das personagens (ou de um narrador, no caso das legendas), mas é igualmente um segmento das próprias formas visuais dessa arte, juntamente com aquelas que definem a caracterização das figuras condutoras da ação: de um lado, temos as formas visuais mesmas que abrigam as incrições (sejam elas da ordem da legenda ou dos balões, que representam a expressão verbal ou o pensamento das personagens); de outro, as inscrições textuais propriamente ditas, e que constituiriam um material à parte da significação icônica, não fossem elas mesmas também investidas de um certo grau de expressividade manifesta numa forma que é propriamente iconico-visual. Quando examinamos a questão da presença do texto no interior da imagem, num caso como o dos quadrinhos, verificamos que a incidência das formas de uma escritura não possui apenas a função de indicar a fala ou o pensamento das personagens (ou de um narrador, no caso das legendas), mas é igualmente um segmento das próprias formas visuais dessa arte, juntamente com aquelas que definem a caracterização das figuras condutoras da ação: de um lado, temos as formas visuais mesmas que abrigam as incrições (sejam elas da ordem da legenda ou dos balões, que representam a expressão verbal ou o pensamento das personagens); de outro, as inscrições textuais propriamente ditas, e que constituiriam um material à parte da significação icônica, não fossem elas mesmas também investidas de um certo grau de expressividade manifesta numa forma que é propriamente iconico-visual. Revista da Associação Nacional dos Programas de Pós-Graduação em C Nesses termos, a presença de formas textuais no contexto dos ícones visuais funciona decerto a títulos diversos, mas sempre sob um aspecto no qual a escritura é inerente à imagem mesma, ela é uma das inscrições pelas quais podemos caracterizar os ícones visuais (do mesmo modo que o são as linhas, contornos, cores), uma vez que estes sejam definidos a partir de sua constituição plástica: isso quer dizer que as funções que o texto preenche, seja no plano informacional, descritivo ou narrativo, De qualquer maneira, trata-se aqui de um modo de endereçar questões sobre a periodização dos estilos e tradições da representação visual que é capaz, ao mesmo tempo, de acolher formas de descrição, digamos assim, mais “internas” ao fenômeno, no momento mesmo em que ele é caracterizado na sua vinculação com seu próprio www.e-compos.org.br \| E-ISSN 1808-2599 \| www.e-compos.org.br \| E-ISSN 1808-2599 \| www.e-compos.org.br \| E-ISSN 1808-2599 \| a variedade dos materiais através dos quais a significação icônica é elaborada. a variedade dos materiais através dos quais a significação icônica é elaborada. que opera de uma forma tal que a análise dos segmentos de escritura jamais estará completamente dissociada das funções que são próprias ao segmento visual de sua apresentação (FRESNAULT-DERUELLE, 1970). O que evidentemente diferencia essas abordagens é decerto a finalidade através da qual se opera essa unificação de métodos de análise, seja na semiótica visual ou nas ciências da arte: já vimos em outras ocasiões que o argumento de Eco sobre a possibilidade de se descrever os princípios estruturais das mensagens visuais investia fortemente contra a subordinação do valor comunicacional das imagens aos princípios da significação descritos pela linguística estrutural; desse modo, poder-se- ia supor que a proposta de Eco investiria numa possível analiticidade ou independência dos códigos icônicos em relação a outras ordens da simbolicidade da compreensão (ECO, 1968). Poderíamos multiplicar aqui os casos em que a relação entre formas escritas e formas plásticas se negociam, no modo de construir um sentido discursivo, que associamos às imagens: mas é necessário que nos detenhamos nesse percurso momentaneamente para reconhecer que a questão da significação textual das imagens não deve se restringir exclusivamente a esse plano de relações entre formas de escritura e formas visuais, mas é algo que pode ser abordado no interior da própria estrutura icônica das representações visuais. O fato de que assimilamos muitas vezes os ícones a enunciados não decorre do fato de que um texto esteja associado a elas, mas decerto ligado a determinantes que são constitutivas da própria plasticidade da imagem. Revista da Associação Nacional dos Programas de Pós-Graduação em Comunicação \| E-compós, Essa suposta característica dos códigos visuais decorreria de um aspecto manifesto da perplexidade das teorias semióticas com respeito ao regime discursivo das imagens, a saber, o das possíveis diferenças entre a descrição verbal e a representação pictórica: é comum entre os semioticistas (mas também entre certos estetas) que se indague sobre as relações estruturais que se pode estabelecer entre uma figuração visual uma descrição verbal. Do ponto de vista das abordagens semióticas de segunda geração, assume-se que o regime de representação no qual as imagens funcionam não pode ser o mesmo daquele que define os enunciados linguísticos; uma decorrência metodológica dessa admissão é Por outro lado, Schapiro distingue na arte paleocristã do medievo dois tipos de tratamento da introdução do texto à imagem, um dos quais prima por inscrever a forma da escrita como motivo figurativo (como inscrições alfabéticas em livros e pergaminhos), e outra, na qual a presença de uma escritura como que prenuncia uma função de representação da expressão verbal das personagens do quadro, e que será característica, por sua vez, do uso dos balões nos quadrinhos. Assim sendo, o uso dos filactérios com o fim de identificar aspectos do que uma personagem ou apóstolo vaticinam (através da fala) implica em um tratamento anti-natural da inserção do verbo na imagem; ainda que os motivos dessa inscrição sejam naturais (um pergaminho que compõe a cena funcionando a título de suporte de uma fala), sua função é a de representar um elemento que a rigor não poderia ser visualizável, na pintura (a não ser através de um recurso arbitrário), isto é, a faculdade da expressão verbal das personagens. Revista da Associação Nacional dos Programas de Pós-Graduação em Comunicaçã Do mesmo modo que Schapiro destaca o valor icônico das inscrições presentes na arte cristã do medievo (pelo modo como a escritura se deixa assimilar pelo contexto plástico da imagem mesma), idenficamos na arte dos quadrinhos o mesmo tipo de assimilação do texto que não o converte em um ente estranho à imagem (como se fosse uma espécie de apêndice complementar à função das imagens): certos especialistas chamam-nos a atenção para a necessidade de considerar a questão do texto visual nos quadrinhos como o efeito de uma relação entre segmentos icônicos e verbais www.e-compos.org.br \| E-ISSN 1808-2599 \| 3 Semiose visual e as estruturas da percepção Num outro caso (como o da semiótica visual de Umberto Eco), a ordem de problemas se altera substancialmente, mas o ponto que defendemos permanece em sua integridade, isto é: identificamos aqui (agora na perspectiva de trabalho própria de um semioticista) preocupações similares àquelas do historiador da arte, ao propor-se integrar os campos da análise de materiais visuais, levando em conta www.e-compos.org.br \| E-ISSN 1808-2599 \| É justamente em sua obra seminal, Art and Illusion (GOMBRICH, 1959), que encontramos um forte discurso sobre a necessidade de se avaliar a pertinência da questão da ilusão e das estratégias da representação figurativa, nem tanto no âmbito das grandes tradições artísticas, mas no que Gombrich designa como sendo o “comércio diário com figuras e imagens de toda espécie”: devemos estar atentos a não recuperar o tema da ilusão como se fosse um motivo para reintroduzir na discussão sobre as artes visuais a questão dos vínculos ontológicos entre representação e realidade. Não obstante, temos em vista ainda hoje uma espécie de franquemento das estratégias de apelo figurativo que pareceriam à tradição artística algo próximo do mágico: o interesse histórico pelo tema da ilusão artística é algo que se põe contemporaneamente na exata medida do barateamento de uma experiência da figuração na atualidade. esquematismo último da representação pictórica, sendo a este propósito que Eco faz menção aos problemas centrais de Art and Illusion, sobretudo na segunda parte de La Strutura Assente, quando discute as relações entre o problema da representação de propriedades visuais do espaço e o desenvolvimento de códigos propriamente estéticos (ECO, 1968). esquematismo último da representação pictórica, sendo a este propósito que Eco faz menção aos problemas centrais de Art and Illusion, sobretudo na segunda parte de La Strutura Assente, quando discute as relações entre o problema da representação de propriedades visuais do espaço e o desenvolvimento de códigos propriamente estéticos (ECO, 1968). nal dos Programas de Pós-Graduação em Comunicação \| E-compós, Brasília, v.11, n.2, maio/ago. 2008. Mais do que isso, o contato (polêmico ou não) entre Eco e Gombrich nos serve como sinalização para esta complementaridade possível entre as abordagens textuais da imagem (de inspiração predominantemente semiótica) e aquelas que valorizam na representação os aspectos, por assim dizer, internos de sua constituição. que os códigos perceptivos aportam, no modo como a representação é capaz de instaurar um mundo visual e de se reportar a ele, através das formas representacionais particulares. Isto tudo significa que a suposta língua das imagens é, em muitos casos, o efeito de uma síntese que os códigos da representação visual realizam, a partir de entidades originárias da experiência perceptiva (esta tomada em seu aspecto estrutural, e não empírico). a de que os regimes textuais da imagem poderiam ser descritos a partir de uma plataforma não necessariamente lingüística. Um aspecto interessante das abordagens semióticas de Eco (1968) sobre o regime discursivo da imagem diz repeito à gênese das figuras de base na formação dos semas visuais, em um hipotético código icônico: quando nos indagamos sobre as entidades que originam essas mesmas figuras de uma estrutura da significação icônica, vemos que as regras para a composição de representações não parecem suficientes para explicar como é que compreendemos a relação entre uma imagem e aquilo que ela representa ou significa (supondo especialmente que a significação da imagem seja um modo que lhe é próprio de dizer alguma coisa). Na melhor das hipóteses, essas mesmas regras são apenas cristalizações momentâneas de um certo modo de pensar a ligação entre as imagens e seu fundamento ou seus temas visuais, mas não exprimem, per se, esse mesmo fundamento pelo qual os ícones se restituem a seus objetos, enquanto parte de uma estrutura inerente a seu funcionamento representacional. Nessas condições, há que considerar que o código das representações se constitui com base em figuras de sentido cuja origem não está no prórpio sistema simbólico das representações visuais, mas sim em um outro tipo de código, mais analítico do que ele: no caso das imagens, portanto, há que se considerar o papel mais fundamental www.e-compos.org.br \| E-ISSN 1808-2599 \| www.e-compos.org.br \| E-ISSN 1808-2599 \| De fato, o recurso a idéias como as de Gombrich nos convida praticamente a uma excursão que nos conduzirá ao estilo de toda uma tradição de reflexão sobre fatos artísticos que poderia ter, por sua vez, enorme valia para uma aproximação à dimensão comunicacional das representações visuais: há um volume considerável de referências a esses pontos de contato entre as disciplinas da interpretação e as ciências da arte, e que parecem justificar o esforço pelo estabelecimento de algum tipo de contato mais produtivo entre esses campos, sobretudo numa área de estudos como a da comunicação.3 10/18 O tratamento isolado que possamos propiciar ao debate entre Eco e Gombrich revelará, em nossa opinião, um aspecto central de uma abordagem ecológica ou perceptualista dos significados visuais (e que instanciará, igualmente, os pontos de contato que propomos aqui entre as abordagens da semiótica, da estética e da história da arte): este ponto diz respeito às teses gombricheanas acerca do 3 Não enumeraremos a imensa quantidade de literatura secundária acerca das relações entre as disciplinas do campo das artes e as teorias da interpretação. Preferimos apenas nos restituir a um dos marcos fundamentais desse possível ponto de encontro entre o valor da obra visual e as condições de sua interpretação: destacamos aqui o papel que a proposição de uma disciplina como a iconologia teve no sentido de dimensionar o valor das obras de uma tradição como decorrência do modo como seus temas e figuras centrais são permanentemente avaliados, através de uma interpretação regrada (de tal modo que sua análise histórica passa a colocar no centro das atenções a questão dos métodos cogitativos empregados nessa mesma avaliação). O programa avançado destas pesquisas é sumariado exemplarmente por Panofsky, na introdução de seu Meaning in the Visual Arts (PANOFSKY, 1979). 3 Não enumeraremos a imensa quantidade de literatura secundária acerca das relações entre as disciplinas do campo das artes e as teorias da interpretação. No caso específico da tradição à qual se encontra vinculado o trabalho de Gombrich, o aspecto mais interessante de sua assimilação às abordagens semióticas da análise das representações visuais parece decorrer justamente de uma falta constitutiva de seu projeto original de pesquisa: certos comentadores de sua obra ressaltam esse dado de tensão que marca a origem mesma da investigação gombricheana sobre os mistérios do estilo na história, justamente quando essa exploração passa a valorizar aspectos da gênese das formas visuais que o afastam da possibilidade de uma compreensão sobre a dimensão temporal (ou histórica) desse mesmo processo (LOPES, 1996). No caso específico da tradição à qual se encontra vinculado o trabalho de Gombrich, o aspecto mais interessante de sua assimilação às abordagens semióticas da análise das representações visuais parece decorrer justamente de uma falta constitutiva de seu projeto original de pesquisa: certos comentadores de sua obra ressaltam esse dado de tensão que marca a origem mesma da investigação gombricheana sobre os mistérios do estilo na história, justamente quando essa exploração passa a valorizar aspectos da gênese das formas visuais que o afastam da possibilidade de uma compreensão sobre a dimensão temporal (ou histórica) desse mesmo processo (LOPES, 1996). gênero de análise histórica em Gombrich, e deixarmos sedimentar-se o horizonte prático dessa indagação é o que nos permitirá endereçarmos o trabalho do historiador da arte em sua validade teórico-metodológica para a análise dos materiais visuais de nossa própria época. gênero de análise histórica em Gombrich, e deixarmos sedimentar-se o horizonte prático dessa indagação é o que nos permitirá endereçarmos o trabalho do historiador da arte em sua validade teórico-metodológica para a análise dos materiais visuais de nossa própria época. Com isso, queremos dizer que certas questões de método do historiador da arte podem ter alguma incidência no modo como podemos explorar, no universo das representações visuais do campo mediático, a presença de uma regência discursiva: assumimos que a origem dessa mesma regência diz respeito, por sua vez, ao modo como a dimensão visual destes ícones é organizada enquanto matriz de significações relativamente independentes das formas que essas constrições assumem no tempo (mas certamente dependentes, por sua vez, de condições dadas no plano de uma estrutura perceptiva). 4 A animação na imagem fixa e os princípios de uma discursividade icônica Preferimos apenas nos restituir a um dos marcos fundamentais desse possível ponto de encontro entre o valor da obra visual e as condições de sua interpretação: destacamos aqui o papel que a proposição de uma disciplina como a iconologia teve no sentido de dimensionar o valor das obras de uma tradição como decorrência do modo como seus temas e figuras centrais são permanentemente avaliados, através de uma interpretação regrada (de tal modo que sua análise histórica passa a colocar no centro das atenções a questão dos métodos cogitativos empregados nessa mesma avaliação). O programa avançado destas pesquisas é sumariado exemplarmente por Panofsky, na introdução de seu Meaning in the Visual Arts (PANOFSKY, 1979). www.e-compos.org.br \| E-ISSN 1808-2599 \| 4 A animação na imagem fixa e os princípios de uma discursividade icônica Curiosamente, entretanto, é desta mesma tensão entre duas dimensões constitutivas dos fatos artísticos (isto é, o apelo que toda representação faz às condições de sua percepção, de um lado, e o fato de que essas mesmas condições se manifestam de modo diverso, conforme condições culturais e históricas específicas) que nos permitirá assimilar o estilo no qual Gombrich formula uma questão de história da cultura, mas sem reter dela os aspectos que são próprios à exploração de um historiador das formas artísticas. Tratemos dessa hipotética proximidade de métodos entre a semiótica e as ciências da arte, a partir de alguns casos concretos: em nossas próprias investigações sobre as modalidades de discursividade visual próprias ao fotojornalismo, temos explorado certos insights de Gombrich sobre a função dos gestos e da atitude corporal na configuração de um sentido narrativo para representações pictóricas como um modelo de análise de nossos próprios materiais (sobretudo no modo como essas imagens se restituem a contextos de uma ação que foi reportada visualmente). A capacidade de fazermos emergir a propriedade dos instrumentais analíticos e descritivos desse www.e-compos.org.br \| E-ISSN 1808-2599 \| Desse modo, a abordagem que Gombrich nos oferece sobre os aspectos ritualísticos e expressionais que encontramos no tratamento da atitude humana em representações de ações nos põe em contato com uma riquíssima chave metodológica para a interpretação destes mesmos motivos, no contexto do fotojornalismo, por exemplo, desde que sejamos capazes de separar da análise dos operadores internos desses materiais (ao menos, momentaneamente) os propósitos que inspiram uma abordagem como a de Gombrich (e o modo como ela se liga a um programa de pesquisas como o da iconologia, a partir de Warburg). No caso da assimilação dos padrões da composição de ícones visuais às regras da imitação na poesia dramática, é necessário considerar-se (na perspectiva do historiador) os tipos de demanda feitos ao campo artístico, em certos períodos: aparentemente, segundo Gombrich (1959), há uma passagem na arte da Antigüidade clássica na qual os motivos da representação começam a deixar de atender a finalidades próprias de uma arte conceitual (a rememoração, o culto à permanência) para exprimir visualmente o sentido da mudança, da transformação (próprios à representação das ações através da narrativa); este aspecto de contextualização temporal de uma interrogação às funções textuais da imagem é algo que devemos ter em vista, em nossas próprias investigações, mas apenas como um dado de pressuposto sobre os limites preliminares entre as abordagens semióticas da imagem e aquelas que definem o interesse das ciências da arte, em geral, sobre o mesmo tópico. Neste caso, temos que negligenciar (mais uma vez, provisoriamente) o fato de que as relações entre imagem e regimes textuais remonta à caracterização do que Gombrich designa como sendo a “revolução grega” nas artes visuais, isto é, o momento em que a cultura artística da Antigüidade clássica passou a assimilar princípios miméticos da construção de representações de ações, agora no contexto da representação pictórica e escultórica: há um aspecto da questão que é tipicamente próprio à indagação sobre uma história dos estilos artísticos e que, justamente por isso, requer que a identificação de certos traços internos da manifestação das formas expressivas estejam permanentemente correlacionados com uma investigação sobre os padrões culturais de certas épocas e períodos. Há, por exemplo, uma mui característica complementaridade entre o modo como o arresto da imagem fixa um valor expressional para o gesto humano, e o caráter indicativo que esse mesmo gesto assume para a disposição de uma personagem para a ação (ou como indicativo das paixões a que ela está submetida), e que demarca, por sua vez, o estilo no qual muitas fotografias se restituem a um contexto narrativo ou dramático dos acontecimentos que elas representam. leva Gombrich a tematizar o problema da representação dos gestos, como que cindido entre a expressividade (inerente aos sintomas) e a ritualidade (própria, por seu turno, aos símbolos): assim sendo, é evidente que a gramaticalidade das expressões gestuais oferece uma espécie de estrutura de base, sobre a qual podemos compreender o modo como o artista apreende a comunicação entre os elementos vivos de uma composição (sejam estes humanos ou não). uação em Comunicação \| E-compós, Brasília, v.11, n.2, maio/ago. 2008. Nesse ponto do argumento gombricheano, muitos comentadores identificam na questão do caráter codificado do gesto o prosseguimento de uma questão permanentemente posta para a história da arte, em seu status de disciplina científica, a saber, o da natureza de determinação dos estilos pictóricos: no caso da linhagem de onde as questões de Gombrich se derivam, esse é o problema da gênese de uma cultura visual clássica, no Renascimento, e o fato de que esta assimilação dos modelos representacionais da Antigüidade se opera através do que Aby Warburg chamará de “fórmulas do patético” (GUINZBURG, 1990). Com isso, dizemos que a mera impregnação indexical, propiciada pela natureza do dispositivo fotográfico, não é capaz de explicar como é que assimilamos essa imagem a um determinado valor de discurso: para que isso se dê, é evidente que o arresto da expressão gestual das personagens tenha se coligado com uma espécie de cânone das representações da atitude e das paixões humanas, na pintura e na escultura. Um modo possível de enquadrar esse aspecto da significação gestual é o de reconhecer, especialmente em seu emprego na representação visual, sua dimensão de ato ritualizado: na perspectiva de certos historiadores da arte, é o caráter fortemente convencionado de certos gestos que oferece à pintura os materiais pelos quais a apresentação dinâmica dos motivos, na percepção, será selecionada para a representação pictórica. É precisamente este aspecto ritualizado das atitudes e das paixões corporais que www.e-compos.org.br \| E-ISSN 1808-2599 \| a da Associação Nacional dos Programas de Pós-Graduação e Por outro lado, é evidente que a novidade de uma abordagem sobre as transformações do estilo figurativo, propostas por Gombrich, implicam em um aprofundamento de questões que a tradição da história da arte não teria podido explorar com fecundidade, se mantivesse seu foco de interesse concentrado nos limites de seus próprios instrumentais conceituais e metodológicos. Vários comentadores dessa tradição que i i i l i di i li é i www.e-compos.org.br \| E-ISSN 1808-2599 \| FRESNAULT-DERUELLE, Pierre. Le verbal dans les bandes dessinées. Communications, n. 15, p. 145- 161, 1970. GOMBRICH, E.H. Art and illusion: a study in the psychology of pictorial representation. New York: Princeton University Press, 1959. GOMBRICH, E.H. Art and illusion: a study in the psychology of pictorial representation. New York: Princeton University Press, 1959. ______. Image and word in 20th century art”. Word and image, n. 1, vol. 3, 1985, p. 213-241. ______. Image and word in 20th century art”. Word and image, n. 1, vol. 3, 1985, p. 213-241. ______. The wit of Saul Steinberg. Art Journal, winter, 1983, p. 377-380. ______. Image and word in 20th century art”. Word and image, n. 1, vol. 3, 1985, p. 213-241. ______. The wit of Saul Steinberg. Art Journal, winter, 1983, p. 377-380. O estudo das assim designadas “imagens demóticas” estabelece que o universo da representação estética mantém uma relação fundamental com regimes outros nos quais encontramos o funcionamento desse tipo de imagens: podemos até mesmo estabelecer uma espécie de paralelo entre o tratamento estético propiciado a elas e aquele que nobilita, na filosofia da linguagem, a importância atribuída ao universo da linguagem ordinária; aqui e ali, as realizações propriamente estéticas e lingüísticas mais “elevadas” (no campo, por exemplo, das metáforas e das alegorias) encontram suas raízes deitadas na estrutura do discurso comum e das relações com as imagens da cultura contemporânea. GUINZBURG, Carlo. De A. Warburg a E.H. Gombrich: notas sobre um problema de método. In: Mitos, emblemas, sinais: morfologia e História. Tradução: Federico Carotti. São Paulo: Cia das Letras, 1990, p. 41-94. LOPES, Dominic. Understanding Pictures. Oxford: Clarendon Press, 1996. PANOFSKY, Erwin Iconografía e Iconología: introducción al estudio del arte del Renacimiento. In: El Significado em las Artes Visuales (trad. Nicanor Ancochea). Madrid: Alianza, 1979. p. 45-75. Referências Bibliográficas BARTHES, Roland. «La rhétorique de l’image». In: Communications, n. 4, p. 40-51, 1964. BARTHES, Roland. «La rhétorique de l’image». In: Communications, n. 4, p. 40-51, 1964. CAUNE, Jean. Esthétique de la Communication. Paris: PUF, 1997. Assim sendo, não se deveria restringir seu campo de observação às imagens figurativas, compostas com o fim de gerar um tipo de experiência puramente fruitiva (como é o caso das imagens artísticas), mas também as funções que caracterizam a compreensão de figurações como as que encontramos em mapas, em imagens de notas de moeda corrente, cenas mitológicas, sinais heráldicos, marcas de produtos e instituições, entre outros. Assim sendo, não se deveria restringir seu campo de observação às imagens figurativas, compostas com o fim de gerar um tipo de experiência puramente fruitiva (como é o caso das imagens artísticas), mas também as funções que caracterizam a compreensão de figurações como as que encontramos em mapas, em imagens de notas de moeda corrente, cenas mitológicas, sinais heráldicos, marcas de produtos e instituições, entre outros. ECO, Umberto. La Struttura Assente. Milano: Bomipiani, 1968. ECO, Umberto. La Struttura Assente. Milano: Bomipiani, 1968. FRESNAULT-DERUELLE, Pierre. Le verbal dans les bandes dessinées. Communications, n. 15, p. 145- 161, 1970. dos estudos sobre fatos artísticos ressaltam o valor próprio que caracteriza a abordagem de Gombrich (muito especialmente esse aspecto da introdução dos saberes psicológicos para a compreensão do modo como os estilos visuais se sedimentam), sobretudo em comparação com o modo como a linhagem intelectual que o gerou havia enfrentado o desafio de uma história dos estilos artísticos; e é precisamente esse aspecto da assimilação de uma psicologia da representação pictórica num projeto de periodização dos estilos que devemos contemplar como chave para a discussão sobre a natureza dos regimes textuais nos quais encontramos a imagem visual funcionando, em nossos dias (GUINZBURG, 1990). capacidade de ser codificada num plano meta- semiósico (como no caso das teorias semiológicas de primeira geração), mas se impõe à experiência como uma presença hipnótica, ainda que haja uma tarefa própria à instância da recepção, na constituição dos percursos visuais através dos quais as formas pictóricas serão restituídas a seu poder de comunicação (COMETTI; MORIZOT; POUIVERT, 2000). Nesse sentido, devemos conceber que se estabelecem entre abordagens semióticas e estéticas um insuspeito vínculo cooperativo: ainda que o efeito da imagem se ofereça no modelo de uma visibilidade de segunda ordem, do ponto de vista da compreensão, esta vicariedade da visão representacional é assimilada como um regime de leitura, no modo como certos semiólogos da segunda geração pensam a respeito de um princípio textual de organização dos materiais visuais na sua forma representacional. Um aspecto essencial do modo como certas teorias semióticas tentaram incorporar à análise do valor discursivo da imagem os aspectos de articulação próprios à matéria icônica das representações visuais é o fato de que essas mesmas teorias (como parece ser o caso de Umberto Eco) incluem problemas de psicologia da percepção na análise da representação visual: nestes termos, concluímos que a questão da compreensão da imagem em termos de seus regimes textuais de significação assume precisamente o estatuto de uma interrogação próprio a uma experiência perceptiva (e, por isso mesmo, estética), esta tomada em seu sentido mais lato possível. Assim, o modo como compreendemos uma representação visual não deriva de sua CAUNE, Jean. Esthétique de la Communication. Paris: PUF, 1997. COMETTI, Jean-Pierre; MORIZOT, Jacques; POUIVET, Roger. Questions d’esthétique. Paris: PUF, 2000. para a análise do discurso visual. Contracampo. vol. 9, n. 2, p. 95-123, 2004. ______. Ícones, Instantaneidade e Interpretação: por uma pragmática da recepção pictórica na fotografia. Galáxia, n. 9,p. 159-184, 2005. ______. Ícones, Instantaneidade e Interpretação: por uma pragmática da recepção pictórica na fotografia. Galáxia, n. 9,p. 159-184, 2005. SCHAPIRO, Meyer. Les mots et les images. Paris : Macula, 2000. ______. On some problems in the semiotics of visual arts: field and vehicle in image-signs. Semiotica, n. 1, vol. 3, p. 223-242, 1969. Revista da Associação Nacional dos Programas de Pós-Graduação em Comunicação \| E-compós, Brasília, v.11, n.2, maio/ago. 2008. 16/18 Palabras clave Análisis de imágenes. Semiótica. Ciencias del Arte. Revista da Associação Nacional dos Programas de Pós-Graduação em Comunicação \| E-compós, B Keywords Image analysis. Semiotics. Art Sciences. Análisis de imágenes. Semiótica. Ciencias del Arte. Recebido em: 29 de julho de 2008 Aceito em: 11 de novembro de 2008 Resumen We intend to present a brief panorama of the points of contact between two approaches towards the analysis of images, one from semiotics, the other from aesthetics: we take as a standpoint the assumption about the discursive regimes that define the uses of visual representation, in the context of mass communications: in acknowledgement of the dominance of the views of semiotics on the matter, we intend to take into account the perspectives brought about by sciences of art (specially the in the methodological questions introduced by some historians of paintings), assuming to find in these theories certain ways of addressing the psychological structures of the experience of interpreting and appreciating visual icons, which could not be disregarded by a theory of image understanding which assumes that visual icons also function as textual forms. Proponemos un panorama breve de los puntos de contacto entre los abordajes semióticos y estéticos del análisis de las imágenes, teniendo en cuenta el régimen discursivo que las demarca, especialmente en el contexto de la comunicación: reconociendo la predominancia histórica de las perspectivas que la semiología ha aportado a la discusión sobre las regencias textuales de las representaciones visuales, buscamos introducir al debate el punto de vista que las ciencias del arte (muy especialmente las cuestiones de método de abordaje histórico de los hechos artísticos), suponiendo encontrar allí ciertos modos de aproximación a la estructura psicológica de la experiencia de los íconos y de su interpretación y fruición que no podrían ser descuidadas por una teoría de la comprensión de la imagen como forma textual. www.e-compos.org.br \| E-ISSN 1808-2599 \| www.e-compos.org.br \| E-ISSN 1808-2599 \| Modos de comprender imágenes: cuestiones de método sobre el análisis textual de las representaciones visuales Modos de comprender imágenes: cuestiones de método sobre el análisis textual de las representaciones visuales Ways of understanding images: method questions on the textual analysis of visual representations Ways of understanding images: method questions on the textual analysis of visual representations Keywords Image analysis. Semiotics. Art Sciences. Recebido em: 29 de julho de 2008 Aceito em: 11 de novembro de 2008 CONSELHO EDITORIAL Afonso Albuquerque Universidade Federal Fluminense, Brasil Alberto Carlos Augusto Klein Universidade Estadual de Londrina, Brasil Alex Fernando Teixeira Primo Universidade Federal do Rio Grande do Sul, Brasil Alfredo Vizeu Universidade Federal de Pernambuco, Brasil Ana Carolina Damboriarena Escosteguy Pontifícia Universidade Católica do Rio Grande do Sul, Brasil Ana Silvia Lopes Davi Médola Universidade Estadual Paulista, Brasil André Luiz Martins Lemos Universidade Federal da Bahia, Brasil Ângela Freire Prysthon Universidade Federal de Pernambuco, Brasil Antônio Fausto Neto Universidade do Vale do Rio dos Sinos, Brasil Antonio Carlos Hohlfeldt Pontifícia Universidade Católica do Rio Grande do Sul, Brasil Arlindo Ribeiro Machado Universidade de São Paulo, Brasil César Geraldo Guimarães Universidade Federal de Minas Gerais, Brasil Cristiane Freitas Gutfreind Pontifícia Universidade Católica do Rio Grande do Sul, Brasil Denilson Lopes Universidade Federal do Rio de Janeiro, Brasil Eduardo Peñuela Cañizal Universidade Paulista, Brasil Erick Felinto de Oliveira Universidade do Estado do Rio de Janeiro, Brasil Francisco Menezes Martins Universidade Tuiuti do Paraná, Brasil Gelson Santana Universidade Anhembi/Morumbi, Brasil Hector Ospina Universidad de Manizales, Colômbia Ieda Tucherman Universidade Federal do Rio de Janeiro, Brasil Itania Maria Mota Gomes Universidade Federal da Bahia, Brasil Janice Caiafa Universidade Federal do Rio de Janeiro, Brasil Jeder Silveira Janotti Junior Universidade Federal da Bahia, Brasil COMISSÃO EDITORIAL evista da Associação Nacional dos Programas COMPÓS \| www.compos.org.br Associação Nacional dos Programas de Pós-Graduação em Comunicação Presidente Erick Felinto de Oliveira Universidade do Estado do Rio de Janeiro, Brasil erickfelinto@uol.com.br Vice-presidente Ana Silvia Lopes Davi Médola Universidade Estadual Paulista, Brasil asilvia@faac.unesp.br Secretária-Geral Denize Correa Araújo Universidade Tuiuti do Paraná, Brasil denizearaujo@hotmail.com Ana Gruszynski \| Universidade Federal do Rio Grande do Sul, Brasil João Freire Filho \| Universidade Federal do Rio de Janeiro, Brasil Rose Melo Rocha \| Escola Superior de Propaganda e Marketing, Brasil Expediente 18/18 John DH Downing University of Texas at Austin, Estados Unidos José Luiz Aidar Prado Pontifícia Universidade Católica de São Paulo, Brasil José Luiz Warren Jardim Gomes Braga Universidade do Vale do Rio dos Sinos, Brasil Juremir Machado da Silva Pontifícia Universidade Católica do Rio Grande do Sul, Brasil Lorraine Leu University of Bristol, Grã-Bretanha Luiz Claudio Martino Universidade de Brasília, Brasil Maria Immacolata Vassallo de Lopes Universidade de São Paulo, Brasil Maria Lucia Santaella Pontifícia Universidade Católica de São Paulo, Brasil Mauro Pereira Porto Tulane University, Estados Unidos Muniz Sodre de Araujo Cabral Universidade Federal do Rio de Janeiro, Brasil Nilda Aparecida Jacks Universidade Federal do Rio Grande do Sul, Brasil Paulo Roberto Gibaldi Vaz Universidade Federal do Rio de Janeiro, Brasil Renato Cordeiro Gomes Pontifícia Universidade Católica do Rio de Janeiro, Brasil Ronaldo George Helal Universidade do Estado do Rio de Janeiro, Brasil Rosana de Lima Soares Universidade de São Paulo, Brasil Rossana Reguillo Instituto Tecnológico y de Estudios Superiores do Occidente, México Rousiley Celi Moreira Maia Universidade Federal de Minas Gerais, Brasil Sebastião Carlos de Morais Squirra Universidade Metodista de São Paulo, Brasil Simone Maria Andrade Pereira de Sá Universidade Federal Fluminense, Brasil Suzete Venturelli Universidade de Brasília, Brasil Valério Cruz Brittos Universidade do Vale do Rio dos Sinos, Brasil Veneza Mayora Ronsini Universidade Federal de Santa Maria, Brasil Vera Regina Veiga França Universidade Federal de Minas Gerais, Brasil COMPÓS \| www.compos.org.br Associação Nacional dos Programas de Pós-Graduação em Comunicação Presidente Erick Felinto de Oliveira Universidade do Estado do Rio de Janeiro, Brasil erickfelinto@uol.com.br Vice-presidente Ana Silvia Lopes Davi Médola Universidade Estadual Paulista, Brasil asilvia@faac.unesp.br Secretária-Geral Denize Correa Araújo Universidade Tuiuti do Paraná, Brasil denizearaujo@hotmail.com Expediente E-COMPÓS \| www.e-compos.org.br \| E-ISSN 1808-2599 Revista da Associação Nacional dos Programas de Pós-Graduação em Comunicação. Brasília, v.11, n.2, maio/ago. 2008. A identificação das edições, a partir de 2008, passa a ser volume anual com três números. Revista da Associação Nacional dos Programas de Pós-Graduação em Comunicação. Brasília, v.11, n.2, maio/ago. 2008. A identificação das edições, a partir de 2008, passa a ser volume anual com três números. A revista E-Compós é a publicação científica em formato eletrônico da Associação Nacional dos Programas de Pós-Graduação em Comunicação (Compós). Lançada em 2004, tem como principal finalidade difundir a produção acadêmica de pesquisadores da área de Comunicação, inseridos em instituições do Brasil e do exterior. Revista da Associação Nacional dos Programas de Pós-Graduação em Comunicação \| E-compós, Brasília, v.11, n.2, maio/ago. 2008. CONSULTORES AD HOC Aníbal Bragança \| Universidade Federal Fluminense, Brasil Gisela Castro \| Escola Superior de Propaganda e Marketing, Brasil Gislene Silva \| Universidade Federal de Santa Catarina, Brasil Maria Helena Weber \| Universidade Federal do Rio Grande do Sul, Brasil Rosana de Lima Soares \| Universidade de São Paulo, Brasil Tania Hoff \| Escola Superior de Propaganda e Marketing, Brasil REVISÃO DE TEXTO E TRADUÇÃO \| Everton Cardoso ASSISTÊNCIA EDITORIAL E EDITORAÇÃO ELETRÔNICA \| Raquel Castedo ASSISTÊNCIA EDITORIAL E EDITORAÇÃO ELETRÔNICA \| Raquel Castedo

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